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Chikungunya and Dengue virus are both prominent in South America. A recent outbreak has caused more talk about this virus.

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Dengue and Chikungunya Outbreaks: Elitza Theel, Ph.D.

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Antibodies from the Chikungunya virus are being found in humans. This study went through many experiments to look and find any type of immunity being formed.

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BMC Infectious Diseases 21, Article number: 1121 (2021) Cite this article

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Abstract

Background

More than 3 million infections were attributed to Chikungunya virus (CHIKV) in the 2014-2016 outbreak in Mexico, Central and South America, with over 500 deaths directly or indirectly related to this viral disease. CHIKV outbreaks are recurrent and no vaccine nor approved therapeutics exist to prevent or treat CHIKV infection. Reliable and robust diagnostic methods are thus critical to control future CHIKV outbreaks. Direct CHIKV detection in serum samples via highly specific and high affinity anti-CHIKV antibodies has shown to be an early and effective clinical diagnosis.

Methods

To isolate highly specific and high affinity anti-CHIKV, Chikungunya virions were isolated from serum of a patient in Veracruz, México. After purification and characterization via electron microscopy, SDS-PAGE and binding to well-characterized anti-CHIKV antibodies, UV-inactivated particles were utilized as selector in a solid-phase panning in combination with ALTHEA Gold Libraries™, as source of antibodies. The screening was based on ELISA and Next-Generation Sequencing.

Results

The CHIKV isolate showed the typical morphology of the virus. Protein bands in the SDS-PAGE were consistent with the size of CHIKV capsid proteins. UV-inactivated CHIKV particles bound tightly the control antibodies. The lead antibodies here obtained, on the other hand, showed high expression yield, > 95% monomeric content after a single-step Protein A purification, and importantly, had a thermal stability above 75 °C. Most of the antibodies recognized linear epitopes on E2, including the highest affinity antibody called C7. A sandwich ELISA implemented with C7 and a potent neutralizing antibody isolated elsewhere, also specific for E2 but recognizing a discontinuous epitope, showed a dynamic range of 0.2-40.0 mg/mL of UV-inactivated CHIKV purified preparation. The number of CHIKV particles estimated based on the concentration of E2 in the extract suggested that the assay could detect clinically meaningful amounts of CHIKV in serum.

Conclusions

The newly discovered antibodies offer valuable tools for characterization of CHIKV isolates. Therefore, the strategy here followed using whole viral particles and ALTHEA Gold Libraries ™ could expedite the discovery and development of antibodies for detection and control of emergent and quickly spreading viral outbreaks.

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Background

Chikungunya fever is a mosquito-borne viral disease caused by Chikungunya virus (CHIKV). Its name derives from a Makonde word translated as 'disease that bends up the joints', which describes the posture of afflicted individuals feeling severe joint pain or arthralgia [ 1]. Other symptoms include abrupt fever, muscle pain, headache, nausea, fatigue, and rash. These symptoms are often mild, and the infection may go unrecognized or not accurately diagnosed, in regions where other infections caused by Dengue virus (DENV) and Zika virus (ZIKV) occur.

CHIKV was first isolated in 1952 in Tanzania [ 1] and reemerged within a span of several years in South East Africa and Asia [ 2]. In December 2013, CHIKV reached the Americas [ 3], first reported on St. Martin in the Caribbean and within weeks in neighboring islands. The first CHIKV report in Mexico occurred in May 2014 from a patient in Jalisco, who showed CHIKV infection symptoms after a trip to the Caribbean region [ 4]. Other reports followed in October 2014 in Chiapas [ 5, 6]. By 2015, over eleven thousand cases occurred mostly in the southern region of the country [ 7] where warm and humid zones are amenable to mosquito growth. Overall, more than three million CHIKV infections have been reported in Mexico, Central and South America, with over 500 deaths directly or indirectly related to this viral disease [ 8].

While CHIKV infection is not currently considered a public health concern, with only three cases reported by mid-2019 ( www.gob.mx/salud/acciones-y-programas/chikungunya-informacion-relevante), chikungunya fever outbreaks are recurrent [ 2, 9]. Some of the factors contributing to such a recurrence are an inadequate disease surveillance and underdiagnosis, virus evolution and mutation, new generations of individuals with no immunity to the virus, supply chains globalization, travelling, and climate change. Since no approved vaccine nor antiviral CHIKV therapeutics exist, early detection and appropriate disease management are the only tools at the disposal of the scientists, health care community and governments to control future CHIKV outbreaks and reduce their devastating social and economic impacts.

