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Unsupervised Learning - Large scale data analysis

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Unsupervised Learning - Large scale data analysis

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teens privecy on internet - Social Sciences

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teens privecy on internet - Social Sciences

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https://ieeexplore.ieee.org/abstract/document/4957844

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Internet and Online Information Privacy: An Exploratory Study of Preteens and Early Teens

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<para xmlns:mml=“http://www.w3.org/1998/Math/MathML” xmlns:xlink=“http://www.w3.org/1999/xlink”> Information security and privacy on the internet are critical issues in our society. In this research, we examine factors that influence internet users’ private-information-sharing behavior. Based on a s…

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Criminology - 2014 - BURT - PULLING BACK THE CURTAIN ON HERITABILITY STUDIES  BIOSOCIAL CRIMINOLOGY IN THE POSTGENOMIC ERA

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הקשר בין מקצוע העבודה הסוציאלית ובין מדיניות רווחה חברתית Schwartz-Tayri 2020

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 Racquel fidler : speech disorder in children with ASD

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 Racquel fidler : speech disorder in children with ASD

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racquel fiedler

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מאמר לעבודה בהתפתחותית

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מאמר קונפליקט לימודים-עבודה-משפחה

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PSYCHOLOGICAL INTERVENTION OF MUSIC THERAPY ON

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Lee Krispel

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Abstract

Abstract
In December 2019, an outbreak of pneumonia of unknown origin was reported in Wuhan, Hubei Province, China. Pneumonia cases were epidemiologically linked to the Huanan Seafood Wholesale Market. Inoculation of respiratory samples into human airway epithelial cells, Vero E6 and Huh7 cell lines, led to the isolation of a novel respiratory virus whose genome analysis showed it to be a novel coronavirus related to SARS-CoV, and therefore named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is a betacoronavirus belonging to the subgenus Sarbecovirus. The global spread of SARS-CoV-2 and the thousands of deaths caused by coronavirus disease (COVID-19) led the World Health Organization to declare a pandemic on 12 March 2020. To date, the world has paid a high toll in this pandemic in terms of human lives lost, economic repercussions and increased poverty. In this review, we provide information regarding the epidemiology, serological and molecular diagnosis, origin of SARS-CoV-2 and its ability to infect human cells, and safety issues. Then we focus on the available therapies to fight COVID-19, the development of vaccines, the role of artificial intelligence in the management of the pandemic and limiting the spread of the virus, the impact of the COVID-19 epidemic on our lifestyle, and preparation for a possible second wave.

1. Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the seventh human coronavirus, was discovered in Wuhan, Hubei province, China, during the recent epidemic of pneumonia in January 2020 [ 1, 2]. Since then, the virus has spread all over the world, and as of 20 May 2020, it has infected 4,806,299 people, and caused 318,599 deaths [ 3]. SARS-CoV-2 as well as SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) cause severe pneumonia with a fatality rate of 2.9%, 9.6% and ∼36%, respectively [ 4-6]. The other four human coronaviruses, OC43, NL63, HKU1 and 229E, generally cause self-limited disease with mild symptoms [ 7].

In this review, we summarize the state of art of COrona VIrus Disease 19 pandemic (COVID-19, as defined by the World Health Organization [WHO] in February 2020), including the origin of SARS-CoV-2, its ability to infect human cells, epidemiology, clinical pathological and laboratory findings, molecular and serological diagnosis and safety issues. We also provide information on available therapies, vaccine development, and the potential role of artificial intelligence in the governance of health care systems and its usefulness in fighting the COVID-19 outbreak.

2. Origin of SARS-CoV-2

SARS-CoV-2 is highly similar to bat SARS-like coronaviruses [ 2] and bat might be the reservoir host. RaGT13 is ∼96% identical to SARS-CoV-2 with some differences in the spike receptor binding domain (RBD) that could explain the differences in ACE2 affinity between SARS-CoV-2 and SARS-like coronaviruses.

The polybasic cleavage site of SARS-CoV-2 is not present in pangolin beta-coronavirus, which share similarities with SARS-CoV-2. Also, the sequence of RBD of the spike protein (S) suggests that it arose from a natural evolutionary process [ 8].

3. Epidemiology

3.1. Disease presentation

The cardiovascular system is often affected, with complications including myocardial injury, myocarditis, acute myocardial infarction, heart failure, dysrhythmias, and venous thromboembolic events, and monitoring with high sensitivity cardiac troponin may be useful [ 20].

Patients presenting with acute respiratory distress syndrome may worsen rapidly and die of multiple organ failure [ 12] induced by the so-called "cytokine storm".

Furthermore, heparin may inhibit proteases involved in virus infectivity [ 25]. Indeed, SARS-CoV-2 entry requires the cleavage of the S1-S2 subunits followed by the fusion of S2 to the cell membrane. The latter requires the action of host proteases such as cathepsins, cell surface transmembrane protease/serine proteases (TMPRSS), furin, trypsin and factor Xa that are inhibited by heparin.

3.2. SARS-COV-2 transmission

As with other respiratory viruses, SARS-CoV-2 transmission occurs with high efficacy and infectivity mainly through the respiratory route. Droplet transmission is the main recognized route, although aerosols may represent another important route [ 28, 29]. Estimates of the reproduction number (R0) of SARS-CoV-2 range from 1.4 to 2.5 [Statement on the meeting of the International Health Regulations (2005) Emergency Committee regarding the outbreak of novel coronavirus (2019-nCoV), https://www.who.int/news-room/detail/23-01-2020-statement-on-the-meeting-of-the-international-health-regulations-(2005)-emergency-committee-regarding-the-outbreak-of-novel-coronavirus-(2019-ncov)] to 2.24-3.58 [ 30].

In some studies, patient urine has been tested for SARS-CoV-2 viral RNA. Amongst these studies, the pooled rate of RNA positivity was about 5-6%; nevertheless, the duration of viral shedding in urine samples as well as the infectivity of urine remains to be established [ 35].

SARS-CoV-2 RNA has also been detected on inanimate surfaces such as door handles and the surface of cell phones in residential sites of patients with confirmed COVID-19. Thus, individuals who have come into contact with infected surfaces could be infected if they touch their eyes, mouth or nose [ 29].

The vertical transmission of SARS-CoV-2 is debated; a series of nine pregnant women with confirmed COVID-19 showed no mother to child transmission. In addition, SARS-CoV-2 was not detected in breast milk, indicating that the virus cannot be transmitted with breastfeeding [ 36]. Nevertheless, a newborn with elevated IgM against SARS-CoV-2 born to a mother with COVID-19 has been recently reported. IgM antibodies along with IgG antibodies were detected 2 h after delivery. Il-6 and IL-10 were also elevated, while polymerase chain reaction (PCR) performed on consecutive nasopharyngeal swabs from 2 h to 16 days of age was always negative. Considering that IgM cannot cross the placenta and be transferred to the fetus, it could be hypothesized that the infant was infected in utero even if amniotic fluid was not tested for SARS-CoV-2 RNA [ 37].

3.3. SARS-CoV-2 incubation period

Determination of the incubation period of SARS-CoV-2 infection is crucial for determining the duration of quarantine, to evaluate the efficacy of entry screening and contact tracing. Based on a Weibull distribution, it was estimated that the mean incubation period is 6.4 days (95% confidence interval (CI): 5.6-7.7), with a range of 2.1-11.1 days (2.5th-97.5th percentile) [ 40]. Similar estimates have been made by other authors. In a study by Lauer et al. [ 41], it was estimated that the median incubation period was 5.1 days (95% CI, 4.5-5.8 days), and that 97.5% of infected individuals would develop symptoms within 11.5 days (CI, 8.2-15.6 days) of infection. Therefore, the 14-day period of active monitoring recommended by health authorities is justified by the evidence [ 42, 43]. Longer monitoring can be required in particular cases. It was estimated that 101 out of every 10,000 cases (99th percentile, 482) may develop symptoms after 14 days of active monitoring or quarantine [ 41].

4. Viral testing

4.1. Reverse real-time PCR assays

Suspected cases of SARS-CoV-2 infection are confirmed by detection of specific and unique viral sequences using a reverse real-time PCR (rRT-PCR) assay. Immediately after the declaration by the Chinese Health Authorities, on 7 January 2020, that the pneumonia outbreak in Wuhan was caused by a novel coronavirus, a European network of laboratories developed an rRT-PCR protocol based on the alignment and comparison of available bat-related coronavirus and SARS-CoV genome sequences plus five sequences from the novel coronavirus SARS-CoV-2 that were released by the Chinese authorities [ 44]. Three rRT-PCR assays were developed. The first line assay targets the E gene common to the coronaviruses belonging to Sarbecovirus subgenus and encoding the envelope protein. The second assay targets the RdRp gene encoding the RNA-dependent-RNA-polymerase. This assay contains two molecular probes: one reacts with the SARS-CoV and SARS-CoV-2 RdRp gene, while the second one (RdRP_SARSr-P2) reacts with SARS-CoV-2 RdRp gene. The third assay targets the N (nucleocapsid) gene. This protocol was adopted in 30 European countries [ 45].

Recently, a new PCR protocol targeting a different region of the RdRp/Hel gene showed a higher sensitivity and specificity than the RdRP_SARSr-P2 assay [ 46].

The US Centers for Disease Control and Prevention (CDC) protocol targets the N gene of SARS-CoV-2. Two primer/probe sets directed toward different regions of N gene were selected. In addition, a primer/probe set that detects the human RNase P gene in control samples and clinical specimens is included ( https://www.fda.gov/media/134922/download. Revision 3, 30 March 2020).

Although amplification tests are sensitive, some infections are missed. The reasons for this may be the quality of the collected specimen, time of collection (very early phase of infection or too late during infection), viral load below the limit of detection of the assay, incorrect handling of the specimen or shipping issues. In the case of low viral load in the upper respiratory tract, a deeper specimen may be required to make a diagnosis [ 47]. Indeed, in SARS and MERS patients, viral RNA in the upper respiratory tract peaked in the first 7-10 days after symptoms onset, while in the lower respiratory tract, viral RNA was still detected 2-3 weeks after disease onset [ 47, 48]. Repeat testing can also be performed in the case of nasopharyngeal swabs initially being negative as this increases the chance of detecting SARS-CoV-2 in the nasopharynx [ 49].

Several real-time PCR assays obtained the CE mark for in vitro diagnostics and are available on the market. Table 1 summarizes the gene targets, the technical features of the assays and the validated specimen types of the real-time PCR assays cleared by the Italian Ministry of Health.

The COVID-19 pandemic

Point of care tests (POCT) such as Xpert ® Xpress SARS-CoV-2 (Cepheid, Sunnyvale, CA, USA) QIAstat-Dx Respiratory 2019-nCoV Panel (QIAGEN, Hilden, Germany), and Simplexa™ COVID-19 Direct kit (DiaSorin Molecular LLC, Cypress, CA, USA) deliver results in about 30-60 min. They do not require skilled technicians and the hands-on time is less than 1 min. These tests can be very useful when clinicians have to make rapid treatment decisions.

A recent paper showed that in confirmed COVID-19 patients, saliva may be a more sensitive specimen for SARS-CoV-2 detection than nasopharyngeal swab. The authors also reported less variability in self-sample collection of saliva compared to nasopharyngeal swabs. This observation could open the way to at-home self-administered sample collection for large-scale screening of SARS-CoV-2 [ 52].

4.2. CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2

Recently, a CRISPR-Cas12-based assay called SARS-CoV-2 DNA endonuclease-targeted CRISPR Trans Reporter (DETECTR) has been developed for the diagnosis of SARS-CoV-2 infection. This assay performs simultaneous reverse transcription and isothermal amplification of RNA extracted from nasopharyngeal or oropharyngeal swabs using loop-mediated amplification (RT-LAMP) [ 55], followed by Cas12 detection of coronavirus sequences. Detection of the virus is confirmed by cleavage of a reporter molecule. The assay targets the E and N regions but unlike the CDC assay, this one does not target the N1 and N3 regions. Amplification of the E region allows the identification of three SARS-like coronaviruses [SARS-CoV-2 (accession NC_045512), bat SARS-like coronavirus (bat-SL-CoVZC45, accession MG772933) and SARSCoV (accession NC_004718)], whereas the N region specifically detects SARS-CoV-2.

The DETECTR assay can be run in 30-40 min and is visualized on a lateral flow strip. The test is positive if both E and N genes are detected or presumptive positive if either E or N gene is detected. The limit of detection (LOD) of this DETECTR assay is 10 copies/µl vs 1 copy/µl for the CDC assay. The positive predictive agreement and the negative predictive agreement of DETECTR assay versus the CDC assay were 95% and 100%, respectively [ 56].

4.3. Viral load in respiratory samples

Viral load determination performed on nasopharyngeal swabs demonstrated that mild clinical cases had a lower viral load in their respiratory specimens compared with severe cases. The mean viral load in severe cases was 60-fold higher than mild cases. Stratification of the data in relation at the time of sampling after disease onset showed that delta cycle threshold (delta Ct) values were significantly lower in severe cases than mild cases in the first 12 days of disease. In mild cases, viral clearance occurred earlier and after 10 days, 90% of the patients repeatedly tested negative by PCR. By contrast, PCR was still positive at day 10 or beyond in all severe cases. These preliminary data suggest that determination of SARS-CoV-2 load may be useful for monitoring the patients with COVID-19 disease and for predicting prognosis and assessing disease severity [ 57].

5. Serology

Several serological assays, including enzyme-linked immunosorbent assays (ELISA), chemiluminescence assays (CLIA), rapid antibody tests, and western blotting, have been developed since the beginning of the SARS-CoV-2 pandemic. The ELISA test developed by Wantai Biological Pharmacy Enterprise Co. (Beijing, China) detects total antibody, IgM and IgG against SARS-CoV-2 [ 58]. Total antibodies were detected based on a double-antigen sandwich immunoassay using recombinant antigens containing the RBD of the S protein of SARS-CoV-2 as the immobilized antigen and horse radish peroxidase (HRP) as the conjugated antigen. The IgM μ-chain capture method was used to detect IgM antibodies (IgM-ELISA), using the same HRP-conjugate RBD antigen as in the double-antigen sandwich immunoassay. The IgG antibodies were detected by an indirect ELISA test (IgG-ELISA) based on the recombinant nucleoprotein antigen. The specificity of the assays was determined by testing plasma samples of healthy individuals collected before the SARS-CoV-2 outbreak, and was 99.1%, 98.6% and 99.0% for total antibody, IgM and IgG, respectively [ 58]. Comparing the results obtained by real-time PCR with those generated by the antibody assays in the first week of illness, PCR showed a higher sensitivity than antibodies assays, 66.7% vs 38.3%. However, from days 8 to 12, the sensitivity of the antibody tests overtook that of the RNA test, and in the late phase of disease, the sensitivity of the antibody tests increased even further compared to the RNA test [ 58]. Nevertheless, to date, all the international professional organizations, the US Food and Drug Administration and the CDC do not recommend that serology tests be used for diagnosis.

Other research groups developed ELISA assays that used as antigens a modified full-length S protein and the RBD of SARS-CoV2. Using this approach, COVID-19 seroconverters were identified as early as 3 days post symptom onset. Similarly, an antibody test designed to detect E and N antigens detected IgM within one week from disease onset. IgM was detectable for about a month and then gradually disappeared, while IgG was detected after 10 days and was detected for a longer period of time [ 59].

Using a magnetic chemiluminescence enzyme immunoassay, 100% of patients with COVID-19 were found to be IgG positive 19 days after symptom onset. The median day for both IgG and IgM seroconversion was 13 days post symptoms. Seroconversion for IgM and IgG occurred simultaneously or sequentially. Three groups of patients were identified: patients with synchronous seroconversion of IgG and IgM, patients with IgM seroconversion earlier than IgG seroconversion, and patients with IgG seroconversion earlier than IgM seroconversion. IgM and IgG plateaued 6 days after the first determination. Interestingly, screening of close contacts of patients with COVID-19 showed that few individuals with negative RT-PCR and no symptoms tested positive to IgG and/or IgM, confirming that serology can help in obtaining better estimates of the spread of SARS-CoV-2 [ 60].

In the case of coronaviruses infection, neutralizing antibodies are elicited by the RBD region of the S protein [ 61, 62]. This subset of antibodies is particularly important because they can prevent the entry of the virus into the cell by binding to epitopes on the surface of the viral particle and blocking their interaction with the receptor. Neutralizing antibodies were first detected in the serum of a hospitalized Chinese female tourist between day 4 and 9 from onset of symptoms [ 61].

In the past, a cocktail of neutralizing antibodies has been used in the treatment of SARS-CoV and Ebola virus infections. The combination of neutralizing antibodies was more effective than a single antibody [ 63, 64]. It is likely that a similar approach will be undertaken in the treatment of SARS-CoV-2 infection. A recently developed human monoclonal antibody, 47D11, against SARS-CoV-2 is able to neutralize the infection of Vero E6 cells by SARS-CoV and SARS-CoV-2 in vitro [ 65]. The capacity of 47D11 to cross-react with SARS-CoV and SARS-CoV-2 suggests that the antibody likely targets the conserved structure of the S1 B RBD [ 65]. It is important to know whether this antibody can be useful in the development of serological assays for SARS-CoV-2 or antigen detection assays. At the clinical level, the 47D11 monoclonal antibody could represent a new therapeutic option for treating COVID-19 disease or for preventing infection.

Detection of neutralizing antibodies against SARS-CoV-2 is also important for identifying individuals who mount a strong immuneresponse against the virus and whose serum/plasma could be used therapeutically to fight SARS-CoV-2 infection.

Major IVD companies [ 68] have introduced SARS-CoV-2 serology tests and offer IgA, IgG, IgM or mixed assays on automated immunoassay analyzers in the clinical laboratory.

Considering the numerous immunoassays (ELISA and CLIA) already developed by several research groups and those that will become available in the near future, it is mandatory to standardize the assays using recognized reference standards or at least to harmonize them. Until standardization/harmonization are realized, it would be appropriate that each laboratory calculate its own cutoff using receiver operating characteristic curves. In many countries, serological assays are also in use to identify people who can return to work safely as soon as the lockdown eases. Indeed, it is possible that we may have to cohabit with the virus for a significant period of time, and it is mandatory to develop strategies for reopening economic activities. A possible algorithm is outlined in Figure 1. According to this algorithm, a serological IgM/IgG assay should be performed on asymptomatic workers before returning to work. In the case of a negative result, the serological assay is repeated after 10 days and if confirmed negative, the subject returns to work. In case of positivity, a real-time PCR assay on a nasopharyngeal swab is performed. If positive, the subject is quarantined for 14 days, and at the end of quarantine, a real-time PCR assay is performed on a new nasopharyngeal swab. A negative result must be confirmed by a second PCR carried out 24 h apart before the individual can return to work.

The COVID-19 pandemic

However, considering the limitations of the current serological tests and the possibility of cross-reaction with SARS-CoV (that shares 82% nucleotide identity with SARS-CoV-2 [ 69]) and other coronaviruses, real-time PCR remains, to date, the main and most effective diagnostic test for COVID-19 worldwide.

5.1. Rapid immunochromatographic tests

Reliable rapid tests could alleviate the pressure on molecular biology laboratories where SARS-CoV-2 RNA is tested and could help in developing countries. In the European Union, licensed rapid tests include both qualitative and semi-quantitative tests [ 70].

There are two types of rapid tests: the SARS-CoV-2 antigen detection tests and antibody detection tests. To date, 10 CE-marked rapid SARS-CoV-2 antigen detection tests conform to the EU legislation FIND ( https://www.finddx.org/), Directive 98/79/EC on IVDs. The sensitivity of these assays is generally low. For instance, the COVID-19 Ag Respi-Strip (Coris BioConcept, Brussels, Belgium), has a sensitivity of ∼60% with 100% specificity (95% CI: 93.5-100%). Its positive predictive value is 100% (95% CI: 86.7-1.00%) and negative predictive value, 85.4% (95% CI: 75.4-91.9%). Agreement with real-time PCR is 88%.

6. Sars-CoV-2 and cellular infection

6.1. SARS-CoV-2 receptor

Several biochemical and structural studies have shown that SARS-CoV-2 binds the human receptor for angiotensin-converting-enzyme 2 (ACE2) [ 75-77]. The spike protein of SARS-CoV-2 contains a polybasic furin cleavage site at the boundary between subunit S1 and S2 of the spike protein that is processed during biogenesis and that distinguishes this virus from SARS-CoV and SARS-related coronavirus. This cleavage site is important for virus infectivity and host range. Six amino acids within the RBD of the spike protein are critical for binding to the ACE2 receptor and for determining the host range of SARS-like coronaviruses. Five of these six amino acids differ between SARS-CoV and SARS-CoV-2.

The intermediate host of SARS-CoV-2 is unknown. Pangolin has been proposed as the intermediate host for coronavirus transmission between bat and humans. The RBD of CoV-pangolin isolated in Guandong shows that it has several amino acids that favor binding to the human ACE2 receptor, supporting the hypothesis of pangolin as the intermediate host [ 78].

Taken together, these observations show that the SARS-CoV-2 RBD may be a useful target for antiviral drugs. By blocking the RBD region, the virus could be prevented from binding to the ACE2 receptor and entering the cell. Another possible target is TMPRSS2, a cellular serine protease used by SARS-CoV-2 for S priming and cell entry. Inhibition of TMPRSS2 by Camostat mesylate blocks SARS-CoV-2 infection in human hair cells [ 79].

6.2. Induction of T-cell lymphocytes apoptosis during human coronavirus infection

SARS-CoV-2 can infect T-cells lymphocytes via S protein mediated endocytosis and internalization [ 80], although the virus is not able to replicate in these cells. Interestingly, the translation of viral RNA proteins induces apoptosis in T-lymphocytes, as demonstrated for MERS coronavirus [ 81]. Induction of apoptosis may represent a mechanism of immune-evasion that contributes to the immune-pathogenicity of the virus as a result of the lymphocytopenia observed in COVID-19 patients.

The apoptosis process has been studied in human coronavirus (HCoV) infected cells from SARS-CoV infected patients. Apoptotic cells have been detected in other tissues such as thyroid tissues [ 85], spleen and kidney [ 86], in addition to lung and upper airway epithelial cells. Apoptosis by human coronaviruses (HCoVs), including SARS-CoV, has been also described in in vitro systems and animals [ 87, 88]. Recently, apoptosis was demonstrated in the spleen and lymph nodes of SARS-CoV-2 patients [ 89].

SARS-CoV infection is able to induce caspase dependent apoptosis, but even if this phenomenon is generated by the infection, apoptosis does not inhibit virus replication [ 90]. Apoptosis has been demonstrated in an in vitro experiment using cultured cell by transfecting single SARS-CoV coding regions, including S, E, M, N, and accessory protein 3a, 3b, 6, 7a, 8a, and 9b. The membrane protein, M, modulates the Akt survival pathway and release of mitochondrial cytochrome c [ 91], protein 3b activates bcl-2-like protein 4 (BAX), and both processes are able to induce apoptosis [ 92]. Even if the ablation of gene 7 from the SARS-CoV genome did not show an effect on replication of the virus in transformed cell lines, it showed a reduction in apoptosis if DNA fragmentation was evaluated [ 93]. The induction of apoptosis by protein 7a is blocked by Bcl-XL [ 94]. The proteins, E and 7a, are able to activate the intrinsic pathway by sequestering antiapoptotic Bcl-XL to the endoplasmic reticulum [ 87]. Protein 3 b is able to induce cell G0/G1 arrest and apoptosis [ 95]. Protein 3a is also pro-apoptotic, and possesses three major protein signatures, the cysteine-rich, Yxxϕ and diacidic domains, that are essential for its apoptotic function [ 96]. Other mechanisms of apoptosis induction by HCoV involve the activation of endoplasmic reticulum stress response and the mitogen-activated protein kinase pathway [ 97].

7. Safety issues

7.1. General safety recommendations

Since the outbreak of SARS-CoV-2, the use of face masks has become ubiquitous. The fear of being infected has caused everyone who can wear face mask to do so and this has contributed to the shortage of this product. Policies on wearing face masks differ among countries. WHO discourages the use of facemask among healthy people unless they are taking care of a person with suspected SARS-CoV-2 infection or with respiratory symptoms. However, the use of face mask is always recommended because it could prevent infection transmission from asymptomatic carriers. In China, national policy encourages the use of face masks among people with low or moderate risk of infection but discourages those with a very low risk of infection from wearing a mask. People in quarantine should wear a face mask if they leave their home for any reason to prevent potential transmission in the asymptomatic phase. In addition, vulnerable populations such as the elderly or those with underlying medical conditions should wear a mask [ 98].

A recent study by Leung et al. showed that wearing face mask significantly reduced the shedding of respiratory viruses such as influenza virus and coronavirus [ 28]. Based on these recent findings and in an attempt to reduce the spread of SARS-CoV-2 in the so-called second phase of the epidemic, many EU governments have made it mandatory to wear face masks in public.

The potential air propagation of SARS-CoV-2 depends on many factors including the particle size, the speed of exhaled air (increased by breathing < speaking < coughing < sneezing) as well as temperature and humidity.

The WHO, CDC and European Center for Disease Prevention and Control strongly recommend that people perform hand hygiene frequently and avoid touching their eyes, nose and mouth.

7.2. Blood specimens handling

Routine blood testing should be performed on automated analyzers using standard practices and procedures at containment level 2 for suspected/confirmed cases of COVID-19. Automated analyzers should be disinfected following sample processing, and actions that could generate infectious aerosols should be avoided. The opening of test tubes is usually not considered a high-risk procedure for generating aerosols. However, risk assessment should be undertaken to determine whether a microbiological safety cabinet should be used.

Blood samples from patients with COVID-19 disease are usually negative for SARS-CoV-2 RNA. Thus, viremia does not seem to be a major problem for SARS-CoV-2 infection. The virus has been detected only in a limited number of cases [11, 31, 99-102]. Furthermore, there is no clear correlation between the presence of viral RNA in the blood and the severity of disease [ 11, 100, 101]. The infectiousness of blood from patients with COVID-19 as well as SARS and MERS has not been demonstrated. No reports that show transmission of these three viruses by blood or blood products exist in the literature.

Based on this evidence, blood samples can be handled using standard laboratory practice at containment level 2. As a precaution, in case of severe COVID-19 disease, if laboratory procedures may result in splashes, spills or aerosol generation, a microbiological safety cabinet can be used to protect the operator [ https://www.gov.uk/government/publications/wuhan-novel-coronavirus-guidance-for-clinical-diagnostic-laboratories/wuhan-novel-coronavirus-handling-and-processing-of-laboratory-specimens].

7.4. Surfaces disinfection

Recently the IFCC Task Force on Covid-19 published a set of recommendations, adapted from official documents of international and national health agencies, on biosafety measures for routine clinical chemistry laboratories [ 105].

8. Treatment

8.1. Determine the treatment site according to the condition of the patient

According to the severity of a patient's symptoms and the medical resources available in a region, different treatment sites may be selected to observe and isolate patients. The specific classification, from Chinese guidelines, is as follows:

Asymptomatic cases: They have not been confirmed and should not be considered as new cases. The main treatment measure is centralized quarantine for 14 days and further monitoring by the local Public Health Department [ 13]. If these cases are in home isolation, household members should stay in a different room, or if this is not possible, maintain a distance for at least 1 meter from the quarantined person [ 106];

Suspected cases: After informed consent, patients who have the ability to self-care, age ≤65 years old, without primary diseases such as respiratory diseases, cardiovascular diseases and mental health issues, should go to a health care facility voluntarily [ 107, 108]. During quarantine observation, the person should in principle stay in a single room and not leave the room at random;

Mild cases: They are treated in a mobile cabin hospital if available or at home if hospitalization is not possible because of the heavy burden on the health care system. In this case, they should be followed up and cared for by family members [ 13]. If patients are in the same room, the space between beds should not be less than 1.2 m, and the room should be equipped with its own facilities. At the same time, family visits and nursing should be declined [ 108];

Severe/critically ill cases: Patients who are initially diagnosed as critically ill should be admitted into the Intensive Care Unit (ICU) immediately for treatment. For patients whose status changes from mild to severe, after hospital triage and prescreening expert consultation in the mobile cabin hospital or at home, they should be transferred to the critical observation and treatment area of a sheltered hospital, and following consultation, they should be transferred to a designated hospital for treatment [ 109].

Facing the COVID-19 pandemic, China creatively adopted large-scale mobile cabin hospitals to prevent and control the epidemic and achieved satisfactory results. Since the epidemic rapidly spread to many other countries around the world, China's experience is recommended and should be considered in their national strategy and at the local level. Thus, countries such as Serbia, Britain, Italy, Spain, France, Iran accelerated the construction of mobile cabin hospitals, which are used mainly to treat mild or asymptomatic patients. According to WHO guidelines, if patient symptoms are mild, providing care at home may be considered as long as patients can be followed up and cared for by family members [ 106]. However, severe cases should be admitted to designated hospitals for treatment at first diagnosis.

8.2. Treatment for mild COVID-19 cases

The clinical symptoms of these cases are mild and there is no pneumonia manifested on chest imaging. Patients should stay in bed, which is the principle for treatment of mild COVID-19 cases. According to the protocol by Prof. Tingbo Liang, blood oxygen saturation monitoring and oxygen therapy with nasal cannula should be conducted regularly for these patients.

8.3. Treatment for moderate COVID-19 cases

The clinical symptoms are moderate, with fever, respiratory tract symptoms and pneumonia manifestations on chest imaging. Treatment principles include bed rest, supportive treatment to maintain energy supply, maintaining water and electrolyte balance and monitoring vital signs and oxygen saturations.

Patients should be given oxygen therapy as needed. Firstly, they can be provided with nasal cannula therapy. If this does not work, patients should be treated with mask oxygen and cannula oxygen therapy. As well, inhalation of hydrogen-oxygen (O 2/H 2: 33.3%/66.6%) can be used.

A randomized double-blind, placebo-controlled, multicenter trial at ten hospitals in Hubei, China, was performed with remdesivir. The treatment was not associated with statistically significant clinical benefits. However, the duration of invasive mechanical ventilation, although also not significantly different between groups, was numerically shorter in remdesivir than placebo recipients [ 111].

8.4. Treatment for severe/critical COVID-19 cases

Severe cases have severe respiratory symptoms such as shortness of breath, a decrease of oxygen levels and a decrease of PaO 2/FiO 2. The general treatment principles for these cases are active prevention and treatment of complications, prevention of secondary infections while treating basic diseases, and organ function support treatment in a timely manner [ 114, 115].

If the respiratory distress and/or hypoxemia cannot be relieved, high-flow nasal cannula oxygen therapy or noninvasive ventilation should be used. If the condition of the patient does not improve or if it worsens within 1-2 h, tracheal intubation and invasive mechanical ventilation should be performed. Sedation and muscle relaxants should be used for patients who have a problem with man-machine synchronization. Closed sputum suction should be considered according to airway secretions. For patients with severe acute respiratory distress syndrome (ARDS), lung expansion is recommended, and prone ventilation should be performed for more than 12 h every day. If it is not effective, extracorporeal membrane pulmonary oxygenation (ECMO) should be considered. The indications of ECMO are as follows: (1) when FiO 2>90%, the oxygenation index is less than 80 mmHg for 3-4 h; (2) patients with simple respiratory failure with plateau ≥35 cm H 2O, the VV-ECMO mode is selected; if circulatory support is needed at the same time, the VA-ECMO mode is preferred. Weaning off ECMO can be considered when the underlying disease is under control and cardiopulmonary function shows signs of recovery [ 116, 117].

In addition, for the severe and critical cases, immunotherapy can be performed. For patients with extensive lung lesions and persistently elevated IL-6, monoclonal antibody therapy can be tried, but attention should be paid to allergic reactions. Immunotherapy is not recommended for patients with active infections, e.g. tuberculosis [ 116].

For patients with deterioration of oxygenation, rapid imaging changes and excessive inflammatory response, glucocorticoids can be used as appropriate. Experts from the Chinese Thoracic Society developed a consensus statement that patients can be treated with glucocorticoids when they meet the following four conditions at the same time: (1) adult (age ≥18 years old); (2) diagnosed with new coronavirus infection by PCR or serum antibodies; (3) symptoms (including fever, cough or other related infection symptoms) occur within 10 days, radiographically confirmed pneumonia and rapid progress; and (4) blood oxygen saturation (SpO2) ≤93% or shortness of breath (breath rate ≥30 breaths/min) or oxygenation index ≤300 mmHg [ 121]. For patients with secondary infection, intestinal microecological regulators and broad-spectrum antibacterial drugs can be used. Immunoglobulin infusion therapy can also be used to improve patient immunity [ 122 ]. It is recommended that pregnant women with severe COVID-19 infection end their pregnancy as soon as possible. In addition, anxiety and fear are often present in severe and critical cases, and the psychiatric service must have a specific approach for dealing with these problems including hospital resource management, mental health intervention, and guidance for family members [ 123].

8.5. Traditional Chinese medicine treatment

Traditional Chinese medicine (TCM) is a unique diagnostic method with a systematic approach, abundant historical literature and materials. It formulates treatment based on symptom-based diagnosis, an approach that is increasingly emphasized by modern medicine. In the treatment of COVID-19, TCM is proposed as an important option by national and provincial guidelines with substantial utilization. According to TCM theory, COVID-19 belongs to the category of "dampness plague", and its core pathogenesis is correlated to "epidemic virus." The "state" of cold and humidity is the basis of the disease, and "stagnation of qi" is the key to disease progression. The corresponding treatment at the early stage targets eliminating dampness, and the strategy focuses on eliminating dampness, releasing lungs and expelling pathogenic factors, to shorten fever duration, relieve symptoms, prevent disease progression, reduce mortality and assist rehabilitation. For TCM treatment, the 7th edition of China National Clinical Guideline divided COVID-19 into a medical observation period and treatment period. The latter is further stratified into four manifestations including mild, intermediate, severe, and critical, followed by a rehabilitation stage [ 124].

8.6. Prospects for COVID-19 drug research

At present, there is no specific drug for the treatment of COVID-19, and the main treatment methods are supportive and symptomatic treatment [ 125]. The mechanisms of different therapeutic drugs mainly include blocking virus invasion, inhibiting virus replication and protein expression and regulating the patient's immunity. Up to 21 April 2020, there were 159 drugs, including 49 antibody drugs, 20 antiviral drugs, 12 cell-based therapy drugs, 5 RNA therapy drugs, and others, in various stages of research [ 126]. In addition to the therapeutic drugs mentioned above, the antimalarial drugs, chloroquine and hydroxychloroquine, are also considered as candidates for the treatment of patients with COVID-19. Both drugs exert an inhibitory effect on the virus by interfering with the binding of ACE2 [ 127]. Chloroquine phosphate drug treatment has been included in clinical trials, and a phase III clinical trial for the drug was started in the United States on 31 March 2020 [ 128].

A potential promising treatment target is cathepsin L (CatL), an endosomal cysteine protease that mediates the cleavage of the S1 subunit of the coronavirus surface spike glycoprotein. While TMPRSS2 acts locally at the host cell plasma membrane and possibly during endocytotic vesicle trafficking, CatL continues S1 subunit degradation in the acidic endosome and lysosome compartments. Therefore, CatL inhibition could provide two sequential blocks for coronavirus infection: blockage of virus entry on the host cell surface and blockage of viral material release and replication inside the host cell endosomes [ 129].

We believe that with the joint efforts of various countries on the research and development of COVID-19 drugs, we will soon find the most effective drugs for its therapy.

9. Prevention and vaccination

According to the infection prevention and control strategies from the WHO, standard precautions for all patients, which are also appropriate for public prevention, include hand and respiratory hygiene, the use of appropriate personal protective equipment, safe injection practices, safe waste management, clean linens, environmental cleaning, and sterilization of patient-care equipment [ 132]. A vaccine to prevent COVID-19 is perhaps the best hope for ending the pandemic, and vaccines are especially needed by health care workers on the front lines and other vulnerable members of the population who have a higher risk of contracting the infection.

Coronaviruses have a spike-like structure, the S protein, on their surface. The S protein is considered to be the key molecule that binds to the ACE2 receptor to attach onto the surface of human cells and that forces the virus through the cell membrane, mediating the process of virus infection and membrane fusion to host cells [ 135]. With the exception of inactivated vaccines and attenuated virus vaccines, other vaccines are designed based on the S protein. The S protein could be expressed in vivo using vectors carrying the S gene, and the expressed protein would induce the synthesis of specific antibodies that could block the entry of the virus to the cells. These approaches are expected to prevent the virus from binding to human cells and to stop the virus from reproducing so as to achieve immune protection. The most advanced candidates that have moved into clinical development are shown in Table 2 [ 133, 136].

The COVID-19 pandemic

09 July 2020

While injection is the most widely used way for vaccine delivery, there are also some novel approaches to deliver vaccine. For example, a fingertip-sized microneedle array vaccine, PittCoVacc, a vaccine from University of Pittsburgh School of Medicine, delivers the spike protein pieces into the skin to stimulate the body to produce antibodies against SARS-CoV-2 [ 137]; INO-4800 delivers DNA into the host's cells by a hand-held smart device, CELLECTRA [ 138]; and Symvivo COVID-19 products are presented as oral lyophilized gel-capsules [ 139].

Different types of technological platforms have their own characteristics regarding speed of research and development, production cycle, equipment requirements, etc. The safety, stability and effectiveness of vaccine candidates still need to be evaluated and compared through clinical experiments. Advantages and disadvantages of different COVID-19 vaccine technology strategies are listed in Table 3 [ 140].

The COVID-19 pandemic

09 July 2020

During the global emergency of the COVID-19 pandemic, the effort in response is unprecedented in terms of scale and speed. However, much work remains unfinished. The vaccine candidates might prove to be not safe or effective in any phase of development; for instance, the Coalition for Epidemic Preparedness Innovations indicates a potential success rate of only 10% for vaccine development [ 133].

Recently a study about the safety, tolerability and immunogenicity of a recombinant adenovirus type-5 vectored COVID-19 vaccine was performed in Wuhan; neutralizing antibodies increased significantly at day 14 and peaked 28 days post-vaccination whereas the T-cell response peaked at day 14 post-vaccination [ 141].

Strong international coordination and cooperation among research groups involved in vaccine research and development will be needed to ensure that promising vaccines can be manufactured in sufficient quantities and equitably supplied to all affected areas, particularly low-resource regions.

A prophylactic vaccine against SARS-CoV-2 is particularly urgent because SARS-CoV-2 is a novel virus of zoonotic origin [ 1, 2] that started spreading among human beings, probably in late November 2019 [ 11], to which the population does not have an existing level of immunity that is able to limit or stop the circulation of the virus. Assuming a R 0 of 3 for SARS-CoV-2, the herd immunity threshold for SARS-CoV-2 is about 67% [ 142]. This means the infection will start declining once the number of individuals with acquired immunity to SARS-CoV-2 exceeds 0.67 [ 142]. Acquisition of herd immunity via natural infection is ethically unacceptable because it would cause millions of deaths around the globe. Furthermore, the unchecked spread of SARS-CoV-2 will quickly paralyze our health care systems, increasing not only the COVID-19 mortality but also all causes mortality. In light of this, and because the development of an effective vaccine will take at least 12-18 months, it will be important to adhere strictly to preventive measures indicated by the WHO and to maintain social distancing.

10. Artificial intelligence and mobile health tools

Artificial intelligence (AI) may have a role in the governance of health care systems and in coping with health emergencies such as the current COVID-19 outbreak. AI may help in analyzing a huge amount of data in a timely way as required during periods of crisis, allowing a prompt response by health authorities. Analysis of medical records, therapies, and laboratory findings may speed up using AI, hasten the decision-making process and improve patient management [ 145]. For instance, it has been proposed that AI could support radiologists in reading CT scans. While a manual read takes about 15 min, AI can complete the reading in a few seconds. Thus, AI could be designed to detect lesions resembling coronavirus pneumonia, to measure the density, volume and shape, and to compare multiple lung lesions from the image. This information should support physician in making a more rapid diagnosis [ 146]. In the work by Li and colleagues, AI allowed the detection of COVID-19 pneumonia and distinguished it from community acquired pneumonia and other lung lesions [ 147].