Three CHIKV major lineages have been described thus far: West African (WA); East, Central, and South African (ECSA); and Asian [ 10, 11]. In addition, an Indian Ocean (IOL) sub lineage emerged within the ECSA and the Asian/American sub lineage emerged within the Asian lineages. The surface of CHIKV particles contains two main structural glycoproteins, E1 and E2, which have been considered targets in the development of diagnosis tools [ 12], therapeutic antibodies [ 13], and vaccines [ 14]. E2 protein shows the highest percentage of amino acid variation among the lineages and strains, impacting in some cases the interaction with the host receptor and hence modifying CHIKV clinical manifestations, virulence and even its epidemiology [ 15]. These changes could also modify the interaction with specific antibodies, limiting the use of such antibodies as diagnostic, therapeutic or prophylactic drugs.

Detection methods of CHIKV infection are based on virus isolation, reverse transcriptase polymerase chain reaction (RT-PCR) [ 16] and IgM/IgG ELISA [ 17]. Since virus isolation requires highly secure (BSL-3 or BSL-2 plus) facilities, PCR-based methods are mostly used for early and accurate diagnosis. Nonetheless, relatively high cost of the PCR-based assay and the requirement of a thermal cycler to conduct the test limit its application in the field and rural regions where outbreaks often occur. Immunochemical methods such as IgM/IgG ELISA can be applied everywhere and are cost-effective, but it takes around a week for the patient to develop an antibody response. Hence, IgM/IgG ELISA as a diagnosis tool is not effective in early stages of the infection.

Alternatively, direct CHIKV detection in serum has been reported to be an early and reliable clinical diagnosis as well as an effective surveillance of CHIKV [ 12, 18, 19]. Some of the tests relies on anti-CHIKV monoclonal antibodies obtained via hybridoma technology after immunization with inactivated CHIKV particles or recombinant proteins derived from the virus [ 12, 20]. Virus-like particles (VLPs) have also been used as selector combined with immune antibody libraries or B-cell sorting as source of human anti-CHIKV antibodies [ 13, 21]. Due to the exquisite specificity of the antibodies however, one of the potential limitations of direct CHIKV detection in serum samples is the differential sensitivity and specificity of the tests for detection of diverse CHIKV lineages. For instance, it has been reported [ 22] that a test with high sensitivity and specificity for clinical samples known to contain ECSA-genotype has low sensitivity with clinical samples obtained from individuals infected with the Asian genotype [ 23]. Obviously, surveillance strategies based on results of tests with low sensitivity for a given CHIKV lineage would be misleading when applied in regions where the outbreak is caused by such a lineage.

Here, with the goal of generating anti-CHIKV antibodies with potential application in the early diagnostic and CHIKV disease management in Mexico, Chikungunya virions were purified from a serum sample from a patient from Veracruz, México, during the 2015 CHIKV outbreak. The integrity of the Chikungunya virions was assessed by structural methods such as electron microscopy as well as functional assays such as binding to diverse well-characterized anti-CHIKV monoclonal antibodies [ 13, 21]. We also determined the whole genome sequence of this CHIKV isolate and a comparison with several strains of different lineages described for CHIKV, e.g., WA, ECSA and IOL lineages was made.

Once qualified, UV-inactivated CHIKV particles were used as selectors to discover high affinity and highly stable antibodies from ALTHEA Gold Libraries™ [ 24, 25]. The specificity of the antibodies was assessed by ELISA against mosquito-transmitted viruses from Venezuelan equine encephalitis virus (VEEV), which belongs to the alphavirus genus of the Togaviridae family, and DENV (DENV-1 and DENV-2) and ZIKV, which belong to Flavivirus genus of the Flaviviridae family. Since DENV and ZIKV could co-exist with CHIKV and generate similar symptoms, it makes difficult the differential diagnostic of CHIKV infection. Finally, a sandwich ELISA with the best antibody, combined with a potent neutralizing antibody reported elsewhere [ 13], was carry out. The results indicate that the newly discovered antibodies and the prototype ELISA developed herein could be valuable and effective tools for CHIKV disease surveillance.

Methods

Cells and viruses

All the virus strains used in this work were propagated and titrated in Vero cells (ATCC CCL-81) using standard plaque assays. Vero cells were grown in complete Eagle's Minimal Essential Medium (ATCC 30-2003) supplemented with 10 % FBS. The CHIKV-033 isolate, was kindly donated by Dra. Rosalia Lira from Unidad de Investigación Médica en Enfermedades Infecciosas y Parasitarias, IMSS, México. The VEEV strain TC-83, derived from ATCC VR-1249TM, was directly amplified in Vero cells from the VEEV vaccine (EQUIVAC TC-83, Productora Nacional de Productos Veterinarios, Mexico). The ZIKV isolate (Yucatán, México), the DENV-1 strain Puerto Rico/PR159-S1/1969 and the DENV-2 strain YUC17438 were kindly donated by Dr. Ma Isabel Salazar Sánchez from the Escuela Nacional de Ciencias Biológicas, National Polytechnic Institute, México.