Block chain and AI could be used for remote patient monitoring and the transfer of clinical information to health authorities [ 145]. A positive SARS-CoV-2 patient could be referred to a quarantine site for monitoring and treatment to limit virus spread. Similarly, information from a certain geographic area could be used for tracking positive patients and/or quarantining that geographic area to limit the spread of the virus.

A mobile-phone based survey along with an AI framework has been proposed to collect travel history along with the common clinical manifestations to identify people with suspected SARS-CoV-2 infection. The collected information should stratify individuals under investigation into no-risk, minimal-risk, moderate-risk, and high-risk of being infected by the virus. Identification of the high-risk individuals should trigger immediate quarantine to contain the spread of the virus [ 148]. In Italy, the mobile-phone app, Immuni, was introduced and can be downloaded to trace contacts of an infected individual using Bluetooth technology. In case of positivity to SARS-CoV-2, the individual uploads the laboratory result on the platform, and an instant message is sent to all close contacts who must remain in quarantine for at least 14 days [ www.salute.gov.it].

A pre-trained deep learning-based drug-target interaction model called Molecule Transformer-Drug Target Interaction (MT-DTI) was used to identify molecules already available on the market that could be used against SARS-CoV-2. Several molecules were identified including atazanavir, remdesivir, efavirenz, ritonavir, and dolutegravir. Atazanavir, the best compound, showed an inhibitory potency with a Kd of 94.94 nmol/L against the SARS-CoV-2 3C-like proteinase, followed by remdesivir (113.13 nmol/L), efavirenz (199.17 nmol/L) and ritonavir (204.05 nmol/L). Lopinavir and ritonavir target proteases, and based on this model could also inhibit the replication components of SARS-CoV-2 with a Kd < 1000 nmol/L [ 149].

DeepMInd developed an AlphaFold algorithm to predict protein structures starting from their amino acid sequences, thus avoiding long and intensive laboratory experiments. Using a deep neural network, the system can make accurate predictions of the distances and angles between amino acid residues and provide more information about the structure than contact predictions. The information obtained might be used as a platform for the development of therapeutics [ 150]. This algorithm works well even on sequences with fewer homologous sequences, as is the case of SARS-CoV-2.

11. Preparedness and lifestyle after COVID-19 pandemic

The COVID-19 pandemic has shown that even the most advanced health care systems cannot sustain a massive influx of critically ill patients in their emergency departments. Italy, with its 3.2 hospital beds per 1000 persons vs 2.8 in the United States had enormous difficulty to meet the needs of critically ill patients arriving in the hospitals in a short time frame [ 151]. As a consequence, elective or semi-elective surgical procedures were canceled or postponed, wards were reorganized to treat COVID-19 patients, and follow up visits were delayed.

To prevent this massive influx to emergency departments in a possible second wave, the implementation of regional assistance is crucial. A large number of nasopharyngeal swabs and serological testing should be performed in the region to identify infected people and to allow contact tracing. Then, patients with mild symptoms or asymptomatic carriers could stay at home and be monitored by health operators, thus reducing visits to clinics. Limiting access to hospitals will save resources, make them available to those who really need them and reduce the risk of exposure. In light of that, the Italian government has recently decided to increase the number of nurses in the region, bringing the ratio to 8 nurses/50,000 persons. Special units of continuing care (Italian, urban search and rescue) have been created to assist patients at home.

In addition, the number of beds in the ICUs will be increased by 70% and to a lesser extent, those in sub-ICUs. Finally, 600 beds will be made available for movable structures in the case of epidemic peaks. Meantime, it is necessary to increase the numbers of ventilators, ECMO machines, and personal protective equipment.

COVID-19 prevalence is currently determined by the number of subjects with positive RT-qPCR nasopharyngeal swabs, but the real prevalence is probably much higher. Testing for the presence of IgG anti-SARS-CoV-2 may identify those who have been exposed and asymptomatic carriers, giving us more reliable case counts and mortality estimates, and a useful tool to control the restarting phase.

Indeed, in a very short time, all countries will be challenged by phase two. However, until the effect of the neutralizing antibodies detected by all the different methods and the antibody serum levels that are needed for individuals to be fully protected against reinfection are known, we could not consider serological positivity as a "license" to stop social distancing rules and the use of protective devices.

Then, during this second phase, it will be important to maintain social distancing, which remains the major preventive measure in the absence of a vaccine and effective antiviral drugs. Social distancing implies a massive reorganization of our society and lifestyle.

Telecommuting (working at a distance) and smart working should be pursued whenever possible. When this is not possible, social distancing and protective measures (protective equipment, hand hygiene, disinfectants) must be provided in the workplace. Since large gatherings will not be permitted, commercial activities such as restaurants, cafes, cinemas, etc. will have to reduce the number of employees with consequent social repercussions and increased poverty. Welfare will be essential for such workers.

Schools and universities will be required to reorganize themselves to provide education and safety for their students. Access to the Internet will be crucial for all students and should be provided to enable them to pursue education and take exams remotely if necessary.

Finally, communication will be important to live through this difficult time. Clear messages from national authorities will be necessary to increase awareness in the population and to support them in following new norms.

Conclusions

The COVID-19 pandemic has stressed our health care systems in an unprecedented way and underlined once more the essential role of laboratory medicine in tackling the spread of new transmissible agents.

Almost all over the world, networks of COVID laboratories have been set up to support the specific needs of citizens and patients, and they will continue to be fundamental during the re-starting of social and work activities.

The COVID-19 pandemic has revealed the weaknesses of our health systems that were unprepared to cope with a very large number of patients requiring respiratory support therapy in a short time frame. This emergency forced health authorities to stop all non-urgent medical procedures and convert wards in ICUs or sub-ICUs. On the other hand, the pandemic has prompted the scientific community to join together in efforts to fight this novel pathogen. Within a few days from the first reported cases of unknown pneumonia, the virus was isolated, sequenced, identified and genetically characterized. It was named SARS-CoV-2 because of its phylogenetic relationship with SARS-CoV and bat SARS-like coronaviruses. Based on its genetic features, molecular and serological assays were developed and have been introduced in routine diagnostics. Furthermore, several vaccine strategies have been developed or are in development, and trials are ongoing or will be begun to determine their effectiveness. In the absence of effective and specific antiviral therapy against SARS-CoV-2, the availability of a prophylactic vaccine is crucial.

Finally, a contribution in the governance of such an emergency could come from AI. Faster processing of clinical data may allow physicians to speed up the decision making process and improve the management of patients. AI and blockchain already used to trace contacts to contain the spread of the virus. In the near future, this technology will become an integral part of our health care systems.

Manufacturer

Specimen type

Method

Gene target

Sensitivity

Vaccine candidate

Technology

Vaccine characteristics

Trail status

Developer

Location

References

Inactivated Novel Coronavirus Pneumonia vaccine (Vero cells)

Inactivated virus

Inactivated SARS-CoV-2 virus (Vero cells)

Phase I(ChiCTR2000031809)

Beijing Institute of Biological Products,
Wuhan Institute of Biological Products

China

-Wuhan Institute of Virology, Wuhan Institute of Biological Products co., LTD. A randomized, double-blind, placebo parallel-controlled phase I/II clinical trial for inactivated Novel Coronavirus Pneumonia vaccine (Vero cells) (Registration number: ChiCTR2000031809) [EB/OL]. (2020-04-13) [2020/04/29]. http://www.chictr.org.cn/showprojen.aspx?proj=52227.
- Tuna Y I. Wuhan Institute of Biological Products' COVID-19 Vaccine Enters Phase II Trials[J]. EqualOcean, 2020.

PiCoVacc

Inactivated virus

Inactivated SARS-CoV-2 virus

Phase I-II(NCT04352608)

Sinovac Biotech Ltd,
Institute of Laboratory Animal Science, China

China

- Sinovac Biotech Co., Ltd. Safety and Immunogenicity Study of Inactivated Vaccine for Prophylaxis of SARS CoV-2 Infection (COVID-19) (ClinicalTrials.gov Identifier: NCT04352608) [EB/OL]. (2020-04-28) [2020/04/29]
- Gao Qiang, Bao Linlin, Mao Haiyan, et al. Rapid development of an inactivated vaccine candidate for SARS-CoV-2[J]. Science, 2020:c1932. DOI:10.1126/science.abc1932.

Ad5-nCov

Non-replicating viral vector

Recombinant adenovirus type 5 vector

Phase I(NCT04313127);
Phase II interventional trial for dosing and side effects (NCT04341389)

CanSino Biologics, Institute of Biotechnology of the Academy of Military Medical Sciences

China

- Institute of Biotechnology, Academy of Military Medical Sciences, PLA of China, CanSino Biologics Inc. A Phase II Clinical Trial to Evaluate the Recombinant Vaccine for COVID-19 (Adenovirus Vector) (CTII-nCoV) (ClinicalTrials.gov Identifier: NCT04341389) [EB/OL]. (2020-04-21) [2020/04/29/]. https://www.clinicaltrials.gov/ct2/show/NCT04341389.
- Institute of Biotechnology, Academy of Military Medical Sciences, PLA of China, CanSino Biologics Inc. Phase I Clinical Trial of a COVID-19 Vaccine in 18-60 Healthy Adults (CTCOVID-19) (ClinicalTrials.gov Identifier: NCT04313127) [EB/OL]. (2020-04-14) [2020/04/29]. https://www.clinicaltrials.gov/ct2/show/NCT04313127.
- Keown A. China's CanSino Prepares to Advance COVID-19 Vaccine Candidate into Phase II[J]. BioSpace, 2020.

ChAdOx1 nCoV-19

Non-replicating viral vector

Adenovirus vector

Phase I-II(NCT04324606)

University of Oxford

United Kingdom

- University of Oxford. A Study of a Candidate COVID-19 Vaccine (COV001) (ClinicalTrials.gov Identifier: NCT04324606) [EB/OL]. (2020-04-24) [2020/04/29]. https://www.clinicaltrials.gov/ct2/show/NCT04324606.
- COVID-19 Oxford Vaccine Trial [EB/OL]. (2020-04-25) [2020/04/29]. https://www.covid19vaccinetrial.co.uk/about.
- U.K. Starts Oxford Coronavirus Vaccine Trial as Germany Green-Lights BioNTech and Pfizer [EB/OL]. (2020-04-23) [2020/04/29/]. https://www.genengnews.com/news/uk-starts-oxford-coronavirus-vaccine-trial-as-germany-green-lights-clinical-trial-for-biontech-and-pfizer.

INO-4800

DNA

DNA plasmid encoding S protein delivered by electroporation (hand-held smart device "CELLECTRA")

Phase I-II(NCT04336410)

Inovio Pharmaceuticals,
CEPI,
Korea National Institute of Health,
International Vaccine Institute

United States, South Korea

- Inovio Pharmaceuticals I. Inovio Accelerates Timeline for COVID-19 DNA Vaccine INO-4800[J]. PR Newswire, 2020.
- Inovio Pharmaceuticals. Safety, Tolerability and Immunogenicity of INO-4800 for COVID-19 in Healthy Volunteers (ClinicalTrials.gov Identifier: NCT04336410) [EB/OL]. (2020-04-24)[2020/04/29]. https://www.clinicaltrials.gov/ct2/show/NCT04336410.

bacTRL-Spike

DNA

Bacterial medium with Bifidobacterium longum engineered to deliver DNA plasmid encoding S protein (oral)

Phase I(NCT04334980)

Symvivo Corporation,
University of British Columbia,
Dalhousie University

Canada

- Symvivo. COVID-19 Program Vision [EB/OL]. (2020-04-29) [2020/04/29/]. https://www.symvivo.com/covid-19.
- Corporation S. Evaluating the Safety, Tolerability and Immunogenicity of bacTRL-Spike Vaccine for Prevention of COVID-19 (ClinicalTrials.gov Identifier: NCT04334980) [EB/OL]. (2020-04-22)[2020/04/29/]. https://clinicaltrials.gov/ct2/show/NCT04334980.

mRNA-1273

RNA

LNP-encapsulated mRNA vaccine encoding S protein

Phase I (NCT04283461)

Moderna,
US National Institute of Allergy and Infectious Diseases

United States

- Moderna Therapeutics, US NIAID. Safety and Immunogenicity Study of 2019-nCoV Vaccine (mRNA-1273) for Prophylaxis SARS CoV-2 Infection (COVID-19) (ClinicalTrials.gov Identifier: NCT04283461) [EB/OL] (2020-04-13) [2020/04/29/]. https://www.clinicaltrials.gov/ct2/show/NCT04283461.
- Grady D. Trial of Coronavirus Vaccine Made by Moderna Begins in Seattle[J]. New York Times, 2020.

BNT162 (a1, b1, b2, c2)

RNA

LNP-encapsulated mRNA vaccine encoding S protein

Phase I-II (UTRN: U1111-1249-4220)

BioNTech,
Fosun Pharma,
Pfizer

Germany,
China,
United States

- Pharmaceuticals B R G. A Multi-site Phase I/II, 2-Part, Dose-Escalation Trial Investigating the Safety and Immunogenicity of four Prophylactic SARS-CoV-2 RNA Vaccines Against COVID-2019 Using Different Dosing Regimens in Healthy Adults (UTRN: U1111-1249-4220) [EB/OL]. (2020-04-20) [2020/04/29]. https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-001038-36/DE.
- Reuters. Germany Approves Trials of COVID-19 Vaccine Candidate[J]. New York Times, 2020.

LV-SMENP-DC

Unknown

DCs modified with lentiviral vector expressing synthetic minigene based on domains of selected viral proteins; administered with antigen specific CTLs

Phase I(NCT04276896)

Shenzhen Geno-Immune Medical Institute

China

Shenzhen Geno-Immune Medical Institute. Immunity and Safety of Covid-19 Synthetic Minigene Vaccine (ClinicalTrials.gov Identifier: NCT04276896) [EB/OL]. (2020-03-19)[2020/04/29]. https://www.clinicaltrials.gov/ct2/show/NCT04276896.

Covid-19/aAPC

Unknown

aAPCs modified with lentiviral vector expressing synthetic minigene based on domains of selected viral proteins

Phase I(NCT04299724)

Shenzhen Geno-Immune Medical Institute

China

Shenzhen Geno-Immune Medical Institute. Safety and Immunity of Covid-19 aAPC Vaccine (ClinicalTrials.gov Identifier: NCT04299724) [EB/OL]. (2020-03-09)[2020/04/29]. https://www.clinicaltrials.gov/ct2/show/record/NCT04299724.

Technology

Characteristics

Advantages

Disadvantages

Inactivated vaccine

Consisting of virus particles with immunogenicity but reduced infectivity (virulence) which have been grown in culture and fully destroyed using heat, chemicals, or radiation.

Similar antigenicity to live virus due to unchanged structure virus particles

No virulence rebound risk

Non-replicatable in host

No virus transmission risk among people

Relatively mature technology

Ease of preparation and production

Multiple-dose and adjuvants may be required due to insufficient immunogenicity

Uncontrollable protective effect

BSL-3 laboratory required

Genetic engineering subunit vaccines

A subunit vaccine is a fragment of a pathogen, typically a surface protein, that is used to trigger an immune response and stimulate acquired immunity against the pathogen from which it is derived. For COVID-19, the subunit vaccine is the recombinant Spike protein or its peptide prepared in vitro through genetic engineering.

Adenovirus vector vaccines

Adenoviruses as vectors for delivering genes or vaccine antigens to the target host

Broad range of tissue tropism

Well-characterized genome

Ease of genetic manipulation including acceptance of large transgene DNA insertions

Inherent adjuvant properties

Ability to induce robust transgene-specific T cell and antibody responses

Non-replicative nature in host

Ease of production at large scale

Pre-existing immunity in humans, inflammatory responses

Sequestering of the vector to liver and spleen

Immunodominance of the vector genes over transgenes.

Nucleic acid vaccines

Genetically engineered plasmid containing the DNA sequence or RNA containing vector which could produce the Spike protein of SARS-COV-2 upon the delivery of the vaccine into the body.

No risk for infection

Immune response focused on antigen of interest

Ease of development and production

Stability for storage and shipping

Cost-effectiveness

Obviates need for peptide synthesis, expression and purification of recombinant proteins and use of toxic adjuvants

Long-term persistence of immunogen

In vivo expression ensures protein more closely resembles normal eukaryotic structure, with accompanying post-translational modifications

Limited to protein immunogens

Risk of affecting genes controlling cell growth

Possibility of inducing antibody production against DNA

Possibility of tolerance to the antigen (protein) produced

Unclear vaccine delivery efficiency and safety problem

Vaccines using attenuated influenza virus as vectors

Attenuated influenza virus as vectors for delivering the Spike protein of SARS-COV-2 to the target host

Protect against COVID-19 and influenza at the same time

Convenient inoculation by nasal drip

The vector is little influence by the human immune system

No risk of integrating vector's DNA into the host genome

Live attenuated virus vaccine

Reducing the virulence of a pathogen, but still keeping it viable.

Activates all phases of the immune system

Provides more durable immunity; boosters are required less frequently

Low cost

Some are easy to transport and administer (for instance orally)

Vaccines have strong beneficial nonspecific effects

Possibilities that the virus can revert to wild type or develop into an entirely new strain

Safety problems.Potentially severe complications

Live strains typically require advanced maintenance, making transport difficult and costly

BSL-3 laboratory required

Disclosure statement

No potential conflict of interest was reported by the author(s).

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The Prevalence of Sleep Disorders in CollegeStudents- Malena

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William Alex Pridemore,1 Matthew C. Makel,2 and Jonathan A. Plucker3

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Replication in Criminology and the Social Sciences

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School of Education and Social Care, Faculty of Health, Education, Medicine and Social Care, Anglia Ruskin University, Chelmsford, United Kingdom

Bullying research has traditionally been dominated by largescale cohort studies focusing on the personality traits of bullies and victims. These studies focus on bullying prevalence, risk and protective factors, and negative outcomes. A limitation of this approach is that it does not explain why bullying happens. Qualitative research can help shed light on these factors. This paper discusses the findings from four mainly qualitative research projects including a systematic review and three empirical studies involving young people to various degrees within the research process as respondents, co-researchers and commissioners of research. Much quantitative research suggests that young people are a homogenous group and through the use of surveys and other large scale methods, generalizations can be drawn about how bullying is understood and how it can be dealt with. Findings from the studies presented in this paper, add to our understanding that young people appear particularly concerned about the role of wider contextual and relational factors in deciding if bullying has happened. These studies underscore the relational aspects of definitions of bullying and, how the dynamics of young people's friendships can shift what is understood as bullying or not. Moreover, to appreciate the relational and social contexts underpinning bullying behaviors, adults and young people need to work together on bullying agendas and engage with multiple definitions, effects and forms of support. Qualitative methodologies, in particular participatory research opens up the complexities of young lives and enables these insights to come to the fore. Through this approach, effective supports can be designed based on what young people want and need rather than those interpreted as supportive through adult understanding.

Introduction

Research on school bullying has developed rapidly since the 1970s. Originating in social and psychological research in Norway, Sweden, and Finland, this body of research largely focusses on individualized personality traits of perpetrators and victims ( Olweus, 1995). Global interest in this phenomenon subsequently spread and bullying research began in the United Kingdom, Australia, and the United States ( Griffin and Gross, 2004). Usually quantitative in nature, many studies examine bullying prevalence, risk and protective factors, and negative outcomes ( Patton et al., 2017). Whilst quantitative research collates key demographic information to show variations in bullying behaviors and tendencies, this dominant bullying literature fails to explain why bullying happens. Nor does it attempt to understand the wider social contexts in which bullying occurs. Qualitative research on the other hand, in particular participatory research, can help shed light on these factors by highlighting the complexities of the contextual and relational aspects of bullying and the particular challenges associated with addressing it. Patton et al. (2017) in their systematic review of qualitative methods used in bullying research, found that the use of such methods can enhance academic and practitioner understanding of bullying.

In this paper, I draw on four bullying studies; one systematic review of both quantitative and qualitative research ( O'Brien, 2009) and three empirical qualitative studies ( O'Brien and Moules, 2010; O'Brien, 2016, 2017) (see Table 1 below). I discuss how participatory research methodologies, to varying degrees, were used to facilitate bullying knowledge production among teams of young people and adults. Young people in these presented studies were consequently involved in the research process along a continuum of involvement ( Bragg and Fielding, 2005). To the far left of the continuum, young people involved in research are referred to as "active respondents" and their data informs teacher practice. To the middle of the continuum sit "students as co-researchers" who work with teachers to explore an issue which has been identified by that teacher. Finally to the right, sit "students as researchers" who conduct their own research with support from teachers. Moving from left to right of the continuum shows a shift in power dynamics between young people and adults where a partnership develops. Young people are therefore recognized as equal to adults in terms of what they can bring to the project from their own unique perspective, that of being a young person now.

Table 1. The studies.

In this paper, I advocate for the active involvement of young people in the research process in order to enhance bullying knowledge. Traditional quantitative studies have a tendency to homogenize young people by suggesting similarity in thinking about what constitutes bullying. However, qualitative studies have demonstrated that regardless of variables, young people understand bullying in different ways so there is a need for further research that starts from these perspectives and focusses on issues that young people deem important. Consequently, participatory research allows for the stories of the collective to emerge without losing the stories of the individual, a task not enabled through quantitative approaches.

What Is Bullying?

Researching school bullying has been problematic and is partly related to the difficulty in defining it ( Espelage, 2018). Broadly speaking, bullying is recognized as aggressive, repeated, intentional behavior involving an imbalance of power aimed toward an individual or group of individuals who cannot easily defend themselves ( Vaillancourt et al., 2008). In more recent times, "traditional" bullying behaviors have been extended to include cyber-bullying, involving the use of the internet and mobile-phones ( Espelage, 2018). Disagreements have been noted in the literature about how bullying is defined by researchers linked to subject discipline and culture. Some researchers for example, disagree about the inclusion or not of repetition in definitions ( Griffin and Gross, 2004) and these disagreements have had an impact on interpreting findings and prevalence rates. However, evidence further suggests that young people also view bullying in different ways ( Guerin and Hennessy, 2002; Cuadrado-Gordillo, 2012; Eriksen, 2018). Vaillancourt et al. (2008) explored differences between researchers and young people's definitions of bullying, and found that children's definitions were usually spontaneous, and did not always encompass the elements of repetition, power imbalance and intent. They concluded, that children need to be provided with a bullying definition so similarities and comparisons can be drawn. In contrast, Huang and Cornell (2015) found no evidence that the inclusion of a definition effected prevalence rates. Their findings, they suggest, indicate that young people use their own perceptions of bullying when answering self-report questionnaires and they are not influenced by an imposed definition.

Nevertheless, differences in children and young people's bullying definitions are evident in the research literature and have been explained by recourse to age and stage of development ( Smith et al., 2002) and their assumed lack of understanding about what constitutes bullying ( Boulton and Flemington, 1996). Naylor et al. (2001) for example, found that younger children think similarly in their definitions of bullying, while Smith et al. (2002) found that 8 year olds did not distinguish as clearly between different forms of behavioral aggression as 14 year olds. Methodological limitations associated with understanding bullying have been identified by Forsberg et al. (2018) and Maunder and Crafter (2018). These authors postulate that quantitative approaches, although providing crucial insights in understanding bullying, are reliant on pre-defined variables, which can shield some of the complexities that qualitative designs can unravel, as individual experiences of bullying are brought to the fore. Indeed, La Fontaine (1991) suggests that unlike standard self-report questionnaires and other quantitative methods used to collect bullying data, analyzing qualitative data such as those collected from a helpline, enables the voice of young people to be heard and consequently empowers adults to understand bullying on their terms rather than relying solely on interpretations and perceptions of adults. Moore and Maclean (2012) collected survey, as well as interview and focus group data, on victimization occurring on the journey to and from school. They found that what young people determined as victimization varied and was influenced by a multifaceted array of circumstances, some of which adults were unaware of. Context for example, played an important role where certain behaviors in one situation could be regarded as victimization while in another they were not. Specific behaviors including ignoring an individual was particularly hurtful and supporting a friend who was the subject of victimization could lead to their own victimization.

Lee (2006) suggests that some bullying research does not reflect individual experiences, and are thus difficult for participants to relate to. Canty et al. (2016) reiterates this and suggests that when researchers provide young people with bullying definitions in which to position their own experiences, this can mask some of the complexities that the research intends to uncover. Such approaches result in an oversight into the socially constructed and individual experiences of bullying ( Eriksen, 2018). Griffin and Gross (2004) further argue that when researchers use vague or ambiguous definitions an "overclassification of children as bullies or victims" (p. 381) ensues. Consequently, quantitative research does not consider children as reliable in interpreting their own lived experiences and therefore some of the interactions they consider as bullying, that do not fit within the conventional definitions, are concealed. This approach favors the adult definition of bullying regarding it as "more reliable" than the definitions of children and young people Canty et al. (2016). The perceived "seriousness" of bullying has also been explored. Overall, young people and adults are more likely to consider direct bullying (face-to-face actions including hitting, threatening and calling names) as "more serious" than indirect bullying (rumor spreading, social exclusion, forcing others to do something they do not want to do) ( Maunder et al., 2010; Skrzypiec et al., 2011). This perception of "seriousness," alongside ambiguous definitions of bullying, has further implications for reporting it. Despite the advice given to young people to report incidents of school bullying ( Moore and Maclean, 2012), the literature suggests that many are reluctant to do so ( deLara, 2012; Moore and Maclean, 2012).

Several factors have been highlighted as to why young people are reluctant to report bullying ( Black et al., 2010). deLara (2012), found apprehension in reporting bullying to teachers due to the fear that they will either not do enough or too much and inadvertently make the situation worse, or fear that teachers will not believe young people. Research also shows that young people are reluctant to tell their parents about bullying due to perceived over-reaction and fear that the bullying will be reported to their school ( deLara, 2012; Moore and Maclean, 2012). Oliver and Candappa (2007) suggest that young people are more likely to confide in their friends than adults (see also Moore and Maclean, 2012; Allen, 2014). However, if young people believe they are being bullied, but are unable to recognize their experiences within a predefined definition of bullying, this is likely to impact on their ability to report it.

Research from psychology, sociology, education and other disciplines, utilizing both quantitative and qualitative approaches, have enabled the generation of bullying knowledge to date. However, in order to understand why bullying happens and how it is influenced by wider social constructs there is a need for further qualitative studies, which hear directly from children and young people themselves. The next section of this paper discusses the theoretical underpinnings of this paper, which recognizes that young people are active agents in generating new bullying knowledge alongside adults.

Theoretical Underpinnings - Hearing From Children and Young People

The sociology of childhood ( James, 2007; Tisdall and Punch, 2012) and children's rights agenda more broadly ( United Nations Convention on the Rights of the Child, 1989) have offered new understandings and methods for research which recognize children and young people as active agents and experts on their own lives. From this perspective, research is conducted with rather than on children and young people ( Kellett, 2010).

Participatory methodologies have proven particularly useful for involving young people in research as co-researchers (see for example O'Brien and Moules, 2007; Stoudt, 2009; Kellett, 2010; Spears et al., 2016). This process of enquiry actively involves those normally being studied in research activities. Previously, "traditional" researchers devalued the experiences of research participants arguing that due to their distance from them, they themselves are better equipped to interpret these experiences ( Beresford, 2006). However, Beresford (2006) suggests that the shorter the distance between direct experience and interpretation, the less distorted and inaccurate the resulting knowledge is likely to be. Jones (2004) further advocates that when young people's voices are absent from research about them the research is incomplete. Certainly Spears et al. (2016), adopted this approach in their study with the Young and Well Cooperative Research Centre (CRC) in Australia. Young people played an active role within a multidisciplinary team alongside researchers, practitioners and policymakers to co-create and co-evaluate the learning from four marketing campaigns for youth wellbeing through participatory research. Through this methodological approach, findings show that young people were able to reconceptualize mental health and wellbeing from their own perspectives as well as share their lived experiences with others ( Spears et al., 2016). Bland and Atweh (2007), Ozer and Wright (2012), highlight the benefits afforded to young people through this process, including participating in dialog with decision-makers and bringing aspects of teaching and learning to their attention.

Against this background, data presented for this paper represents findings from four studies underpinned by the ethos that bullying is socially constructed and is best understood by exploring the context to which it occurs ( Schott and Sondergaard, 2014; Eriksen, 2018). This socially constructed view focusses on the evolving positions within young people's groups, and argues that within a bullying situation sometimes a young person is the bully, sometimes the victim and sometimes the bystander/witness, which contrasts the traditional view of bullying ( Schott and Sondergaard, 2014). The focus therefore is on group relationships and dynamics. For that reason, Horton (2011) proposes that if bullying is an extensive problem including many young people, then focusing entirely on personality traits will not generate new bullying knowledge and will be problematic in terms of interventions. It is important to acknowledge that this change in focus and view of bullying and how it is manifested in groups, does not negate the individual experiences of bullying rather the focus shifts to the process of being accepted, or not, by the group ( Schott and Sondergaard, 2014).

The Studies

This section provides a broad overview of the four included studies underpinned by participatory methodologies. Table 1 presents the details of each study. Young people were involved in the research process as respondents, co-researchers and commissioners of research, along a continuum as identified by Bragg and Fielding (2005). This ranged from "active respondents" to the left of the continuum, "students as co-researchers" in the middle and "students as researchers" to the right of the continuum. Young people were therefore recognized as equal to adults in terms of what they can bring to the project from their own unique perspectives ( Bradbury-Jones et al., 2018).

A key finding from study one ( O'Brien, 2009) was the lack of voice afforded to young people through the research process and can be seen to reflect the far left of Bragg and Fielding (2005) continuum, as young people were not directly involved as "active respondents" but their views were included in secondary data analysis and informed the studies that followed. For example, the quantitative studies used an agreed academic definition of bullying which may or may not have influenced how young participants defined bullying within the studies. On the other hand, the qualitative study involved a group of students in deciding which questions to ask of the research participants and in interpreting the findings.

In contrast, study two ( O'Brien and Moules, 2010) was commissioned and led by a group of young people called PEAR (Public health, Education, Awareness, Researchers), who were established to advise on public health research in England. PEAR members were based in two large English cities and comprised 20 young people aged between 13 and 20 years. The premise of the study was that PEAR members wanted to commission research into cyber bullying and the effects this has on mental health from the perspectives of young people rather than adult perspectives. This project was innovative as young people commissioned the research and participated as researchers ( Davey, 2011) and can be seen to reflect the middle "students as co-researchers" as well as moving toward to right "students as researchers" of Bragg and Fielding (2005) continuum. Although the young people did not carry out the day-to-day work on the project, they were responsible for leading and shaping it. More importantly, the research topic and focus were decided with young people and adults together.

Study three ( O'Brien, 2016) involved five self-selecting students from an independent day and boarding school who worked with me to answer this question: What do young people in this independent day and boarding school view as the core issue of bullying in the school and how do they want to address this? These students called themselves R4U (Research for You) with the slogan researching for life without fear. Three cycles of Participatory Action Research (PAR) ensued, where decision making about direction of the research, including methods, analysis and dissemination of findings were made by the research team. As current students of the school, R4U had a unique "insider knowledge" that complemented my position as the "academic researcher." By working together to generate understanding about bullying at the school, the findings thus reflected this diversity in knowledge. As the project evolved so too did the involvement of the young researchers and my knowledge as the "outsider" (see O'Brien et al., 2018a for further details). Similar to study two, this project is situated between the middle: "students as co-researchers" and the right: "students as researchers" of Bragg and Fielding (2005) continuum.

Study four ( O'Brien, 2017) was small-scale and involved interviewing four young people who were receiving support from a charity providing therapeutic and educational support to young people who self-exclude from school due to anxiety, as a result of bullying. Self-exclusion, for the purposes of this study, means that a young person has made a decision not to go to school. It is different from "being excluded" or "truanting" because these young people do not feel safe at school and are therefore too anxious to attend. Little is known about the experiences of young people who self-exclude due to bullying and this study helped to unravel some of these issues. This study reflects the left of Bragg and Fielding (2005) continuum where the young people were involved as "active respondents" in informing adult understanding of the issue.

A variety of research methods were used across the four studies including questionnaires, interviews and focus groups (see Table 1 for more details). In studies two and three, young researchers were fundamental in deciding the types of questions to be asked, where they were asked and who we asked. In study three the young researchers conducted their own peer-led interviews. The diversity of methods used across the studies are a strength for this paper. An over-reliance on one method is not portrayed and the methods used reflected the requirements of the individual studies.

Informed Consent

Voluntary positive agreement to participate in research is referred to as "consent" while "assent," refers to a person's compliance to participate ( Coyne, 2010). The difference in these terms are normally used to distinguish the "legal competency of children over and under 16 years in relation to research." ( Coyne, 2010, 228). In England, children have a legal right to consent so therefore assent is non-applicable ( Coyne, 2010). However, there are still tensions surrounding the ability of children and young people under the age of 18 years to consent in research which are related to their vulnerability, age and stage of development ( Lambert and Glacken, 2011). The research in the three empirical studies (two, three and four) started from the premise that all young participants were competent to consent to participate and took the approach of Coyne (2010) who argues that parental/carer consent is not always necessary in social research. University Research Ethics Committees (RECs) are nonetheless usually unfamiliar with the theoretical underpinnings that children are viewed as social actors and generally able to consent for themselves ( Lambert and Glacken, 2011; Fox, 2013; Parsons et al., 2015).

In order to ensure the young people in these reported studies were fully informed of the intentions of each project and to adhere to ethical principles, age appropriate participant information sheets were provided to all participants detailing each study's requirements. Young people were then asked to provide their own consent by signing a consent form, any questions they had about the studies were discussed. Information sheets were made available to parents in studies three and four. In study two, the parents of young people participating in the focus groups were informed of the study through the organizations used to recruit the young people. My full contact details were provided on these sheets so parents/carers could address any queries they had about the project if they wished. When young people participated in the online questionnaire (study two) we did not know who they were so could not provide separate information to parents. Consequently, all participants were given the opportunity to participate in the research without the consent of their parents/carers unless they were deemed incompetent to consent. In this case the onus was on the adult (parent or carer for example) to prove incompetency ( Alderson, 2007). Favorable ethical approval, including approval for the above consent procedures, was granted by the Faculty Research Ethics Committee at Anglia Ruskin University.

In the next section I provide a synthesis of the findings across the four studies before discussing how participatory research with young people can offer new understandings of bullying and its impacts on young people.

Findings

Although each study was designed to answer specific bullying research questions, the following key themes cut across all four studies :

* Bullying definitions

◦ Behaviors

* Impact of bullying on victim

* Reporting bullying

Bullying Definitions

Behaviors

Young people had various understandings about what they considered bullying to be. Overall, participants agreed that aggressive direct behaviors, mainly focusing on physical aggression, constituted bullying:

"...if someone is physically hurt then that is bullying straight away." (Female, study 3).

"I think [cyber-bullying is] not as bad because with verbal or physical, you are more likely to come in contact with your attacker regularly, and that can be disturbing. However, with cyber-bullying it is virtual so you can find ways to avoid the person." (Female, study 2).

Name-calling was an ambiguous concept, young people generally believed that in isolation name-calling might not be bullying behavior or it could be interpreted as "joking" or "banter":

"I never really see any, a bit of name calling and taking the mick but nothing ever serious." (Male, study 3).

The concept of "banter" or "joking" was explored in study three as a result of the participatory design. Young people suggested "banter" involves:

"...a personal joke or group banter has no intention to harm another, it is merely playful jokes." (Female, study 3).

However, underpinning this understanding of "banter" was the importance of intentionality:

"Banter saying things bad as a joke and everyone knows it is a joke." (Male, study 3).

"Banter" was thus contentious when perception and reception were ambiguous. In some cases, "banter" was considered "normal behavior":

"...we've just been joking about, but it's never been anything harsh it's just been like having a joke..." (Male, study 3).

The same view was evident in relation to cyber-bullying. Some participants were rather dismissive of this approach suggesting that it did not exist:

"I don't really think it exists. If you're being cyber-"bullied" then there is something wrong with you- it is insanely easy to avoid, by blocking people and so on. Perhaps it consists of people insulting you online?" (Male, study 2).

When young people considered additional factors added to name calling such as the type of name-calling, or aspects of repetition or intention, then a different view was apparent.

"...but it has to be constant it can't be a single time because that always happens." (Male, study 3).

Likewise with words used on social media, young people considered intentionality in their consideration of whether particular behaviors were bullying, highlighting important nuances in how bullying is conceptualized:

"Some people they don't want to sound cruel but because maybe if you don't put a smiley face on it, it might seem cruel when sometimes you don't mean it." (Female, study 2).

Study one also found that young people were more likely to discuss sexist or racist bullying in interviews or focus groups but this information was scarce in the questionnaire data. This is possibly as a result of how the questions were framed and the researchers' perspectives informing the questions.

Evident across the four studies was the understanding young people had about the effects of continuous name-calling on victims:

"...you can take one comment, you can just like almost brush it off, but if you keep on being bullied and bullied and bullied then you might kind of think, hang on a minute, they've taken it a step too far, like it's actually become more personal, whereas just like a cheeky comment between friends it's become something that's more serious and more personal and more annoying or hurtful to someone." (Female, study 3).

"Cyber-bullying is basically still verbal bullying and is definitely psychological bullying. Any bullying is psychological though, really. And any bullying is going to be harmful." (Female, study 2).

Aspects of indirect bullying (social exclusion) were features of studies one and three. For the most part, the research reviewed in study one found that as young people got older they were less likely to consider characteristics of social exclusion in their definitions of bullying. In study three, when discussing the school's anti-bullying policy, study participants raised questions about " isolating a student from a friendship group." Some contested this statement as a form of bullying:

".... there is avoiding, as in, not actively playing a role in trying to be friends which I don't really see as bullying I see this as just not getting someone to join your friendship group. Whereas if you were actually leaving him out and rejecting him if he tries to be friends then I think I would see that as malicious and bullying." (Male, study 3).

"Isolating a student from a friendship group - I believe there are various reasons for which a student can be isolated from a group - including by choice." (Female, study 3).

Cyber-bullying was explored in detail in study two but less so in the other three studies. Most study two participants considered that cyber-bullying was just as harmful, or in some cases worse than, 'traditional' bullying due to the use of similar forms of "harassment," "antagonizing," "tormenting," and 'threatening' through online platforms. Some young people believed that the physical distance between the victim and the bully is an important aspect of cyber-bullying:

"I think it's worse because people find it easier to abuse someone when not face to face." (Male, study 2).

"I think it could be worse, because lots of other people can get involved, whereas when it's physical bullying it's normally just between one or two or a smaller group, things could escalate too because especially Facebook, they've got potential to escalate." (Female, study 2).

Other participants in study two spoke about bullying at school which transfers to an online platform highlighting no "escape" for some. In addition, it was made clearer that some young people considered distancing in relation to bullying and how this influences perceptions of severity:

"...when there's an argument it can continue when you're not at school or whatever and they can continue it over Facebook and everyone can see it then other people get involved." (Female, study 2).

"I was cyber-bullied on Facebook, because someone put several hurtful comments in response to my status updates and profile pictures. This actually was extended into school by the bully..." (Male, study 2).

Impact of Bullying on Victim

Although bullying behaviors were a primary consideration of young people's understanding of bullying, many considered the consequences associated with bullying and in particular, the impact on mental health. In these examples, the specifics of the bullying event were irrelevant to young people and the focus was on how the behavior was received by the recipient.

In study two, young people divulged how cyber-bullying had adversely affected their ability to go to school and to socialize outside school. Indeed some young people reported the affects it had on their confidence and self-esteem:

"I developed anorexia nervosa. Although not the single cause of my illness, bullying greatly contributed to my low self-esteem which led to becoming ill." (Female, study 2).

"It hurts people's feelings and can even lead to committing suicide...." (Female, study 2).

Across the studies, young people who had been bullied themselves shared their individual experiences:

"....you feel insecure and it just builds up and builds up and then in the end you have no self-confidence." (Female, study 2).

"...it was an everyday thing I just couldn't take it and it was causing me a lot of anxiety." (Male, study 4).

"I am different to everyone in my class .... I couldn't take it no more I was upset all the time and it made me feel anxious and I wasn't sleeping but spent all my time in bed being sad and unhappy." (Male, study 4).