Virus assays

The alphaviruses (CHIKV-033 and VEEV strain TC-83) and flaviviruses (ZIKV, DENV-1 and DENV-2) were routinely propagated in our laboratory. Briefly, Vero cells were grown in 75 cm ² flasks until a confluence of 80% was reached. Afterwards, the growth media was removed, and the cells were infected with alphaviruses at a MOI of 0.001, or Flaviviruses at a MOI of 0.1 using 1 mL of viral inoculum per 75 cm ² bottle. After an adsorption period of 2 h, the viral inoculum was removed and maintenance media (EMEM supplemented with 3% FBS) was added. The infection was left to proceed for 3 days for the alphaviruses, and 4 days for the Flavivirus ZIKA and 7 days for DENV-1 and DENV-2, afterwards the infected cultures were frozen and thawed once, and the viral lysates were supplemented with 20% FBS, clarified and kept at −80 ºC until further use.

CHIKV purification

Before virus purification, CHIKV-033 lysates were inactivated by three cycles of UV light for periods of 30 min. The inactivation of final preparations was verified using standard plaque assays in accordance with Warter and co-workers [ 21]. CHIKV-033 particles were precipitated from the inactivated viral lysates with cold PEG-NaCl at least 12 h. The mixture was centrifugated at 3000xg for 30 min at 4 ºC. The precipitated CHIKV particles were resuspended in TNE buffer (50 mM Tris-HCl, 100 mM NaCl, 0.1 mM EDTA, pH 7.4) at 4 °C. The CHIKV stock from PEG precipitation (25 mL) was then layered over 10 mL of a 60−30 % (w/v) sucrose step gradient prepared in ultracentrifuge tubes (Beckman Coulter, Cat No. 349622) on ice. Ultracentrifugation was carried out at 121,000× g for 3 h at 4 °C using a Beckman SW-28 rotor. After centrifugation, the CHIKV particles were observed as a white band above the 60% (w/v) sucrose layer. The particles were collected using a Pasteur pipette and diluted in TNE buffer. To pellet down the virus, 10 mL of sample with TNE buffer was layered over 2 mL of 30% (w/v) sucrose to a 12 mL ultracentrifuge. A second ultracentrifugation step was carried out at 281,000× g for 1 h at 4 °C using a Beckman SW41Ti rotor. The supernatant was removed, and the pellet was resuspended with 200 µL of TNE buffer.

Transmission electron microscopy (TEM)

Negative staining TEM was used to analyze the shape and size of the purified viral particles. Chikungunya particles (10 µL) were fixed for 1 min on copper grids coated with Formvar-carbon (Electron Microscopy Sciences, Cat No. CF200-Cu) and stained with 2.5% (v/v) uranyl acetate for 15 s. After air dry, samples were observed in a Zeiss Libra 120 transmission electron microscope at 80 kV. The images of the CHIKV were displayed using ImageJ software (National Institutes of Health, Bethesda, MD).

Control antibodies

Three control antibodies were used in this work: two human antibodies (4J21 and 4N12) reported by [ 13] and another antibody (5F10) reported by [ 21]. 4J21 and 4N12 were cloned as human IgG1/kappa molecules from the V regions disclosed in the patent WO 2016/168,417 and Protein-A purified in house-See IgG conversion below. 5F10 was purchased at Novus Biologicals (Clone E26D9.02, Cat No. DDX9 100P-100).

Direct binding ELISA

Nunc MaxiSorp™ flat-bottom plates (Thermo Scientific; Cat No. 44-2404-21) were coated with purified CHIKV particles at 3 µg/ml in PBS overnight at 4 °C. After blocking with 3% skim milk prepared in PBS (MPBS) for 1 h, IPTG-induced cultures, purified scFvs or IgGs in MPBS were added to the wells in 3-fold serially dilutions and incubated for 1 h at 37 °C. Bound samples were detected with recombinant Protein-A conjugated to HRP (1:8000, Invitrogen, Thermo Fisher Scientific; Cat No. 101123). The assay was revealed with TMB Substrate Reagent Set (BD OptEIA, BD Biosciences; Cat No. 555214). The reaction was stopped by the addition of 1 M phosphoric acid. The absorbance was read at 450/570 nm using an automated BioTek´s 2 microplate reader.

ALTHEA Gold Libraries™ and panning using inactivated CHIKV particles

ALTHEA Gold Libraries™ consist of two semisynthetic libraries built with synthetic human germline genes combined with natural human HCDR3/J H (H3J) fragments obtained from peripheral blood mononuclear cells (PBMCs) of a large pool of 180 donors. One of the libraries, called SL1, was built with a V L scaffold assembled with the human IGKV3-20*01 germline gene combined with the human IGJV4*01 joining region. The other library, called SL2, was made with a V L scaffold built with the human IGKV4-01*01 germline gene, also combined with the IGJV4*01 germline gene. Both libraries have a universal V H scaffold, which was partially built with the human IGHV3-23*01 germline gene. SL1 has a short LCDR1, whereas SL2 has a long one. By changing the length of LCDR1 from short to long, antibodies alter the preference to bind protein or peptide targets, respectively [ 26, 27]. Therefore, by using the library with the proper V L scaffold, antibodies against protein or peptide targets can be selected. When used in combination, SL1 and SL2 would potentially produce antibodies that bind diverse epitopes on a given target.