Young people who had not experienced bullying themselves agreed that the impact it had on a person was a large determiner of whether bullying had happened:

"When your self-confidence is severely affected and you become shy. Also when you start believing what the bullies are saying about you and start to doubt yourself." (Female, study 3).

"...it makes the victim feel bad about themselves which mostly leads to depression and sadness." (Male, study 2).

Further evidence around the impact of bullying was apparent in the data in terms of how relational aspects can affect perceived severity. In the case of cyber-bullying, young people suggested a sense of detachment because the bullying takes place online. Consequently, as the relational element is removed bullying becomes easier to execute:

"...because people don't have to face them over a computer so it's so much easier. It's so much quicker as well cos on something like Facebook it's not just you, you can get everyone on Facebook to help you bully that person." (Female, study 2).

"Due to technology being cheaper, it is easier for young people to bully people in this way because they don't believe they can be tracked." (Male, study 2).

"The effects are the same and often the bullying can be worse as the perpetrator is unknown or can disguise their identity. Away from the eyes of teachers etc., more can be done without anyone knowing." (Female, study 2).

Relational aspects of bullying were further highlighted with regards to how "banter" was understood, particularly with in-group bullying and how the same example can either be seen as "banter" or bullying depending on the nature of the relationship:

"...we've just been joking about, but it's never been anything harsh it's just been like having a joke. well, I haven't done it but I've been in a crowd where people do it, so I don't want to get involved just in case it started an argument." (Female, study 3).

"But it also depends...who your groups with, for example, if I spoke to my friends from [School]... I wouldn't like use taboo language with them because to them it may seem inappropriate and probably a bit shocked, but if I was with my friends outside of school we use taboo language, we'll be ourselves and we'll be comfortable with it, and if a stranger walked past and heard us obviously they'd be thinking that we're being bullied ourselves." (Female, study 3).

Furthermore, how individuals are perceived by others tended to influence whether they were believed or not. In study four for example, participants suggested that who the bullies were within the school might have impacted how complaints were acted upon by school officials:

"When I went to the school about it, the students said I had attacked them - all eight of them! I just realized that no one believes me...." (Female, study 4).

While in study three, a characteristic of bullying was the influence the aggressor has over the victim:

"When the victim starts to feel in danger or start to fear the other person. Consequently he or she tries to avoid the bad guy (or girl!)" (Male, study 3).

These relational and contextual issues also influenced a young person's ability to report bullying.

Reporting Bullying

Young people were more likely to report bullying when they considered it was 'serious' enough. Just under half of participants in study two sought emotional/practical support if they worried about, or were affected by cyber-bullying, with most talking to their parents. In study three, young people were less likely to seek support but when they did, most went to their teachers. In study four, all participants reported bullying in school where they did not feel supported.

Fear of making the bullying worse was captured across the studies as a reason for not reporting it:

"I'm scared that if I tell then the bullying will still go on and they will do more." (Female, study 3).

"The bully might bully you if he finds out." (Male, study 3).

Being able to deal with the incident themselves was also a reason for non-reporting:

"...it's embarrassing and not necessary, my friends help me through it, adults never seem to understand." (Female, study 2).

"I don't tend to talk to anyone about it, I just keep it to myself and obviously that's the worst thing you should ever do, you should never keep it to yourself, because I regret keeping it to myself to be honest...." (Female, study 3).

"...but I think I'd deal with it myself 'cos. I was quite insecure but now I'm quite secure with myself, so I'll sort it out myself. I think it's just over time I've just sort of hardened to it." (Male, study 3).

Most young people seeking support for bullying said they spoke to an adult but the helpfulness of this support varied. This finding is important for understanding relationships between young people and adults. Those who felt supported by their teachers for example, suggested that they took the time to listen and understood what they were telling them. They also reassured young people who in turn believed that the adult they confided in would know what to do:

"So I think the best teacher to talk to is [Miss A] and even though people are scared of her I would recommend it, because she's a good listener and she can sense when you don't want to talk about something, whereas the other teachers force it out of you." (Female, study 3).

"My school has had assemblies about cyber-bullying and ways you can stop it or you can report it anonymously.... you can write your name or you can't, it's all up to YOU." (Male, study 2).

Others however had a negative experience of reporting bullying and a number of reasons were provided as to why. Firstly, young people stated that adults did not believe them which made the bullying worse on some level:

"I went to the teachers a couple of times but, no, I don't think they could do anything. I did sort of go three times and it still kept on going, so I just had to sort of deal with it and I sort of took it on the cheek...." (Male, study 3).

Secondly, young people suggested that adults did not always listen to their concerns, or in some cases did not take their concerns seriously enough:

"...I had had a really bad day with the girls so I came out and I explained all this to my head of year and how it was affecting me but instead of supporting me he put me straight into isolation." (Male, study 4).

"I could understand them thinking I maybe got the wrong end of the stick with one incident but this was 18 months of me constantly reporting different incidents." (Female, study 4).

"If cyber-bullying is brought to our school's attention, usually, they expect printed proof of the situation and will take it into their own hand depending on its seriousness. However this is usually a couple of detentions. And it's just not enough." (Female, study 2).

Finally, some young people suggested that teachers did not always know what to do when bullying concerns were raised and consequently punished those making the complaint:

"I think I would have offered support instead of punishment to someone who was suffering with anxiety. I wouldn't have seen anxiety as bad behavior I think that's quite ignorant but they saw it as bad behavior." (Male, study 4).

It is worth reiterating, that the majority of young people across the studies did not report bullying to anybody, which further underscores the contextual issues underpinning bullying and its role in enabling or disabling bullying behaviors. Some considered it was "pointless" reporting the bullying and others feared the situation would be made worse if they did:

"My school hide and say that bullying doesn't go on cos they don't wanna look bad for Ofsted." (Male, study 2).

"My school is oblivious to anything that happens, many things against school rules happen beneath their eyes but they either refuse to acknowledge it or are just not paying attention so we must suffer." (Female, study 2).

"That's why I find that when you get bullied you're scared of telling because either, in most cases the teacher will - oh yeah, yeah, don't worry, we'll sort it out and then they don't tend to, and then they get bullied more for it." (Female, study 3).

Young people were concerned that reporting bullying would have a negative impact on their friendship groups. Some were anxious about disrupting the status quo within:

"I think everyone would talk about me behind my back and say I was mean and everyone would hate me." (Female, study 3).

Others expressed concern about the potential vulnerability they were likely to experience if they raised concerns of bullying:

"I was worried it might affect my other friendships."(Boy, study 2).

"I'm scared that if I tell, then the bullying will still go on and they will do more." (Female, study 3).

"....because they might tell off the bullies and then the bullies will like get back at you." (Female, study 3).

These findings underscore the importance of contextual and relational factors in understanding bullying from the perspectives of young people and how these factors influence a young person's ability or willingness to report bullying.

Finally one young person who had self-excluded from school due to severe bullying suggested that schools:

"...need to be looking out for their students' mental wellbeing - not only be there to teach them but to support and mentor them. Keep them safe really... I missed out on about three years of socializing outside of school because I just couldn't do it. I think it's important that students are encouraged to stand up for each other." (Female, study 4).

Discussion

The studies presented in this paper illustrate the multitude of perceptions underpinning young people's understandings of what constitutes bullying, both in terms of the behavior and also the impact that this behavior has on an individual. In turn, the ambiguity of what constitutes bullying had an impact on a young person's ability to seek support. Discrepancies in bullying perceptions within and between young people's groups are shown, highlighting the fluid and changing roles that occur within a bullying situation. Findings from quantitative studies have demonstrated the differing perceptions of bullying by adults and young people (see for example Smith et al., 2002; Vaillancourt et al., 2008; Maunder et al., 2010; Cuadrado-Gordillo, 2012). However, by combining findings from participatory research, new understandings of the relational and contextual factors important to young people come to the fore.

Young people participating in these four studies had unique knowledge and experiences of bullying and the social interactions of other young people in their schools and wider friendship groups. The underpinning participatory design enabled me to work alongside young people to analyze and understand their unique perspectives of bullying in more detail. The research teams were therefore able to construct meaning together, based not entirely on our own assumptions and ideologies, but including the viewpoint of the wider research participant group ( Thomson and Gunter, 2008). Together, through the process of co-constructing bullying knowledge, we were able to build on what is already known in this field and contribute to the view that bullying is socially constructed through the experiences of young people and the groups they occupy ( Schott and Sondergaard, 2014).

With regards to understanding what bullying is, the findings from these studies corroborate those of the wider literature from both paradigms of inquiry (for example Naylor et al., 2001; Canty et al., 2016); that being the discrepancies in definitions between adults and young people and also between young people themselves. Yet, findings here suggest that young people's bullying definitions are contextually and relationally contingent. With the exception of physical bullying, young people did not differentiate between direct or indirect behaviors, instead they tended to agree that other contextual and relational factors played a role in deciding if particular behaviors were bullying (or not). The participatory research design enabled reflection and further investigation of the ideas that were particularly important to young people such as repetition and intentionality. Repetition was generally seen as being indicative of bullying being "serious," and therefore more likely to be reported, and without repetition, a level of normality was perceived. This finding contradicts some work on bullying definitions, Cuadrado-Gordillo (2012) for example found that regardless of the role played by young people in a bullying episode (victim, aggressor or witness), the criteria of 'repetition' was not important in how they defined bullying.

Relational factors underpinning young people's perception of bullying and indeed it's "seriousness" were further reflected in their willingness or otherwise to report it. Fear of disrupting the status quo of the wider friendship group, potentially leading to their own exclusion from the group, was raised as a concern by young people. Some were concerned their friends would not support them if they reported bullying, while others feared further retaliation as a result. Friendship groups have been identified as a source of support for those who have experienced bullying and as a protective factor against further bullying ( Allen, 2014). Although participants did not suggest their friendship groups are unsupportive it is possible that group dynamics underscore seeking (or not) support for bullying. Other literature has described such practices as evidence of a power imbalance ( Olweus, 1995; Cuadrado-Gordillo, 2012) but young people in these studies did not describe these unequal relationships in this way and instead focused on the outcomes and impacts of bullying. Indeed Cuadrado-Gordillo (2012) also found that young people in their quantitative study did not consider "power imbalance" in their understanding of bullying and were more likely to consider intention. This paper, however, underscores the relational aspects of definitions of bullying and, how the dynamics of young people's friendships can shift what is understood as bullying or not. Without such nuances, some behaviors may be overlooked as bullying, whereas other more obvious behaviors draw further attention. This paper also shows that contextual issues such as support structures can shift how young people see bullying. Contextual factors were evident across the four studies through the recognition of bullying being enabled or disabled by institutional factors, including a school's ability to respond appropriately to bullying concerns. Young people suggested that schools could be influenced by bullies, perceiving them as non-threatening and consequently not dealing appropriately with the situation. Indeed some young people reported that their schools placed the onus on them as victims to change, consequently placing the "blame" on victims instead. These findings raise questions about who young people feel able to confide in about bullying as well as issues around training and teacher preparedness to deal with bullying in schools. Evidenced in these four studies, is that young people feel somewhat disconnected from adults when they have bullying concerns. Those who did report bullying, identified particular individuals they trusted and knew would support them. Novick and Isaacs (2010) identified teachers who young people felt comfortable in approaching to report bullying and described them as "most active, engaged and responsive." (p. 291). The bullying literature suggests that as young people get older they are more likely to confide in friends than adults ( Moore and Maclean, 2012; Allen, 2014). However, findings from this paper indicate that although fewer young people reported bullying, those who did confided in an adult. Young people have identified that a variety of supports are required to tackle bullying and that adults need to listen and work with them so nuanced bullying behaviors are not recognized as "normal" behaviors. Within the data presented in this paper, "banter" was portrayed as "normal" behavior. Young people did not specify what behaviors they regarded as "banter," but suggested that when banter is repeated and intentional the lines are blurred about what is bullying and what is banter.

Exploring bullying nuances in this paper, was enhanced by the involvement of young people in the research process who had a unique "insider" perspective about what it is like to be a young person now and how bullying is currently affecting young people. In studies one and four, young people were "active respondents" ( Bragg and Fielding, 2005) and provided adults with their own unique perspectives on bullying. It could be argued that study one did not involve the participation of young people. However, this study informed the basis of the subsequent studies due to the discrepancies noted in the literature about how bullying is understood between adults and young people, as well as the lack of young people's voice and opportunity to participate in the reviewed research. Accordingly, young people's data as "active respondents" informed adult understanding and led to future work involving more active research engagement from other young people. Participation in study four provided an opportunity for young people to contribute to future participatory research based on lived experiences as well as informing policy makers of the effects bullying has on the lives of young people ( O'Brien, 2017). In studies two and three, young people were involved further along Bragg and Fielding (2005) continuum as "co-researchers" and "students as researchers" with these roles shifting and moving dependent on the context of the project at the time ( O'Brien et al., 2018a). These young researchers brought unique knowledge to the projects ( Bradbury-Jones et al., 2018) that could not be accessed elsewhere. Perspectives offered by the young researchers supported adults in understanding more about traditional and cyber-bullying from their perspectives. Furthermore, this knowledge can be added to other, quantitative studies to further understand why bullying happens alongside bullying prevalence, risk and protective factors, and negative outcomes.

Conclusion

Findings from the four studies offer an alternative perspective to how bullying is understood by young people. Complexities in defining bullying have been further uncovered as understanding is informed by individual factors, as well as wider social and relational contexts ( Horton, 2011; Schott and Sondergaard, 2014). This has implications for the type of support young people require. This paper highlights how definitions of bullying shift in response to relational and contextual aspects deemed important to young people. Because of this, further nuances were uncovered through the research process itself as the respective studies showed discrepancies in bullying perceptions within and between young people's groups.

These understandings can act as a starting point for young people and adults to collaborate in research which seeks to understand bullying and the context to which it occurs. Furthermore, such collaborations enable adults to theorize and understand the complexities associated with bullying from the perspective of those at the center. There is a need for additional participatory research projects involving such collaborations where adults and young people can learn from each other as well as combining findings from different methodologies to enable a more comprehensive picture of the issues for young people to emerge. Further research is needed to unravel the complexities of bullying among and between young people, specifically in relation to the contextual and relational factors underscoring perceptions of bullying.

Data Availability

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

Ethics Statement

Ethical approval was granted for all four studies from the Faculty of Health, Education, Medicine and Social Care at the Anglia Ruskin University. The research was conducted on the premise of Gillick competency meaning that young people (in these studies over the age of 12 years) could consent for themselves to participate. Parents/carers were aware the study was happening and received information sheets explaining the process.

Author Contributions

The author confirms being the sole contributor of this work and has approved it for publication.

Funding

These four studies were conducted at the Anglia Ruskin University. Study one was part of a wider masters degree funded by the Anglia Ruskin University, Study two was funded by a group of young people convened by the National Children's Bureau with funding from the Wellcome Trust (United Kingdom). Study three was a wider Doctoral study funded by the Anglia Ruskin University and Study four was also funded by the Anglia Ruskin University.

Conflict of Interest Statement

The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

I would like to thank Dr. Grace Spencer, Ruskin Fellow at the Anglia Ruskin University for providing the critical read of this manuscript and offering constructive feedback. I would also like to thank the two independent reviewers for their feedback on the drafts of this manuscript.

Footnotes

References

O'Brien, N. (2017). An Exploratory Study of Bullied Young People's Self-Exclusion from School. Evidence: Presented at Meetings of the All Party Parliamentary Group on Bullying 2011-2016. Project Report. All Party Parliamentary Group on Bullying. Available at: http://arro.anglia.ac.uk/id/eprint/702024 (accessed September 20, 2018).
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Understanding Alternative Bullying Perspectives Through Research Engagement With Young People

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Bullying research has traditionally been dominated by largescale cohort studies focusing on the personality traits of bullies and victims. These studies focus on bullying prevalence, risk and protective factors, and negative outcomes. A limitation of this approach is that it does not explain why bullying happens.

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Abstract

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that partly evade neutralizing antibodies raises concerns of reduced vaccine effectiveness and increased infection. We previously demonstrated that the SARS-CoV-2 spike protein vaccine adjuvanted with AS03 (CoV2 preS dTM-AS03) elicits robust neutralizing antibody responses in naïve subjects. Here we show that, in macaques primed with mRNA or protein-based subunit vaccine candidates, one booster dose of CoV2 preS dTM-AS03 (monovalent D614 or B.1.351, or bivalent D614 + B.1.351 formulations), significantly boosts the pre-existing neutralizing antibodies against the parental strain from 177- to 370-fold. Importantly, the booster dose elicits high and persistent cross-neutralizing antibodies covering five former or current SARS-CoV-2 variants of concern (Alpha, Beta, Gamma, Delta and Omicron) and, unexpectedly, SARS-CoV-1. Interestingly, we show that the booster specifically increases the functional antibody responses as compared to the receptor binding domain (RBD)-specific responses. Our findings show that these vaccine candidates, when used as a booster, have the potential to offer cross-protection against a broad spectrum of variants. This has important implications for vaccine control of SARS-CoV-2 variants of concern and informs on the benefit of a booster with the vaccine candidates currently under evaluation in clinical trials.

Introduction

Emergence of severe acute respiratory-syndrome coronavirus-2 (SARS-CoV-2) variants of concern (VOC), such as Alpha (B.1.1.7), Beta (B.1.351), and Delta (B.1.617.2) in late 2020 and lastly Omicron (B.1.1.529) in November 2021 have sparked concerns due to their higher transmissibility and/or pathogenicity1,2,3,4. These VOC harbor specific mutations in the spike proteins impacting the transmission and antigenicity reflecting growing immune pressure at positions shown to involve antibody recognition5. Some VOC (Beta, Gamma, Delta, and Omicron) have also demonstrated partial evasion of natural and vaccine-elicited neutralizing antibodies6,7,8,9,10 and are associated with reduction of vaccine effectiveness11,12,13. In response to the rapid evolution of SARS-CoV-2 and the global circulation of VOC since the end of 2020, vaccines are being developed using a modified spike antigen containing the mutations identified in Beta, Delta, and Omicron variants (NCT04785144)14,15,16. This strategy is based on the influenza-vaccine model where the vaccine is modified yearly according to the epidemiology, likewise, a combination of spike antigens covering multiple cocirculating strains may be necessary. In early 2021, the Beta spike (B.1.351) was selected, as it displayed the greatest breakthrough infections against the parental (D614) vaccines11,12, before Omicron was shown to cause even higher levels of vaccine escape. Considering the risk of waning immunity after natural infection or immunization and the risk of vaccine escape by emerging variants, the key attributes of future vaccines will be the ability to boost, prolong, and broaden protective immunity against new emerging variants.

Here we formulated soluble prefusion-stabilized spike trimers (CoV2 preS dTM) with the well-characterized adjuvant AS03, an oil-in-water emulsion composed of α-tocopherol, squalene, and polysorbate 8017. AS03 has been shown to potently induce antibody responses, increase vaccine durability, promote heterologous strain cross-reactivity, and has dose-sparing effects18,19. We previously reported that CoV2 preS dTM-AS03 (D614) confers protective efficacy against SARS-CoV-2 (D614 strain) challenge in nonhuman primates (NHPs) and the vaccine-induced IgG mediated protection from SARS-CoV-2 challenge following passive transfer to hamsters20. We also showed safety and immunogenicity of an optimized vaccine formulation in humans in a phase-2 clinical trial21 and the vaccine formulation is being assessed as a booster vaccine (NCT04762680) and for efficacy (NCT04904549) in phase-3 clinical studies.

In this work, the objective was to assess a protein-based subunit vaccine booster in macaques vaccinated 7 months before with either mRNA-LNP or subunit CoV2 preS dTM-AS03 Sanofi vaccine candidates. Various formulations, namely AS03-adjuvanted parental (D614), variant (B.1.351), and bivalent (D614 + B.1.351) CoV2 preS dTM or non-adjuvanted CoV2 preS dTM (B.1.351), were evaluated for their ability to boost neutralizing antibodies (NAbs) against the parental strain and to induce cross-neutralization against the five VOC described so far (Alpha, Beta, Gamma, Delta, and Omicron) and SARS-CoV-1 (from the 2003 outbreak) to further explore the breadth of neutralization.

Results

Boosting with homologous or heterologous formulations of CoV2 preS dTM led to significant increase in neutralizing antibodies against parental SARS-CoV-2, with a significant AS03-adjuvant effect

Following primary vaccination with either mRNA-LNP or CoV2 preS dTM-AS03 (Fig. 1a), we assessed the neutralizing antibody (NAb) responses against the parental D614G strain. Using a GFP-based pseudovirus-neutralization assay, the mean NAb titers at day 35 (D35, 2 weeks post-dose 2) against the parental pseudovirus were 2.6 log 10 for the mRNA-primed cohort and 2.9 log 10 for the subunit-primed cohort (Fig. 1b). Seven months post prime (baseline pre-boost), mean parental NAb titers declined to 1.4 log 10 for the mRNA-primed cohort and to 2.2 log 10 for the subunit-primed cohort. For comparison, the mean D614 NAb titer in a panel of 93 human convalescent sera (collected within 3 months after positive PCR test) was 2.0 log 10 (D614 and D614G NAb titers were shown to be similar in a concordance analysis, Supplementary Fig. 1) and the WHO International Standard for anti-SARS-CoV-2 immunoglobulin (human) (National Institute for Biological Standards and Control (NIBSC) code: 20/136) had a titer of 2.8 log 10.

a Schematic representation of the study schedule. In mRNA-primed cohort, 4 groups of 4 cynomolgus macaques were immunized intramuscularly with mRNA COVID-19 (D614) vaccine candidates on day 0 (D0) and on day 21 (D21). In subunit-primed cohort, 5 groups of 4-5 rhesus macaques were immunized intramuscularly with CoV2 preS dTM-AS03 (D614) vaccine candidates on D0 and D21. Both cohorts were boosted 7 months post-dose 1 with nonadjuvanted monovalent (B.1.351) CoV2 preS dTM ( n = 4) or AS03-adjuvanted monovalent (D614 or B.1.351) ( n = 5) or bivalent (D614 + B.1.351) CoV2 preS dTM ( n = 5). Humoral immune responses were assessed at different timepoints throughout the study. b Pseudovirus-neutralizing antibody titers against the parental SARS-CoV-2 and c Beta variant were assessed at different timepoints after the priming phase and after the booster immunization in macaques. Individual macaque data are shown. Connecting lines indicate mean responses and horizontal dotted lines the limits of quantification of the assay. *timepoints relative to boosters. A human convalescent panel of sera (Human Conv. sera) ( n  = 93) was assayed against D614 and is plotted as a comparator. Dotted line represents the World Health Organization (WHO) International Standard for anti-SARS-CoV-2 immunoglobulin (human) NIBSC code: 20/136.

A booster dose with non-adjuvanted CoV2 preS dTM (B.1.351) significantly increased parental NAb titers compared with baseline as soon as 7 days after injection, reaching a plateau between 14 and 28 days in both mRNA-primed (mean of 3.3 log 10) and subunit-primed (mean of 3.5 log 10) cohorts ( p-value < 0.001), and then slightly decreased up to 3 months post booster (mean of 2.6 and 2.8 log 10 in mRNA-primed and subunit-primed cohorts, respectively). As in each cohort, macaques were randomized for their baseline titers 6 months post-primary vaccination and peak titers post- prime (D35), we analyzed the fold-increase of NAb titers 14 days post-booster compared with baseline or post-prime. Compared with baseline, mean increase in NAbs were 60-fold ( p-value < 0.001) in mRNA-primed and 16-fold ( p-value < 0.001) in subunit-primed cohorts. Of note, baseline titers in the mRNA cohort were lower than in the subunit cohort. Compared with post-prime, the mean parental NAb titers 14 days post-booster were also significantly higher than the peak titer at D35 (3.2-fold, p-value < 0.05 and 3.8-fold, p-value < 0.01 for mRNA-primed and subunit-primed cohorts, respectively).

A booster dose with CoV2 preS dTM-AS03 adjuvanted vaccine candidates (monovalent or bivalent formulations) also significantly increased parental NAb titers compared with baseline in both cohorts as early as 7 days after injection, and by 14 days, the mean titers were 4.0 log 10 in mRNA-primed (mean fold increase of 370, p-value < 0.001) and 4.4 log 10 in subunit-primed (mean fold-increase of 177, p-value < 0.001) animals. The titers were stable up to D28, and then slightly declined up to D84 (mean titers at 3.3 log 10 and 3.7 log 10 in the mRNA- and subunit-primed cohorts, respectively).

In both the mRNA- and subunit-primed cohorts, after the AS03-adjuvanted booster, the parental NAb titers (D7-D28 post-boost) were significantly higher than the peak titers post-priming vaccination at D35 (mean fold increase of 21 in the mRNA-primed cohort and 31 in the subunit-primed cohort, p-value < 0.001). There were no statistically significant differences in parental NAb titer increase (from D35 post primary vaccination or from baseline pre-booster) between the various AS03-adjuvanted vaccine formulations.

In both cohorts, a significant adjuvant effect was observed on the fold increase from baseline to 2 weeks post-booster with CoV2 preS dTM-AS03 (B.1.351) vaccine compared with nonadjuvanted CoV2 preS dTM (B.1.351). AS03 induced a 6.5-fold and an 8.1-fold increase ( p-value < 0.01) in the mRNA-primed cohort and the subunit-primed cohort, respectively.

Up to one month post booster, the parental NAb titers elicited by nonadjuvanted CoV2 preS dTM (B.1.351) booster were all higher than the mean NAb titers of human convalescent sera (2.2 log 10), and all AS03-adjuvanted candidate vaccines induced titers above the highest titer observed in human convalescent sera in the subunit-primed cohort (the lowest NAb titers were 3.6 log 10 after the AS03-adjuvanted boost, and the highest NAb titer in human convalescent sera panel was 3.3 log 10).

Boosting with homologous and heterologous formulations of CoV2 preS dTM led to significant increase in neutralizing antibodies against the Beta variant, with a significant AS03-adjuvant effect

We next assessed NAb titers against the Beta variant using the lentivirus-based neutralization assay. After the primary immunization, Beta NAb titers were low to undetectable in the mRNA-primed cohort (mean titer of 1.4 log 10) and low in the subunit-primed cohort (mean titer of 1.6 log 10) at D35 (Fig. 1c). Seven months post-prime, Beta NAb titers were undetectable in the mRNA-primed cohort (<limit of quantification) and low to undetectable in the subunit-primed cohort (mean titer of 1.3 log 10).

A booster dose with the non-adjuvanted CoV2 preS dTM (B.1.351) vaccine induced Beta NAb titers within 7 days after injection, with a mean titer of 2.6 log 10 in the mRNA-primed and 2.9 log 10 in the subunit-primed cohorts 14 days post-injection that were stable until D28, and then slightly decreased up to 3 months post-booster (mean titers of 2.3 log 10 in both cohorts at D84).

The increase in Beta NAb titers from baseline was higher for the AS03-adjuvanted CoV2 preS dTM (B.1.351) vaccine candidate compared with non-adjuvanted CoV2 preS dTM (B.1.351) in the mRNA-primed cohort (8.5-fold, p-value = 0.057) and significantly higher in the subunit-primed cohort (11.8-fold, p-value < 0.001). No statistical differences were observed between the various AS03-adjuvanted vaccine formulations with mean Beta NAb titers of 3.3 log 10 in the mRNA-primed animals (mean fold-increase of 180 from baseline) and 3.7 log 10 in the subunit-primed animals (mean fold-increase of 280 from baseline) 14 days post-booster. In the two cohorts, after the AS03-adjuvanted booster immunization, the mean Beta NAb titers observed up to D28 were significantly higher than the peak titers post-primary vaccination on D35 whatever the vaccine formulations ( p-values < 0.001, mean fold-increase of 46 in the mRNA-primed and 140 in the subunit-primed cohort). Over the 3-month period post-booster, the Beta NAb responses declined slightly in the AS03-adjuvanted groups, with mean titers of 2.5 log 10 and 3.0 log 10 in the mRNA- and subunit-primed cohorts, respectively, on D84.

Boosting with CoV2 preS dTM led to significant increase in cross-neutralizing antibodies against Alpha, Gamma, Delta, Omicron and SARS-CoV-1, with a significant AS03-adjuvant effect

The breadth of neutralization was further assessed against the other known SARS-CoV-2 VOC, Alpha, Gamma, Delta, Omicron, and SARS-CoV-1. NAb cross-reactivity against the VOC was observed 14 days post-injection with all vaccine formulations in both cohorts (Fig. 2, Supplementary Figs. 2 and 3).

Pseudovirus-neutralizing antibody assays against the parental SARS-CoV-2, variants of concern (Alpha, Beta, Gamma, Delta, and Omicron), and SARS-CoV-1 was assessed at day 14 after the booster injection in primed-macaques a mRNA-primed cohort ( n = 4/group) and b protein subunit-primed cohort ( n  = 4 or 5/group). Individual macaque data are shown and are linked by connecting lines. Horizontal dotted lines indicate the limits of quantification of the assay.

The nonadjuvanted CoV2 preS dTM (B.1.351) vaccine booster induced, in the mRNA-primed cohort, mean NAb titers of 3.1 log 10 (Alpha), 2.9 log 10 (Gamma), 2.8 log 10 (Delta), and 1.9 log 10 (Omicron) (Fig. 2a) and, in subunit-primed cohort, mean VOC NAb titers of 3.5 log 10 (Alpha), 3.0 log 10 (Gamma) and 3.2 log 10 (Delta), and 2.5 log 10 (Omicron) (Fig. 2b).

Overall, the various AS03-adjuvanted vaccine boosters induced mean VOC NAb titers of 4.0 log 10 (Alpha), 3.6 log 10 (Gamma), 3.7 log 10 (Delta), and 2.4 log 10 (Omicron) (Fig. 2a) in the mRNA-primed cohort and mean VOC NAb titers of 4.4 log 10 (Alpha), 4.0 log 10 (Gamma) and 4.1 log 10 (Delta), and 3.4 log 10 (Omicron) (Fig. 2b) in the subunit-primed cohort. As observed with the parental strain and the Beta variant, a significant adjuvant effect was observed with the CoV2 preS dTM B.1.351 monovalent vaccine booster in both cohorts ( p-value < 0.05) for Alpha, Gamma, Delta, and Omicron NAb titers (mean fold-increases in the mRNA-primed cohort: 7.4, 11.9, 7.1, and 13.8 respectively, and in the subunit-primed cohort: 5.7, 8.6, 6.6, and 12.6, respectively).

Unexpectedly, we also observed a significant enhanced neutralizing breadth against SARS-CoV-1 14 days post-injection with all booster-vaccine formulations ( p-value < 0.001). The nonadjuvanted vaccine booster induced significant SARS-CoV-1 neutralizing titers in all macaques except one (mean NAb titers of 1.9 log 10 and 2.3 log 10 in the mRNA-primed and subunit-primed cohorts, respectively, p-value < 0.001). Overall, AS03-adjuvanted vaccine formulations induced significant SARS-CoV-1 NAb in all macaques, with mean titers of 2.7 log 10 and 3.3 log 10 in the mRNA-primed cohort and subunit-primed cohort, respectively ( p-value < 0.001; Fig. 2 and Supplementary Fig. 4). The AS03 adjuvant effect was confirmed on SARS-CoV-1 NAb-titer increase from baseline when comparing the AS03-adjuvanted and the non-adjuvanted B.1.351 monovalent vaccine candidates (9.1-fold and 7-fold higher booster effect for mRNA-primed and subunit-primed cohort, respectively, p-value < 0.01).

Booster immunization with CoV2 preS dTM led to higher functional to binding antibody ratio in both mRNA- and subunit-primed macaques

To better assess the impact of booster on the quality of antibody responses, we measured spike- and receptor-binding domain (RBD)-binding antibodies in sera collected at D35 post-prime and 14 days post-booster injection (Supplementary Fig. 5). We then calculated the individual ratio between neutralizing and RBD-binding antibodies for the parental strain and Beta variant (Fig. 3). The ratios increased in all groups, except for the Beta ratio in one group (CoV2 PreS dTM D614-AS03 monovalent group in the mRNA-primed cohort). The mean fold-increases for parental and Beta ratios reached 4.4- and 23-fold in the mRNA-primed cohort and reached 10- and 29-fold in the subunit-primed cohort, respectively. The consistent increase indicates that the booster dose increases functional antibody responses in both mRNA- and subunit-primed groups through induction of a higher proportion of neutralizing antibodies among RBD-specific antibodies.

Pseudovirus NAb/RBD-binding IgG ratio for parental D614G ( a) and for Beta ( b), post prime (D35) and 2 weeks post-boost (D224 or D216) for mRNA-primed cohort ( n = 4/group) and subunit-primed cohort ( n  = 4-5/group). Individual animals are shown; bars indicate means.

Discussion

We previously reported that our protein-based CoV2 preS dTM-AS03 subunit vaccine provided robust protection against challenge with SARS-CoV-2 in rhesus macaques and hamsters and provided robust immunity in humans. In this study, we show that a booster with various formulations of CoV2 preS dTM in macaques previously vaccinated with Sanofi's mRNA (D614) or CoV2 preS dTM-AS03 (D614) subunit-vaccine candidates induced a robust increase in neutralizing antibody titers against the parental strain, as early as 7 days post-booster, and achieved significantly higher NAb titers than the D35 vaccination peak with AS03 adjuvant. Importantly, booster injection with CoV2 preS dTM formulations, induced cross-neutralization covering the five SARS-CoV-2 VOC, Alpha, Beta, Gamma, Delta, and Omicron, and broadened the NAb response to SARS-CoV-1. The VOC display from 10 (Alpha) to more than 30 (Omicron) mutations in the spike protein (Table 1), with some key mutations occurring in the RBD for VOC displaying the highest resistance to prior immunity (Beta, Gamma, and Omicron). In contrast, the SARS-CoV-1 spike protein displays about 350 amino acid differences compared with SARS-CoV-2 but still, was neutralized at similar levels as Omicron after the booster. The increase in neutralizing antibodies lasted over the 3-month follow-up period post-booster and was more pronounced on the neutralizing antibodies than on RBD-binding antibodies.

Table 1 Description of the reporter virus particles (RVPs) used in the pseudovirus neutralization assay.

To our knowledge, this is the first report of SARS-CoV-2 booster-subunit vaccine formulations demonstrating broad cross-neutralizing antibodies covering the five VOC (Alpha, Beta, Gamma, Delta, and Omicron) in macaques primed with different vaccine platforms.

Furthermore, in our studies, we observed an extended breadth of neutralization to SARS-CoV-1, which was responsible for the more severe respiratory disease during the 2002-2003 outbreak22. Notably, the neutralizing titers induced against SARS-CoV-1 were similar to the titers against Omicron, although the SARS-CoV-1 spike bares only 72% homology with the SARS-CoV-2 spike. This confirms that the cross-neutralization is not a linear function of the number of mutations and suggests that Omicron might display the greatest antigenic distance from the parental D614 within SARS-CoV-2.

Although there was a trend for a more balanced neutralization profile with the monovalent B.1.351 or bivalent vaccine formulations, it is noteworthy that all booster-vaccine formulations tested induced NAb titers with similar breadth, suggesting that the booster vaccine mainly targets cross-reactive B cells as it was shown for mRNA-1273 booster vaccine in NHPs23.

We observed a significant adjuvant effect of AS03, which increased the titers of neutralizing antibodies against the parental strain, all VOC and SARS-CoV-1 when compared with nonadjuvanted formulation (6- to 8-fold on parental NAb titers, and 8- to 11.8-fold on Beta NAb titers). This is in line with previous findings in the context of pandemic influenza vaccination, where the AS03 adjuvant effect consistently resulted in increased magnitude and breadth of the response (including heterotypic antibody responses) in primed individuals24,25,26.

Interestingly, the non-adjuvanted B.1.351 monovalent formulation was also capable of boosting and broadening the neutralizing responses, suggesting that the AS03 adjuvant might not be as necessary for a booster compared with primary vaccination, where it was required to induce functional antibody responses. For comparison, the NAb titers induced by the non-adjuvanted monovalent B.1.351 vaccine were in the same range as those induced in NHPs with a third dose of mRNA-1273 vaccine for Beta, Gamma, and Delta variants measured using similar neutralization assays27.

The greater impact of the booster on the neutralization of VOC and SARS-CoV-1 compared with the neutralization of the parental strain suggests that the booster vaccine can remodel the antibody specificity in addition to increasing the antibody levels. Improvement of the antibody quality was also observed when calculating the ratios between functional (neutralizing) and RBD-binding antibodies (parental and Beta), which increased after the booster immunization. Broader neutralization, coupled to the higher proportion of neutralizing antibody after the booster, is probably due to activation of preexisting memory B cells in primed animals and further affinity maturation, possibly identifying a key role for memory B cells in mounting recall responses to SARS-CoV-2 antigens and generating cross-reactive NAbs28.

The stability of the NAb titers observed over 3 months after the booster immunization holds promise for an improved durability of the circulating antibodies.

As our macaques were not challenged, our study does not define mechanistic correlates of protection against SARS-CoV-2 variants, but various studies previously reported that NAb levels are highly predictive of immune protection from symptomatic SARS-CoV-2 infection29,30,31. Moreover, a recent report with mRNA vaccine established the critical role of NAbs as a correlate of protection against infection in NHPs32. Based on these studies, and on the levels of NAb titers observed here after the booster against the VOC and SARS-CoV-1, one can hypothesize that a booster with CoV2 preS dTM vaccine will augment and extend the protection against existing and newly emerging SARS-CoV-2 variants. In particular, AS03-adjuvanted CoV2 preS dTM booster immunization resulted in higher neutralizing titers than those observed in NHPs with a third dose of mRNA-1273 vaccine for the Beta, Gamma, and Delta variants27, hinting to a beneficial effect of an adjuvanted subunit booster strategy for protection, especially against distant variants such as Omicron, and, potentially, durability of the response.

These data have important implications for the utility of current vaccines and inform boosting strategies to address the risks associated with SARS-CoV-2 VOC and waning immunity. Based on these encouraging results, non-adjuvanted and AS03-adjuvanted vaccine formulations, using a dose range of antigen and adjuvant, are being evaluated as booster vaccines in a clinical trial, and the preliminary results confirm our observations in primed NHPs33.

The study has several limitations. First, the study included a small number of animals, specifically 4-5 macaques per group. Second, as adenovirus-vector vaccine-primed macaques were not available, the booster was evaluated in mRNA- and subunit-primed macaques only. However, the adenovirus priming platform is included in the clinical trial (NCT04762680). Third, the interval between the primary vaccination and the booster was set at 7 months, evaluating shorter and longer intervals from 4 to 10 months is part of the clinical trial. Finally, the mechanism underlying the cross-neutralization conferred by the booster, i.e., broadening of the repertoire of antibodies or selection of preexisting cross-reactive specificities, would need to be further defined. While the antibody repertoire is not easily accessible due to limitation of the immunological tools in macaques, analysis of the Ab quality using system serology, epitope mapping, and memory B-cell population at later timepoints after the booster immunization is ongoing to better characterize the mechanism for cross-protection.

In conclusion, a single dose of the CoV2 preS dTM recombinant vaccine (non-adjuvanted monovalent B.1.351, AS03-adjuvanted monovalent D614 or B.1.351, or bivalent D614 + B.1.351) used as a booster in macaques previously vaccinated with mRNA or subunit COVID-19 vaccines induced high and persistent cross-reactive neutralizing antibodies against the currently known SARS-CoV-2 variants of concern (Alpha, Beta, Gamma, Delta, and Omicron) and the two-decade-old SARS-CoV-1, as early as 7 days post-boost injection. Our findings show that CoV2 preS dTM vaccine candidates when used as a booster have the potential to offer cross-protection against a broad spectrum of variants and have important implications for vaccine control of SARS-CoV-2 VOC. Preliminary results in humans with monovalent CoV2 preS dTM-AS03 (D614) vaccine booster in subjects previously primed with adenovirus-vector-based and mRNA-based COVID-19 vaccines33 confirm the value of the primed-NHP model used here.