Selection of anti-CHIKV antibodies was performed in solid phase. Nunc MaxiSorp™ flat-bottom plates (Thermo Scientific; Cat No. 44-2404-21) were coated with purified CHIKV-033 at a concentration of 5 µg/mL in PBS. Aliquots of 10 ¹¹-10 ¹² cfu from ALTHEA Gold Libraries™, covering 10 to 100 times the initial library diversity, were used in the first round of panning. Subsequent rounds were performed with the output of the previous round at 10 ¹² cfu.

Screening for positive and unique anti-CHIKV scFvs

Soluble scFvs were induced with IPTG (1 mM) and tested in plates with CHIKV or BSA as negative control. As reporter reagent, Protein-A/HRP (Thermo Fisher; Cat No. 101,123) was used. PCR of the positive clones were submitted to Sanger sequencing and unique clones were expressed as soluble scFvs, purified using Protein-A and re-tested for binding to CHIKV and BSA in direct ELISA assays.

IgG conversion

The scFvs identified in the ELISA-based screening were converted to human IgG1 via PCR of the V regions and cloning in TGEX expression vectors (Antibody Design Labs, Inc). The V regions of the sequences identified by NGS were PCR amplified with a reverse primer matching the HCDR3 sequences and a universal forward primer hybridizing in the leader peptide. The IgGs were expressed in HEK 293 cells (ATCC CRL-3216) by co-transfection of plasmid DNA containing the heavy and light chains. After a 4-day incubation, the supernatants containing the IgGs were harvested, filtrated and purified using Protein A MabSelect SuRe column (5 mL, GE Healthcare). The IgGs were captured in PBS (20 mM, 150 mM NaCl, pH 7.4) and eluted with 20 mM citrate buffer pH 3.5. The monomeric content of the purified IgGs was estimated by UPLC BEH200 150 mm Size Exclusion Chromatography (SEC) columns (Waters). The integrity of the IgG was evaluated by SDS-PAGE and Mass Spectrometry (Intact Mass).

Specificity assay

The cross-reactivity experiment was performed in MaxiSorp 96-well plates coated with 100 µL of purified particles from alphavirus genus: CHIKV (3 µg/mL) and VEEV (1 µg/mL) in PBS, pH 7.4, for 16 h at 4 °C. Additionally, inactivated flaviviruses lysates from ZIKV, DENV-1 and − 2 (30 µg/mL) were used. The plates were then incubated with MPBS for 1 h at RT, washed with 0.1% Tween-20 in PBS (PBS-T), and then further incubated with the anti-CHIKV antibodies (10 µg/mL) for 1 h at 37 °C, followed by further incubation with HRP-conjugated Protein A (1:8000, Invitrogen, Thermo Fisher Scientific) for 1 h at 37 °C. The positive controls: VEEV, hyperimmune serum 1:10 (RIH Research); DENV-1, monoclonal antibody D2-1F1-3 1:10 (Biotem, Le Rivier d'Apprieu, France); DENV-2 and ZIKV, mouse Anti-flavivirus Envelope Protein Antibody (4G2, 1:10; Native Antigen Company, UK) were included. All measurements were made at least three times. Statistical analysis was performed by one-way ANOVA followed by post hoc Tukeys's multiple comparisons test.

Western blot

CHIKV (2 µg/mL) were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (0.2 μm; Biorad, CA, USA) at 25 V and 1.3 A. After blocking with 3% skim milk in PBS-T, the membranes were incubated with the anti-CHIKV antibodies (10 µg/mL) overnight at room temperature. Afterwards, the membranes were washed three times with PBS-T and further incubated with goat anti-human IgG Fc-HRP (1:5,000, Abcam, US) for 1 h. The immunoreactive bands were detected using ECL Western Blotting Substrate (Pierce, Thermo Scientific) and visualized in a Chemidoc ™ MP System.

Detection of CHIKV by a sandwich ELISA

Nunc MaxiSorp™ flat-bottom plates were coated with 100 µL of 10 µg/mL of the C7 antibody in carbonate buffer pH 9.4 overnight at 4 °C and blocked with 3% MPBS for 60 min at 37 °C. Serial dilutions of the CHIKV purified and UV-inactivated extract, starting at a concentration of 40 µg/mL, were incubated for 1 h at 37 °C. After washing the non-bond CHIKV, biotinylated 4N12 antibody (0.1 µg/mL) was added and incubated at 37 °C for 1 h. The assay was revealed with Streptavidin conjugated to HRP (1:1,000; Invitrogen Cat. No. S911) and TMB Substrate Reagent Set (BD OptEIA, BD Biosciences; Cat No. 555214). The reaction was stopped with 1 M phosphoric acid, and the absorbance was read at 450/570 nm by using automated BioTek´s 2 microplate reader.

Number of virions in the purified CHIKV preparation

To estimate the number of CHIKV particles per mL in the UV-inactivated extract, we first determined the mass of E2 protein in our preparation as follows. Three volumes: 10, 5 and 2 µL of the CHIKV stock at a total protein concentration of 691 µg/mL 2D-Quant Kit (Sigma Aldrich) were loaded into an SDS-PAGE gel and ran side-by-side with a standard of known mass (0.5 µg of BSA). The density of the E2 bands was quantified by comparison with the density of the BSA standard. The concentration of E2 was estimated in 36 µg/mL, which is 5.15% of the CHIKV UV-inactivated stock. The number of CHIKV particles per mL (CHIKV/mL) in 40 µg/mL of our CHIKV preparation was calculated in 1.2 × 10 ¹⁰ CHIKV/mL as follows: # of molecules of CHIKV/mL = {[(40 µg/mL ×  0.0515)/ 240)] / 46,000 g/mol} ×   6.022 × 10 ²³ number of particles/mol x 10 − 6; where 40 µg/mL is the concentration of the CHIKV UV-inactivated in the starting dilution (see above); 0.0515 is the fraction of E2 protein; 46,000 g/mol is the molecular weight of E2; 240 is number of E2 copies per CHIKV particle [ 28]; 6.022 × 10 ²³ number of particles/mol is Avogadro's number; and 10 − 6 is a factor to convert µmol into mol.

Statistical analysis

Data are shown as the average ± standard error of the mean (SEM). To determine the affinity constants by direct binding ELISA, the results were adjusted following the 4-Parameter Logistic (4PL) curve model and each experiment was carried out at least three times with independent samples. Data were analyzed by one-way ANOVA followed by Tukey's multiple comparisons tests for the cross-reactivity experiments. Differences were considered statistically significant with a p value < 0.05. Statistical analyses were performed in Prism 9.2.0 (Graphpad Software, LLC).

Results

CHIKV-033 purification and characterization

A detailed characterization of CHIKV-033, including sequencing of its whole genome, will be published elsewhere (Rosalia Lira et al. personal communication). In this work, to identify the phylogenic origin of this CHIKV strain, we compared the whole genome and E2 of CHIKV-033 with the sequences of fifty-four CHIKV strains including 10 sequences from WA, 18 from Asian, five from ECSA and 26 from IOL lineages (Additional file 1: Fig. S1). The phylogenetic trees of both the whole genome and E2 indicated that the CHIKV-033 belongs to the Asian lineage.

Electron microscopy of CHIKV-033 purified virions, SDS-PAGE and direct ELISA using three known anti-CHIKV antibodies [4N12, 4J21 and D9 (5F10)] is shown in Fig. 1. The purified viral particles had the proper size (~ 70 nm) and the spherical shape previously described for CHIKV [ 29, 30] (Fig. 1A). The SDS-PAGE showed the typical pattern of the spike proteins, i.e., E1 ∼48 kDa, E2 ∼46 kDa and capsid protein (CP) ∼30 kDa [ 31] (Fig. 1B). In addition, the UV-inactivated CHIKV particles were specifically recognized by the three well-characterized anti-CHIKV antibodies (Fig. 1 C), indicating that the CHIKV capsid proteins were properly folded after the UV-inactivation process.

Identity and functional analysis of the CHIKV-033 isolate. A Representative electron micrographs of negative staining of CHIKV-033 particles purified by sucrose gradient. Images were captured using the Zeiss Libra 120 transmission electron microscope at 80 kV (Scale bars, 2 μm, 0.5 μm, 100 nm and 50 nm). B SDS-PAGE pattern of purified CHIK-033 virions. MiniProtean Stain Free gels (Biorad) showed the typical pattern of the spike proteins, i.e., E1 at ∼48 kDa, E2 at ∼46 kDa and capsid protein (CP) at ∼30 kDa. Precision Plus Protein Unstained Standards (Biorad) was used like molecular weight marker. C Direct ELISA of the anti-CHIKV 4N12, 4J21 and D9 (5F10) control antibodies. Each experiment was carried out at least three times with independent samples. Representative numbers are presented as average ± standard error of the mean (SEM)

Panning with viral particles and screening for CHIKV specific clones

Having qualified CHIKV-033 isolate, the UV-inactivated purified particles were used as selectors to isolate human antibodies from ALTHEA Gold Libraries™ [ 24]. The panning was performed in solid phase, e.g., ELISA plates. The screening for specific anti-CHIKV antibody fragments was conducted following two strategies: (1) direct binding to CHIKV particles and BSA, the latter as specificity control; and (2) NGS and data mining.