Methods

Vaccines

For the primary immunization, the mRNA vaccines were SARS-CoV-2 prefusion spike constructs 2 P, GSAS, 2 P/GSAS, 2 P/GSAS/ALAYT and 6 P/GSAS described in Kalnin et al.34, the subunit vaccines were AS03-adjuvanted CoV2 preS dTM vaccines, where the antigens were produced using the phase-I/-II manufacturing process, 1.3- and 2.6-µg doses, or using an intermediate manufacturing process, 2.4 µg dose.

For the booster, the CoV2 preS dTM derived from the parental D614 strain and the B.1.351 variant was produced using an optimized purification process to ensure a minimum of 90% purity.

The antigens were formulated in monovalent or bivalent formulations with AS03 adjuvant, the monovalent CoV2 preS dTM (B.1.351) was also formulated without AS03. The CoV2 preS dTM was produced from a Sanofi proprietary cell culture technology based on the insect cell-baculovirus system, referred to as the Baculovirus Expression Vector System (BEVS). The CoV2 preS dTM (D614) sequence was designed based on the Wuhan YP_009724390.1 strain S sequence, modified with 2 prolines in the S2 region, deletion of the transmembrane region, and addition of the T4 foldon trimerization domain. The CoV2 preS dTM (B.1.351) was designed based on the B.1.351 sequence (GISAID Accession EPI_ISL_1048524) and contains the same modifications.

AS03 is a proprietary adjuvant system composed of α-tocopherol, squalene, and polysorbate 80 in an oil-in-water emulsion manufactured by GSK. Vaccine doses were formulated by diluting the appropriate dose of preS dTM with PBS-tween to 250 µL, then mixing with 250 µL of AS03, followed by inversion five times for a final volume of 500 µL. Each dose of AS03 contains 11.86 mg of α-tocopherol, 10.69 mg of squalene, and 4.86 mg of polysorbate-80 (Tween 80) in PBS.

Animals and study design

Animal experiments were carried out in compliance with all pertinent US National Institutes of Health regulations and were conducted with approved animal protocols from the Institutional Animal Care and Use Committee (IACUC) at the research facilities. NHP studies were conducted at the University of Louisiana at Lafayette New Iberia Research Center.

Two cohorts of vaccinated NHPs received a booster immunization after randomizing each group within a cohort based on their baseline characteristics (Fig. 1a).

In the mRNA-primed cohort, 16 adult male and female Mauritius cynomolgus macaques ( Macaca fascicularis) aged 4-10 years, selected based on their responses to the primary vaccination, were randomly allocated to 4 groups of 4 animals according to their baseline characteristics.

In the subunit-primed cohort, 24 adult-male Indian rhesus macaques ( Macaca mulatta) aged 4-7 years were randomly allocated to 5 groups of 4 or 5 animals. In the priming phase, animals received two immunizations of either Sanofi's mRNA COVID (D614) experimental candidate vaccines or CoV2 preS dTM-AS03 (D614) vaccine through the intramuscular route in the deltoid at day 0 and day 21. Seven months after the primary immunization, both cohorts were immunized with CoV2 preS dTM (B.1.351) without adjuvant, CoV2 preS dTM (D614)-AS03, CoV2 preS dTM (B.1.351)-AS03, and a bivalent CoV2 preS dTM (D614 + B.1.351)-AS03. All groups received a total dose of 5 µg of CoV2 preS dTM antigen. An additional group in subunit-primed cohort received a bivalent CoV2 preS dTM (D614 + B.1.351)-AS03 high-antigen dose (5 + 5 µg). All immunologic analyses were performed blinded on serum collected at 7, 14, 21, 28, 56, and 84 days post-boost injection. Animal studies were conducted in compliance with all relevant local, state, and federal regulations, and were approved by the New Iberia Research Center.

Convalescent human sera

Convalescent serum panel ( N  = 93) was obtained from commercial vendors (Sanguine Biobank, iSpecimen, and PPD). The serum samples were collected within 3 months following PCR-positive diagnosis of COVID-19.

The WHO International Standard for anti-SARS-CoV-2 immunoglobulin (human) (NIBSC code: 20/136) was also used.

Pseudovirus-based virus neutralization assays

Serum samples were diluted 1:4 or 1:20 in media (FluoroBrite™ phenol-red-free DMEM + 10% FBS + 10 mM HEPES + 1% PS + 1% GlutaMAX™) and heat-inactivated at 56 °C for 30 min. Further, 2-fold, 11-point, dilution series of the heat-inactivated serum were performed in media. Diluted serum samples were mixed with reporter-virus particle (RVP)-GFP (Integral Molecular) listed in Table 1 diluted to contain ~300 infectious particles per well and incubated for 1 h at 37 °C. 96-well plates of ~50% confluent 293T-hsACE2 clonal cells (Integral Molecular, Cat# C-HA102) in 75 µL volume were inoculated with 50 µL of the serum + virus mixtures and incubated at 37 °C for 72 h. At the end of the 72-hour incubation, plates were scanned on a high-content imager and individual GFP-expressing cells were counted. The neutralizing antibody titer was reported as the reciprocal of the dilution that reduced the number of virus plaques in the test by 50%.

Enzyme linked immunosorbent assay (ELISA)

Nunc microwell plates were coated with SARS-CoV S-GCN4 protein (GeneArt, expressed in Expi 293 cell line) at 0.5 µg/mL or SARS-CoV2 variant RBD protein purchased from SinoBiological (D614: 40592-v08h; Beta: 40592-v08h85; Delta: 40592-v08h90; SARS-CoV-1: 10583CV100) at 1 µg/mL in PBS at 4 °C for overnight. Plates were washed 3 times with PBS-Tween 0.1% before blocking with 1% BSA in PBS-Tween 0.1% for 1 h. Samples were heat-inactivated at 56 °C for 30 min and plated at a 1:450 initial dilution followed by 3-fold, 7-point serial dilutions in blocking buffer. Plates were washed 3 times after a 1-hour incubation at room temperature before adding 50 µL of 1:8000 Goat anti-human IgG (Jackson Immuno Research, CAT# 109-036-098) to each well. Plates were incubated at room temperature for 1 h and washed thrice. Plates were developed using Pierce 1-Step Ultra TMB-ELISA Substrate Solution for 6 min and stopped by TMB STOP solution. Plates were read at 450 nm in SpectraMax ® plate reader, and the data analyzed using Softmax ® Pro 6.5.1 GxP software and the proprietary software, Sanofi Universal Exporter 2.1. Antibody titers were reported as the highest dilution that is equal to 0.2-OD cutoff.

Statistical analyses

For both, mRNA-primed and CoV2 preS dTM-AS03-primed cohorts, at the time of the assignment, the characteristics at baseline (sex, age, and weight) were balanced to have comparable groups. The pseudovirus-neutralizing titers were also taken into account as well as the previous vaccine groups. ELISA titers and neutralizing titers were log 10-transformed prior to statistical analysis. All statistical tests were two-sided, and the nominal level of statistical significance was set to α = 5%. The analyses were performed on SEG SAS v9.4 ®.

To use the ANOVA models, the normality of the residual of the model was checked using Normal Probability Plot, and the results were considered acceptable. Although the number of animals is small, we assumed that the readouts analyzed (IgG and pseudovirus-neutralizing titers) follow a log-normal distribution based on past experience. The biostatisticians concluded that the use of ANOVA test was thus acceptable. Since nonparametric tests allow to conclude only on the median or on the rank, one-way ANOVA test was preferred to estimate and compare the mean fold-changes from baseline between the different groups.

Statistical comparisons were performed among different groups and conducted as follows for each readout and each strain separately: one way ANOVA with the group as factor was performed to analyze the IgG ratio, PsV ratio post-booster, and post prime. Mixed-effect models for repeated measures, including group, day, and their interactions, where day was specified as repeated measure, were used for PsV neut/IgG ratio and to evaluate the boost effect.

Using the data pooled from both cohorts (i.e., primed with mRNA or protein subunit), a concordance analysis was performed between the D614G and D614 PsV-neutralizing titers using a regression. The concordance was considered verified if the 90% confidence interval of the slope is included in [0.80; 1.25].

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

Accession codes for the publicly available datasets are provided in the paper. The source data generated in this study are provided as Supplementary Material. Source data are provided with this paper.

Acknowledgements

The authors thank Jon Smith for coordinating the production and providing the vaccine antigens for the study, Caroline Patriarca Ruat for project management, Tong-Ming Fu for coordinating the sharing of mRNA-primed macaques, and Carlos Diaz-Granados, Stephen Savarino, and Saranya Sridhar for critical discussions on the study designs and data analysis. The authors thank Julie Piolat for statistic analyses support. The authors also thank Isabel Grégoire, Anirban Sanyal, Saili Dharadhar, and Atharva Moghe for providing editorial assistance and paper coordination. This work was done in collaboration with GlaxoSmithKline, who provided access to, and use of, the AS03 adjuvant system.

Author information

Author notes

These authors contributed equally: Vincent Pavot, Catherine Berry.

Affiliations

Sanofi, Marcy l'Etoile, France

Vincent Pavot, Catherine Berry, Alice Raillard, Sylviane Gautheron & Valerie Lecouturier

Sanofi, Cambridge, MA, USA

Michael Kishko, Natalie G. Anosova, Dean Huang, Tim Tibbitts & Roman M. Chicz

GSK, Rixensart, Belgium

Cindy Gutzeit

GSK, Wavre, Belgium

Marguerite Koutsoukos

Contributions

V.L., C.B., V.P., T.T., N.A., A.R., S.G., R.C., C.G., and M.K. contributed to the concept or design of the study. M.K., D.H. and N.A. collected and analyzed data. T.T. provided study coordination. C.G. and M.K. contributed critical reagents. N.A., C.B., V.P., V.L., T.T., A.R., S.G., R.C., C.G., and M.K. were involved in the analysis and interpretation of the data. V.L., R.C. provided supervision. V.P. and C.B. drafted the first paper and all authors critically reviewed paper versions. All authors had full access to the data and approved the paper before it was submitted by the corresponding authors.

Funding

This study was funded by Sanofi and by federal funds from the Biomedical Advanced Research and Development Authority, part of the office of the Assistant Secretary for Preparedness and Response at the US Department of Health and Human Services in collaboration with the US Department of Defense Joint Program Executive Office for Chemical, Biological, Radiological, and Nuclear Defense under contract number W15QKN-16-9-1002. The views presented here are those of the authors and do not purport to represent those of the Department of the Army.

Corresponding author

Correspondence to Valerie Lecouturier.

Ethics declarations

Competing interests

All authors have declared the following interests: C.B., V.P., M.K., D.H., N.A., T.T., A.R., S.G., V.L., and R.C. are Sanofi employees and may own Sanofi shares. M.K. and C.G. are employees of the GSK group of companies and report ownership of GSK shares.

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Nature Communications thanks Nanda Kishore Routhu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Pavot, V., Berry, C., Kishko, M. et al. Protein-based SARS-CoV-2 spike vaccine booster increases cross-neutralization against SARS-CoV-2 variants of concern in non-human primates. Nat Commun 13, 1699 (2022). https://doi.org/10.1038/s41467-022-29219-2
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Protein-based SARS-CoV-2 spike vaccine booster increases cross-neutralization against SARS-CoV-2 variants of concern in non-human primates - Nature Communications

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The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that partly evade neutralizing antibodies raises concerns of reduced vaccine effectiveness and increased infection. We previously demonstrated that the SARS-CoV-2 spike protein vaccine adjuvanted with AS03 (CoV2 preS dTM-AS03) elicits robust neutralizing antibody responses in naïve subjects.

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SARS-CoV-2 viruses and cells used for viral replication

The 2019n-CoV/USA_WA1/2020 isolate of SARS-CoV-2 (NR-52281) and the SARS-CoV-2 isolate hCoV-19/Japan/TY7-503/2021 (Brazil P.1) (NR-54982) were obtained from BEI Resources, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH). Vero E6 (CRL-1586; ATCC) were cultured at 37 °C in DMEM supplemented with 10% FBS, 1 mM sodium pyruvate, 1× nonessential amino acids and 100 U ml −1 of penicillin-streptomycin. SARS-CoV-2 was propagated in Vero E6 cells. The supernatant was collected on observation of cytopathic effect. Debris was removed by centrifugation and passage through a 0.22-μm filter. The supernatant was then aliquoted and stored at −80 °C. Virus titers were quantitated by a plaque-forming assay using Vero E6 cells.

Inoculation of SARS-CoV-2 in K18-hACE2 transgenic mice and administration of sACE22.v2.4

Biosafety level 3 (BSL-3) protocols for animal experiments with live SARS-CoV-2 were performed by personnel equipped with powered air-purifying respirators in strict compliance with NIH guidelines for humane treatment and approved by the University of Illinois Institutional Animal Care & Use Committee (University of Illinois at Chicago protocol no. 18-076). Hemizygous K18-hACE2 mice with a C57BL/6J background (strain no. 034860: B6.Cg-Tg(K18-ACE2)2Prlmn/J) were purchased from The Jackson Laboratory. Animals were housed in groups and fed standard chow. Mice (8-10 weeks old) were anesthetized with ketamine/xylazine (50/5 mg kg −1, intraperitoneal). Mice were then inoculated intranasally with 1 × 10 5 PFU of SARS-CoV-2 isolate WA or 1 × 10 4 PFU of SARS-CoV-2 isolate WA or P.1 variant suspended in 20 μl of sterile PBS. sACE2 2.v2.4 was administered intravenously (retro-orbital injection) either 12 h before virus inoculation (single dose) or 12/24 h post virus inoculation (once a day for 7 d) to anesthetized K18-hACE2 transgenic mice. Genotyping of mice was performed by PCR using tail DNA. We conducted an a priori power analysis to calculate the number of animals needed to achieve statistically significant results. We calculated power and sample sizes according to data from pilot experiments; variations within each group of data and variance similarities between groups were compared statistically. Animals with sex- and age-matched littermates were included randomly in the experiments. No animals were excluded due to illness after the experiments. Animal experiments were carried out in a blinded fashion whenever feasible.

Plaque-forming assay

Vero E6 cells were seeded at a density of 2.5 × 10 5 cells per well in flat-bottom, 12-well tissue culture plates. The following day, the medium was removed and replaced with 200 μl of tenfold serial dilutions of the material to be titered, diluted in DMEM + 2% FBS. At 1 h, 1 ml of 0.8% agarose overlay was added. Plates were incubated for 72 h, then fixed with 4% paraformaldehyde (PFA, final concentration) in PBS for 30 min. Plates were stained with 0.05% (w/v) Crystal Violet in 20% methanol and washed twice with distilled, deionized water.

Generation of pseudoviruses

Recombinant vesiculovirus (rVSV) Indiana serotype expressing the SARS-CoV-2 S protein was generated as described previously. HEK 293T cells were grown to 80% confluency before transfection with pCMV3-SARS-CoV-2-spike (Sino Biological) using Lipofectamine 3000 (Invitrogen). Cells were cultured overnight at 37 °C with 5% CO 2. The next day, the medium was removed and VSV-G pseudotyped ΔG-luciferase (a gift from D. D. Ho, Columbia University) was used to infect the cells in DMEM at an MOI of 3 for 2 h. After infection, cells were washed with PBS twice and new complete DMEM culture medium was added. DMEM supplemented with 2% FBS, 100 IU ml −1 penicillin and 100 μg ml −1 streptomycin. The supernatant was collected and clarified by centrifugation at 800 g for 10 min and filtered by 0.45-µm pore size (Merck Millipore) before aliquoting and storing at −80 °C. Before infection of target cells, we incubated viral stock with 20% I1 hybridoma (anti-VSV-G) supernatant (CRL-2700; ATCC) for 1 h at 37 °C to neutralize contaminating G*ΔG rVSV-luciferase particles.

Virus entry determined by luciferase assay

For the in vitro studies, cultured cells were preincubated with 5 and 25 µg ml −1 soluble sACE2 2-IgG1 (WT) and the sACE2 2.v2.4-IgG1 variant for 1 h. SARS-CoV-2 pseudovirus (MOI = 0.1) was then added to the cultured cells. Cells were collected for the luciferase assay after 24 h. For the in vivo studies, 10 mg kg −1 sACE2 2.v2.4-IgG1 (WT), sACE2 2.v2.4-IgG1 and control buffer were administrated intravenously into K18-hACE2 mice for 30 min before SARS-CoV-2 pseudo-entry virus (10 6 PFU) intraperitoneal injection for 1 d postinoculation. Tissue lysates were prepared and virus entry in the selected organs was evaluated by luciferase activity using the luciferase assay system (Promega Corporation) and a microplate luminometer. Data are shown as the mean ± s.e.m. of n  = 4 replicates.

mRNA expression by RT-PCR and real-time qPCR

RNA was extracted from homogenized lung tissues using TRIzol Reagent (catalog no. 15596026; Thermo Fisher Scientific) according to the manufacturer's protocol. RNA was quantified by NanoDrop 1000 (Thermo Fisher Scientific). One-step PCR with reverse transcription (RT-PCR) amplification was performed using the SuperScript One-Step RT-PCR system with Platinum Taq DNA polymerase (catalog no. 10966026; Invitrogen). One microgram total RNA isolated from human cells was used as a template for subsequent one-step RT-PCR. (The primer information is included in Supplementary Table 6.) The one-step RT-PCR products were analyzed by electrophoresis on 1.2% agarose gels containing ethidium bromide. For real-time qPCR, RNA was reversely transcribed into complementary DNA using the High-Capacity cDNA Reverse Transcription Kit (catalog no. 4368814; Thermo Fisher Scientific). FastStart Universal SYBR Green Master (catalog no. 4913850001; Sigma-Aldrich) was used for the relative quantification of cDNA on the ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). (The primer information is included in Supplementary Table 6.)

Measurement of viral burden

Tissues were weighed and homogenized in 1 ml TRIzol solution. Tissue homogenates were clarified by centrifugation at 10,000 r.p.m. for 5 min and stored at −80 °C. RNA was extracted according to the TRIzol protocol. RNA was reverse-transcribed with Superscript III Reverse Transcriptase (Invitrogen) using random primers. Synthesized cDNA samples were amplified in the ABI PRISM 7000 Sequence Detection System (Applied Biosystems) thermocycler with SYBR Green using primers designed to target a highly conserved region of the S and NSP genes. (The primer information is included in Supplementary Table 6.)

Reagents

Polyclonal anti-SARS-CoV-2 S glycoprotein was obtained from BEI Resources. The luciferase assay system (E1500) was purchased from the Promega Corporation. Enhanced chemiluminescence (ECL) western blotting detection reagents and nitrocellulose membranes (Hybond ECL) were obtained from Amersham Biosciences Corp. The Lipofectamine 3000 transfection reagents were sourced from Invitrogen. Antibodies for flow cytometry are described elsewhere in the Methods.

Lung vascular permeability measurement in mice

EBA pulmonary transvascular flux measurements were performed to measure lung vascular leakage. Briefly, 200 μl EBA (1% Evans blue dye (EBD) and 4% albumin in PBS) was administered intravenously (retro-orbital injection) into anesthetized mice and allowed to circulate for 30 min. Mice were euthanized and lungs were perfused by 10 ml PBS. Lung tissues were then excised, weighed, homogenized in 1 ml PBS and extracted overnight in 2 ml formamide at 60 °C. Samples were centrifuged at 10,000 g for 5 min. The Evans blue concentration in the supernatants of lung homogenates was quantified spectrophotometrically at an absorbance of 620 nm. Tissue EBD content (μg EBD g −1 fresh lung tissue) was calculated by comparing tissue supernatant OD 620 readings with EBD standard curve. The concentration of EBD was determined in micrograms per gram of wet lung tissue. The ratio of wet to dry lung weight for edema measurement was calculated.

Histology and imaging

Animals were euthanized before the collection and fixation of tissues. Lung lobes were fixed with 4% PFA for 48 h before further processing. Tissues were embedded in paraffin and sections were stained with hematoxylin and eosin, trichrome blue and Sirius Red for collagen deposition. Images were taken with a ZEISS microscope and analyzed with the Zen software version 2 (blue edition, ZEISS).