Direct ELISA of 88 colonies chosen at random from the third round (R3) of panning showed eight positive and specific IPTG-induced supernatants (8/88; 10 %). Sanger sequencing of these eight clones resulted in four unique sequences, called C10, C7, A11 and F2, giving a hit rate of 4.5 %. This hit rate compared well with commonly observed hit rates obtained with other targets and diverse phage display platforms [ 25]. C10 was found 4 times, whereas, C7 was found twice, and A11 and F2 were found once. To enrich for additional specific scFvs, a fourth round (R4) of panning was performed. Out of 83 IPTG-induced supernatants tested in ELISA, 68 (68/85; Analysis 82%) resulted positive and specific. C10 represented 60% of the positive clones, whereas C7 and A11 showed up 32 % and 8%, respectively. F2 was not seen in R4 and was not processed further.

The NGS statistics are shown in Additional file 1. Over 1.5 million reads were processed from round 2 (R2) to R4. The frequencies and enrichment factors of the HCDR3 sequences of C7, C10 and A11 are shown in Table 1. These clones contributed close to 80% of all the unique sequences in R4. As expected, an enrichment in the frequency of C10, C7 and A11 HCDR3 sequences occurred as the rounds of selection proceeded. Consistent with the ELISA-based screening, C10 was enriched over three orders of magnitude in R4 with respect to R2, whereas C7 was enriched two orders of magnitude and A11 increased its frequency 20-fold from R2 to R4. Additional 6 HCDR3 amino acid sequences not seen in the ELISA-based screening were also enriched within the 50 most frequent HCDR3 sequences in R4. The scFvs having these HCDR3 sequences were amplified using specific primers matching the HCDR3 nucleotide sequences and cloned as human IgG1/kappa antibodies.

Table 1 Frequency per round of panning of the antibodies selected via ELISA-based screening and Enrichment Factor (EF)

Characterization of the anti-CHIKV antibodies

Four out the six antibodies coming out of the NGS screening did not show significant binding and were excluded from this study. The characterization of the remining five antibodies isolated from ALTHEA Gold Libraries™ is summarized in Table 2. As a reference, the three control antibodies: 4N12, 4J12 and D9(5F10) are also included in the Table.

Table 2 Summary of the characteristics of the anti-CHIKV antibodies obtained from ALTHEA Gold Libraries™

One out the five antibodies was isolated from the library called SL1 (second column of Table 2). The other four were obtained from SL2. The SL1 library has a V L scaffold assembled with the human IGKV3-20*01 germline gene combined with the human IGJV4*01 joining region. The SL2 library was made with a V L built with the human IGKV4-01*01 germline gene also combined with the IGJV4*01 germline gene. Both libraries have a universal V H scaffold, which was partially built with the human IGHV3-23*01 germline gene. Therefore, all antibodies but C10 have the same V H:V L combination (VH3-23:VL3-20), whereas, C10 has the VH3-23:VL4-01 combination. Interestingly, all the antibodies obtained in the ELISA-based screening have a relatively short HCDR3 sequence (seven to eight residues, third column of Table 2) as compared to the two NGS-obtained antibodies, which have a relatively long HCDR3 (fourteen and seventeen residues).

The integrity of the purified anti-CHIKV antibodies was verified by SDS-PAGE and analytical SEC (Additional file 1: Fig. S2). The expression yield in HEK 293 cells (last column of Table 2) of the antibodies selected based on ELISA was five-fold or higher than that of the NGS antibodies. The expression yield of C7, C10 and A11 was also higher than the control antibodies (4N12 and 4J21). A higher expression yield of C7, C10 and A11 may explain because, although both ELISA-based and the NGS-identified antibodies were enriched during the panning, only C7, C10 and A11 were identified in the ELISA-based screening.

The Tm of the antibodies is also reported in Table 2 (eighth column, see also Additional file 1: Fig. S3). All the five antibodies obtained in this work have Tm values ≥ 69 °C, with C10, C7 and NGS2 having two unfolding transitions. The second transition is close or above 80 °C. In contrast, the control antibodies (4N12 and 4J21) have a single transition at 61 °C or 65 °C, respectively. The control antibodies were isolated from a CHIKV convalescent patient, although they present high affinity, they did not exhibit high thermostability.

Binding of the antibodies to UV-inactivated CHIKV particles-determined as the EC ₅₀ of direct ELISA curves-is reported in the seventh column of Table 2. The dose-response curves are shown in Fig. 2. Conversion from scFv to IgG1 improved binding to the CHIKV particles by C7, C10 and A11 antibodies (compare the fifth and seventh column, Table 2). One of the two antibodies isolated by NGS, NGS4, showed a similar binding profile than two of the antibodies identified by ELISA-based screening: C10 and A11. NGS2 bound CHIKV particles poorly and was not further characterized.

Direct binding ELISA of the anti-CHIKV antibodies isolated from ALTHEA Gold Libraries ™. The control antibodies were assayed side-by-side with our antibodies. Each experiment was carried out at least three times with independent samples, and representative numbers are presented as average ± SEM. The curves were adjusted to a 4-Parameter Logistic (4PL) curve to determine the EC ₅₀ and apparent affinity values

C7 happened to be the highest affinity antibody isolated from ALTHEA Gold Libraries™, with a binding profile close to the control antibodies D9(5F10) and 4J21. We estimated 0.016 and 0.045 nM for 4N12 and 4J21, respectively. C7 gave an apparent affinity in the ELISA of 0.09 nM which is twofold the value measured for 4N12. The K D values reported for the control antibodies, 4N12 and 4J21, when measured by BIAcore using the recombinant CHIKV capsid proteins are 0.95 and 0.54 nM, respectively [ 13]. Therefore, we can safely assume that the K D of C7 if measured in the BIACore with recombinant CHIKV proteins would be in the single nM or sub-nM range. The difference with the affinity measured by ELISA and BIAcore could be due to a number of factors including: (1) mutations of CHIKV-033 affecting binding to these antibodies, (2) configuration and arrangement of the E2 protein in the UV-inactivated CHIKV with respect to isolated recombinant proteins, (3) isotype of the antibodies-notice that we used different isotypes than the original publication [ 13] and (4) assay conditions.

Cross-reactivity with VEEV, DENV and ZIKV

Cross-reactivity of C7, C10, A11 and NGS4 with VEEV in shown in Fig. 3. C7, C10 and A11 had a slight cross-reactivity with VEEV, whereas NGS4 did not show cross-reactivity. Interestingly, cross-reactive immune response with VEEV after immunization with CHIKV has been reported [ 32]. None of the antibodies showed cross-reactivity with DENV1, DENV2 and ZIKV.

Specificity of the anti-CHIKV human antibodies. Inactivated arbovirus (CHIKV, VEEV, DENV-1, DENV-2, and ZIKV) were coated onto ELISA plates. After the blocking, all the antibodies were used at the same concentration and detected with HRP-conjugated Protein A and enhanced by chemiluminescence (ECL) substrate. For the recognition of each virus, a hyperimmune serum or monoclonal antibodies specific for each virus were used as positive controls (see details in " Method" section). BSA was used as negative control. Corrected OD values are shown. All measurements were caried out in triplicate. Data are shown as average ± standard error of the mean (SEM). Statistical analysis was performed by one-way ANOVA followed by post hoc Tukeys's multiple comparisons test. *** indicates p  < 0.001

Reactivity of the antibodies with CHIKV proteins

To identify the CHIKV proteins recognized by C7, C10, A11 and NGS4, a western blot with the UV-inactivated CHIKV particles was performed (Fig. 4 above). All the antibodies recognized specifically the structural protein E2, with C7 showing a band of higher intensity than that of C10, NGS4 and A11 (Fig. 4 below). A higher E2 binding by C7 with respect to the other antibodies correlates with the binding profile obtained by ELISA except for A11 (see above). A11 showed a similar ELISA binding profile than C10 and NGS4 but barely recognized CHIKV in the western blot. Likewise, 4N12 gave a weak signal in the western blot, despite being the highest affinity antibody by ELISA. This result suggested that 4N12 (and perhaps A11) recognizes a conformational epitope on E2, whereas C7, C10 and NGS4 bound continuous epitopes.

Representative western blot analysis of the antibodies isolated in this work. Inactivated CHIKV-033 proteins were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The blocked membranes were probed with the anti-CHIKV human antibodies from ALTHEA Gold Libraries ™, followed by incubation with HRP-conjugated anti-human IgG. The 4N12 and 4J21 antibodies were used for comparison. In the lower panel, the E2 band densitometry values recognized by the anti-CHIKV human antibodies are shown. All measurements were made at least in duplicate. Data are shown as average ± SEM

Detection of CHIKV particles using a sandwich ELISA

Based on the above results and the binding profile of the antibodies (Fig. 2), we decided to generate a sandwich ELISA with C7 and 4N12 to explore the potential of this assay in quantifying CHIKV particles in biological samples. To this end, both antibodies were biotinylated and used in alternative formats, i.e., C7 as capture reagent and 4N12 as reporter, and vice versa. The format with C7 as capture reagent and 4N12 as reporter gave the higher dynamic range and sensitivity (data not shown) and was thus used in the subsequent experiments.

To determine how serum affected binding of the antibodies to CHIKV particles, serial dilutions of the UV-inactivated and purified CHIKV preparation in both 1:2 serum and assay buffer (MPBS) were prepared and assayed in the sandwich ELISA. Both dilution matrixes gave similar curves (Fig. 5A; Additional file 2). The minimum concentration of CHIKV particles per mL detected was 1.9 × 10 ⁸, estimated as twofold the assay background. The curves plateaued at 1.8 × 10 ¹⁰ CHIKV/mL, for a dynamical range of 1.9 × 10 ⁸-1.8 × 10 ¹⁰ CHIKV/mL.