Mouse cell isolation

Mouse cells were isolated as described previously. Briefly, flushed mouse lung tissue was minced and digested with 3 ml Collagenase A (catalog no. 10103586001, Roche; 1 mg ml −1 in PBS) in a 37 °C water bath for 1 h. Mixtures were titrated with no. 18-G needles and then pipetted through a 40-μm disposable cell strainer. After centrifuging at 300 g for 5 min and washing with PBS, isolated cells were treated with red blood cell lysis buffer (catalog no. 00-4300-54; eBioscience) for 5 min on ice to lyse red blood cells.

Flow cytometry

After isolation, cells were incubated with anti-mouse CD16/CD32 (1:50 dilution, catalog no. 553142; BD Biosciences) to block endogenous Fc for 10 min on ice. Cells were stained with antibodies including CD45-EF450 (1:200 dilution, catalog no. 48-0451-82; eBioscience) and CD31-allophycocyanin (APC) (1:100 dilution, catalog no. 17-0311-82; eBioscience) for 45 min at 4 °C. After washing, cells were resuspended in 500 μl buffer and analyzed on an LSRFortessa (BD Biosciences) cell cytometer. Data were analyzed with the Kaluza software version 2.1 (Beckman Coulter). The gating information for the population of mouse lung vascular endothelial cells is shown in Supplementary Fig. 1e-g.

Cell culture

The 2019n-CoV/USA_WA1/2019 isolate and P.1 variant of SARS-CoV-2 and ACE2-expressing A549 cell lines were obtained from BEI Resources. Vero E6, Vero (catalog no. CCL81; ATCC), ACE2-expressing A549 cells and standard A549 cells were cultured at 37 °C in DMEM supplemented with 10% FBS, 10 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 1× nonessential amino acids and penicillin-streptomycin. Primary hLMVECs from Lonza were cultured in endothelial basal medium-2 supplemented with 10% endotoxin-free FBS (Omega Scientific). Infectious stocks were grown by inoculating Vero cells and collecting the supernatant on observation of cytopathic effect. Debris was removed by centrifugation and passing through a 0.22-μm filter. Supernatant was then aliquoted and stored at −80 °C.

Expi293F cells (Thermo Fisher Scientific) were cultured in Expi293 Expression Medium (Thermo Fisher Scientific) at 37 °C, 125 r.p.m. and 8% CO 2. Expi293F cells were transfected at a density of 2 × 10 6 per ml with 400 (pCEP4-myc-ACE2 plasmids) or 500 ng (pCEP4-myc-S and pcDNA3.1-SARS-spike plasmids) per ml of culture using ExpiFectamine 293 (Thermo Fisher Scientific) according to the manufacturer's instructions. For in vitro binding assays using flow cytometry, transfection enhancers were excluded and cells were collected 24-30 h post-transfection. To produce monomeric sACE2-8 h or RBD-8 h, ExpiFectamine 293 transfection enhancers (Thermo Fisher Scientific) were added approximately 20 h post-transfection and the medium was collected for protein purification 4-6 d later.

Dimeric sACE2 2-IgG1 proteins were expressed in ExpiCHO-S cells (Thermo Fisher Scientific) cultured in ExpiCHO Expression Medium (Thermo Fisher Scientific) at 37 °C, 125 r.p.m. and 8% CO 2. Cells were transfected with ExpiFectamine CHO (Thermo Fisher Scientific) according to the manufacturer's instructions, using 500-1,000 ng plasmid per ml of culture. ExpiFectamine CHO Enhancer (6 μl per ml of culture) and ExpiCHO Feed (240 μl per ml of culture) (Thermo Fisher Scientific) were added 18-22 h post-transfection and the temperature was decreased to 33 °C. At day 5 post-transfection, ExpiCHO Feed (240 μl per ml of culture) was again added. On days 9 and 11, CO 2 was stepped down to 7% and 6%, respectively. The medium was collected on days 12-14 for protein purification.

Plasmids

Residue numbers for proteins begin with amino acid 1 as the start methionine. Plasmids for the expression of myc-tagged human ACE2 (pCEP4-myc-ACE2, plasmid no. 141185; Addgene), 8 histidine-tagged monomeric sACE2 (ACE2 amino acids 1-615; pcDNA3-sACE2(WT)-8his, plasmid no. 149268 and pcDNA3-sACE2v2.4-8his, plasmid no. 149664; Addgene), SARS-CoV-1 S protein with C-terminal C9 tag (pcDNA3.1-SARS-spike, plasmid no. 145031; Addgene), 8his-tagged RBD (pcDNA3-SARS-CoV-2-S-RBD-8his, plasmid no. 145145; Addgene) and human IgG1-Fc fused dimeric sACE2 2 (ACE2 amino acids 1-732; pcDNA3-sACE2-WT(732)-IgG1, plasmid no. 154104 and pcDNA3-sACE2v2.4(732)-IgG1, plasmid no. 154106; Addgene) have been described previously. We used the nG1m1 isoallotype for the IgG1 Fc sequence, which is less immunogenic in humans since it shares an epitope at a polymorphic residue pair (E356/M358) common to other IgG subclasses. Human codon-optimized S (GenBank ID YP_009724390.1) was subcloned into pCEP4 (Invitrogen) from pUC57-2019-nCoV-S(Human); distributed by Molecular Cloud on behalf of H. Yu, Chinese Academy of Medical Sciences) with a N-terminal hemagglutinin leader (MKTIIALSYIFCLVFA), myc-tag (EQKLISEEDL) and linker (GSPGGA) upstream of the mature polypeptide (amino acids V16-T1273). All mutations were made by extension overlap PCR and confirmed by Sanger sequencing (ACGT). Compared to the SARS-CoV-2 S protein reference sequence (GenBank ID YP_009724390.1), the variant S and RBD proteins cloned in this study have the following mutations: P.1 (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I); B.1.351 (L18F, D80A, D215G, ΔL242-L244, K417N, E484K, N501Y, D614G, A701V); B.1.1.7 (ΔH69-V70, ΔY144, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H); B.1.429 (W152C, L452R, D614G); and B.1.617.2 (T19R, G142D, A222V, L452R, T478K, D614G, P681R).

Purification of sACE22-IgG1

The ExpiCHO-S expression medium was centrifuged (800 g, 4 °C, 10 min) to remove cells. Tris base (1 M) was added to the supernatant until the pH reached approximately 7.5. The medium was centrifuged again (15,000 g, 4 °C, 20 min). KanCapA resin (equilibrated in PBS; Kaneka Corporation) was incubated with the supernatant (2 ml resin per 100 ml medium) for 2 h while the sample was rotated at 4 °C. The resin was collected by passing the sample through an empty chromatography column and was washed with 10 column volumes of Dulbecco's PBS. Protein was eluted with 4 column volumes 60 mM sodium acetate (pH 3.7) into a vessel containing 2 column volumes 1 M Tris (pH 8.0), yielding a final pH of approximately 6. The pH was raised to 7 by the addition of 1 M Tris base (1-2 column volumes). The eluate was concentrated with a 50-kD molecular weight cutoff centrifugal filtration unit (Merck Millipore) and injected on a HiLoad Superdex 200 pg 16/600 gel filtration column (GE Healthcare) with PBS as the running buffer. Peak fractions at the expected molecular weight of a dimer were pooled, concentrated (>30 mg ml −1) and aliquots were frozen in liquid nitrogen and stored at −80 °C. Purity was routinely >98% based on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Concentrations were determined by absorbance at 280 nm using the calculated molar extinction coefficient for the mature, monomeric polypeptide. For the mouse studies, protein aliquots were thawed, diluted to 5 mg ml −1 with PBS and filter-sterilized (0.2 μm) ready for use.

Purification of sACE2-8 h and RBD-8 h

The Expi293F expression medium was centrifuged to remove cells (800 g, 4 °C, 10 min) and smaller particulates (15,000 g, 4 °C, 20 min). HisPur Ni-NTA Resin (Thermo Fisher Scientific) equilibrated in PBS was added (0.5 ml resin per 100 ml medium). The sample was rotated at 4 °C for 90 min. The resin was collected by passing through an empty chromatography column and then washed with >20 column volume PBS. Proteins were eluted using a step gradient of PBS containing 20 mM, 50 mM and 250 mM imidazole (pH 8.0, 12 column volumes for each fraction). Imidazole fractions containing pure protein based on Coomassie-stained SDS-PAGE were pooled and concentrated using 10 (RBD-8 h) or 30 kD (sACE2-8 h) molecular weight cutoff centrifugal filtration units (Merck Millipore). Proteins were separated on a Superdex 200 Increase 10/300 GL column (GE Healthcare) with PBS as the running buffer. Peak fractions at the expected molecular weight were pooled, concentrated (approximately 5 mg ml −1) and aliquots were snap-frozen and stored at −80 °C. Concentrations were based on ultraviolet (280 nm) absorbance using the molar extinction coefficient for the mature, monomeric polypeptides.

Toxicology

Animal experimental procedures were reviewed and approved by the University of Illinois Institutional Animal Care and Use Committee (University of Illinois at Chicago protocol no. 20127, 18-076/21-055). The toxicity of each protein was assessed in five female and five male CD-1 IGS mice (8 weeks old). For sACE2 2.v2.4 without any tags or fusion partner (corresponding to ACE2 residues 19-732; protein provided by Orthogonal Biologics), mice were administered (tail vein, 0.5 mg kg −1) protein intravenously twice daily for 5 consecutive days (days 0-4) and euthanized on day 7. Multiple doses were administered due to the anticipated short serum half-life. To assess the toxicity of sACE2 2.v2.4-IgG1, a single protein dose was administered intravenously (tail vein, 2.0 mg kg −1) and mice were euthanized on day 7. Toxicity was assessed by body weight, complete blood count, serum chemistry (Charles River Laboratories) and necropsy/histopathology. No differences were observed compared to vehicle-treated mice.

PK

Eight-week-old CD-1 IGS mice (three females and three males per time point) were administered protein solutions intravenously. Mice were euthanized and blood was drawn for serum analysis at the designated times. sACE2 2.v2.4 (without any tags or fusion partner) and sACE2 2.v2.4-IgG1 were injected via the tail vein at doses of 0.5 mg kg −1 and 2.0 mg kg −1, respectively. Catalytic activity in serum was measured using the Fluorometric ACE2 Activity Assay Kit (BioVision), sACE2 concentrations were measured by Human ACE-2 ELISA Kit (RayBiotech) and concentrations of the IgG1 Fc moiety were measured by Human IgG ELISA Kit (Immunology Consultants Laboratory). To characterize different routes side by side, proteins were administered to C57BL/6 mice, three males per time point. Animals weighed 25-35 g and were 8-10 weeks old. Administration was via tail vein injection (2.0 mg protein kg −1), intubation-mediated intratracheal instillation (1.0 mg protein kg −1) or inhalation, where protein solution was nebulized into the animal-holding chamber. Protein solutions were provided sterile-filtered in PBS at 0.4 mg ml −1 (intravenous), 1.0 mg ml (intratracheal) or 50 mg ml −1 (inhalation). For intratracheal delivery, mice were anesthetized with 2-3% isoflurane/oxygen and protein solution (50 µl of air (150 µl)) was delivered with a blunt 20-G catheter. At each time point, whole blood was collected by terminal bleeds from the orbital sinus into K2EDTA microtainers for plasma preparation. Lung tissue (50-100 mg) was extracted with T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific) at 1/20 (weight/volume of reagent) with protease inhibitor. Lung tissue samples were homogenized using the FastPrep-24 5G Instrument, centrifuged (10,000 g, 5 min) and the supernatants were analyzed. For immunoblot analysis of protein degradation, samples were prepared in reducing (for the ACE2 blots) or nonreducing (for the human IgG1 blots) SDS load dye and separated by PAGE, transferred to polyvinylidene fluoride membrane, blocked with 5% skimmed milk and stained with 1:2,000 horseradish peroxidase (HRP)-conjugated antihuman ACE2 (OTI1D2 clone; Origene) or 1:5,000 HRP-conjugated donkey antihuman IgG (Jackson ImmunoResearch) in Tris-buffered saline containing 0.1% Tween 20 and 1% skimmed milk. Blots were developed with the Clarity ECL Substrate (Bio-Rad Laboratories) and imaged on a ChemiDoc XRS+ system (Bio-Rad Laboratories).

In vitro binding assays to S variants

Expi293F cells were transfected with pCEP4-myc-S plasmids or with pcDNA3.1-SARS-spike as described above. Four volumes of culture were centrifuged (600 g, 4 °C, 120 s), the pellet was washed with 0.8 volumes of ice-cold PBS-BSA and then cells were resuspended in 1 volume of PBS-BSA. Serial dilutions of purified sACE2 or mAb proteins were prepared in round bottom 96-well trays using PBS-BSA as the diluent. Equal volumes of cells and protein solution were then mixed and incubated at 4 °C on a rotator for 30 min. Plates were centrifuged (600 g, 4 °C, 90 s) and the supernatant was removed. Cells were washed twice with ice-cold PBS-BSA, then resuspended in PBS-BSA containing 1:100 anti-His fluorescein isothiocyanate (FITC) (chicken polyclonal; Immunology Consultants Laboratory) plus 1:250 anti-myc Alexa 647 (9B11 clone; Cell Signaling Technology) to detect bound sACE2-8 h proteins or 1:250 antihuman IgG-APC (HP6017 clone; BioLegend) plus 1:150 anti-myc-FITC (chicken polyclonal; Immunology Consultants Laboratory) to detect bound sACE2 2-IgG1 or mAbs. Plates were incubated for another 30 min on a rotator at 4 °C, centrifuged, cell pellets were washed twice and finally cells were resuspended in PBS-BSA for analysis on a BD Accuri C6 flow cytometer. Data were analyzed using CFlow version 1.0.264.15. In all analyses, cells were gated by side scatter-forward scatter to remove debris. To assess protein binding, cells positive for the myc tag on S (excluding cells expressing S from SARS-CoV-1, which was untagged at the extracellular N terminus; in this case there was no further gating) were gated and the mean fluorescence signal for bound sACE2 within the myc-positive gate was measured (Supplementary Fig. 8j-l). To assess surface levels of S, the mean fluorescence signal for the myc tag was measured for the entire cell population. The background fluorescence of negative controls was subtracted to calculate the Δ mean fluorescence. All replicates were from independent, distinct samples and data were normalized based on the total measured fluorescence in each independent experiment.

MD simulations

For apo and RBD-bound complexes, protein structures were obtained from the Protein Data Bank ID 6M17 (ref. ). Extracellular ACE2 residues 21-730 were considered in the simulations. For the apo simulations, the RBD was removed from the system. Mutations were introduced using PyMOL 2.2.3 ( https://pymol.org/2/) and the systems were solvated using transferable intermolecular potential 3P water and 150 mM NaCl using PACKMOL 18.169. Protein residues and glycosylation sites were parameterized using AMBER ff14SB and GLYCAM06 (ref. ) force fields. The parameters for the Zn 2+ center of ACE2 were collected using the cationic dummy atom method.

The systems were first minimized and equilibrated before the production runs. Minimization was performed with the steepest descent and conjugate gradient algorithm for 15,000 steps. The minimized systems were heated in NVT ensemble with backbone atoms restrained with a restraining force ( k( x) 2) and a spring constant of 10 kcal/(mol-Å 2). The temperature was increased to 300 K in two steps (0-10 K, 10-300 K). Each step was performed for 2 ns. To increase the pressure of the system to 1 bar, the heated systems were simulated in NPT ensemble keeping the restraint on the backbone for another 2 ns. After this step, the restraints were removed to equilibrate the system at 300 K and 1 bar for 44 ns. To maintain the temperature of the systems, Langevin dynamics were used with 2 ps −1 collision frequency. A Berendsen barostat was used to maintain the pressure; a 2-fs time step was used for the simulation. To avoid instability of the bonds involving hydrogen atoms, the SHAKE algorithm was used. To consider nonbonded interactions, a 10-Å cutoff distance was selected. Long-range electrostatic interactions were considered using the particle mesh Ewald method. Minimization and equilibration were done using the AMBER18 engine.

After equilibration, production runs were performed with adaptive sampling to utilize parallel computing power. Adaptive sampling is a parallel iterative process to sample the protein conformational space by running many short-length simulations. In adaptive sampling, the protein conformational space is characterized by important features, such as distances, angles and root mean square deviation (RMSD), and the feature space is clustered into microstates. The starting points for the next round of simulation are selected from the least populated states. The first five rounds of adaptive sampling were performed using the AMBER engine at the Blue Waters supercomputing facility. The features selected for the adaptive sampling were backbone RMSD. Data collected over 5 rounds of adaptive sampling were clustered into 500 clusters, from which 500 structures were selected to submit to Folding@Home. Each structure was given 20 different starting velocities to begin 20 different trajectories. Simulations were run in the OpenMM engine. For each apo system, approximately 400 µs of data were collected; for each RBD-bound (original/Wuhan variant) complex, approximately 530 µs of data were gathered. For the Delta and Gamma RBD-bound systems, approximately 10 µs of data were collected for each system.

An MSM was built on MD data to obtain unbiased estimates of the canonical ensemble of interface movements. The MSM calculates the canonical ensemble distribution from the eigenvector space of the transition probability matrix. To estimate the transition probability matrix, we featurized the MD simulation data with interresidue distances between the human and viral protein interfaces. The featured space was linearly transformed into TICs. The TIC space was clustered into microstates using the k-means algorithm. The probability of a jump between one microstate ( i) to another ( j) with a particular lag time (\(\tau\)) corresponds to the T ij element of the transition probability matrix, T. Feature calculations were performed with MDTraj 1.9.3. The TIC calculation, clustering and MSM building steps were done using PyEMMA 2.5.6. The lag time for MSM was selected by logarithmic convergence of implied timescales. To build the optimal MSM, VAMP2 scores of the MSMs were calculated using different hyperparameters (TIC variance and numbers of clusters). Feature calculations and analysis of the trajectories were performed using MDTraj and CPPTRAJ 18.01. For trajectory visualization, the VMD 1.9.3 software was used.

Error analysis in the MD data was performed with bootstrapping. In each bootstrapped sample, 80% of the total trajectories were used to make MSMs with the same clustering levels. Twenty bootstrapped samples were calculated for each system with the same MSM hyperparameters.

Anti-SARS-CoV-2 mAbs

The sequences for mAbs that had received emergency use authorization from the U.S. Food and Drug Administration were pulled from the Kyoto Encyclopedia of Genes and Genomes (accession nos. REGN10933, D11938; REGN10987, D11939; VIR-7831, D12014; LY-CoV555, D11936). H and L chains were cloned with CD5 leader sequences into pcDNA3.1(+) and expressed in Expi293F cells. Proteins were purified using the KanCapA resin (Kaneka Corporation) and size exclusion chromatography with PBS as the running buffer. Peak fractions at the expected molecular weight of approximately 150 kD were concentrated, frozen in liquid nitrogen and stored at −80 °C. Concentrations were determined using absorbance at 280 nm and the calculated extinction coefficient for mature protein.

BLI

BLI kinetics were collected on an Octet RED96a and analyzed with a 1:1 binding model (global fit) using instrument software (Molecular Devices). mAbs and sACE2 2.v2.4 were immobilized at 100 nM for 60 s to antihuman IgG Fc capture biosensors (Molecular Devices). The assay buffer was 10 mM HEPES (pH 7.6), 150 mM NaCl, 3 mM EDTA, 0.05% polysorbate 20 and 0.5% nonfat dry milk (Bio-Rad Laboratories). Loaded sensors were equilibrated for 30 s in buffer, then dipped in RBD-8 h solution for 60 s to measure association and transferred back to the buffer to measure dissociation over 300 s.

Statistics and reproducibility

Quantification of replicate experiments is presented as the mean ± s.e.m. A Student's t-test, one-way analysis of variance (ANOVA) and two-way ANOVA with Tukey post hoc test were used to determine statistical significance, with a P value threshold <0.05. Significance levels are indicated in the figures as * P < 0.05, ** P < 0.01 and *** P < 0.001. Based on our experience, we expected changes in the gene/protein expression and function measurements to be detected with 4 mice per group, so the effect size was determined as n  = 4 independent mice. The variance between groups that were being statistically compared was similar.

Study approval

All aspects of this study were approved by the office of Environmental Health and Safety at the University of Illinois at Chicago before the start of this study. Working with SARS-CoV-2 was performed in a BSL-3 laboratory by personnel equipped with powered air-purifying respirators.

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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