A Sandwich ELISA using C7 as capture antibody and biotinylated 4N12 as reporter. The dose/response curves were prepared with 1:2 serial dilutions of the UV-inactivated CHIKV preparation in CHIKV-free serum or assay buffer. The concentration of CHIKV is expressed in terms of CHIKV particles per mL, based on the concentration of E2 as described in detail in the " Method" section. B Recovery assay showing samples of CHIKV spiked at known concentration in serum and measured in the sandwich ELISA

To determine the recovery of CHIKV particles in serum we spiked known concentrations of the UV-inactivated and purified CHIKV preparation in CHIKV-free serum and compared the expected and measured value. The R ² was 0.99 and a slope of the regression curve was close to one (Fig. 5 B), indicating a good recovering of the CHIKV in serum.

Discussion

In the previous sections we reported the characterization of a panel of anti-CHIKV antibodies. Our strategy to obtain these antibodies differed from other methods currently described to isolate CHIKV antibodies [ 13, 21]. These methods have used VLPs or recombinant capsid proteins as immunogens to obtain polyclonal or monoclonal antibodies from diverse species. In other instances, VLPs or recombinant proteins have been employed as selectors combined with immune libraries or B-cell sorting methods as source of antibodies. Here, we used UV-inactivated CHIKV particles as selectors and ALTHEA Gold Libraries™ [ 24, 25] as source of antibodies. Different from immune libraries, which are prepared with B-cells isolated from patients infected with a given virus, ALTHEA Gold Libraries™ are general purpose libraries that can be used to obtain antibodies against any given target.

After three rounds of panning with UV-inactivated CHIKV particles, the screening by ELISA of a relatively small number of clones (< 100) yielded three unique anti-CHIKV antibodies. Two additional antibodies were discovered by a complementary screening strategy based on NGS and data mining. Interestingly, the antibodies selected by ELISA and those obtained by NGS differed in several respects, including expression and cross-reactivity with VEEV, a virus closely related CHIKV. At least two different epitopes on the CHIKV E2 protein were recognized by these antibodies. Most of the antibodies recognized linear epitopes on E2, whereas, one antibody, A11, seemed to bind a non-linear epitope. The best antibody, C7, showed an affinity in the single digit nM or sub-nM range, comparable to some of the best anti-CHIKV antibodies described to date [ 13, 21].

To explore the value of C7 as substrate for development of CHIKV detection methods, a sandwich ELISA with C7 and 4N12 was performed. Both antibodies bind E2 but via different epitopes; C7 binds a linear epitope, whereas 4N12 binds a conformational one. The dynamic range of the sandwich ELISA was 0.2-40 mg/mL of UV-inactivated and purified CHIKV preparation. Based on the concentration of E2 in the CHIKV preparation, we estimated that this range of concentrations translates into 10 ⁸-10 ¹⁰ CHIKV particles per mL. Although this estimation rests on a number of assumptions, some authors [ 33] have reported that the CHIKV load during the acute phase of the viremia is 10 ⁹-10 ¹² CHIKV/mL of blood. Other authors [ 34] have measured concentrations of CHIKV in samples drawn on day 0 of the infection around 10 ⁹ RNA copies/mL, with viral loads decreasing over time. Yet another report [ 35] has determined that the median viral load ranges from 2.9 × 10 ⁴ to 1.6 × 10 ⁸ plaque forming units (PFU) per mL during days 1 through 4 of the viremia period. Considering that PFU underestimate the actual number of viral particles by one or two orders of magnitude due to variations of in the infectivity of the virus, values of 10 ⁹ CHIKV/mL or RNA copies/mL or 1.6 × 10 ⁸ PFU/mL fall well within the 10 ⁸-10 ¹⁰ CHIKV/mL dynamical range of the sandwich ELISA. Therefore, although optimizable, this assay could serve as the basis for detection of meaningful CHIKV loads during the acute phase of the CHIKV infection.

It should be noted that our antibodies showed superior expression, highly pure preparations after a single purification step and higher stability than the best known CHIKV antibodies [ 13, 21]. These favorable pharmaceutical properties could translate in diagnostic tests produced at lower costs, with longer shelf stability and higher robustness. Nevertheless, these antibodies have not been tested in independent clinical samples from CHIKV RNA-positive patients or from different CHIKV strains, which limit their immediate uses. This information will be essential to future investigations, above all, in the case of new CHIKV infections appear.

Conclusions

We report here the discovery and characterization of new anti-CHIKV human antibodies. These antibodies showed specificity and high affinity to the CHIKV virions, with a superior expression, highly pure preparations after a single-step purification and high stability. These characteristics make the antibodies described here valuable tools in developing cost-effective assays for early stages detection and disease control of the CHIKV infection in future outbreaks.

Availability of data and materials

The datasets generated and/or analysed during the current study are available in the GenBank repository, [Accession numbers: OK247726, OK247727, OK247728, OK247729, OK247730, OK247731, OK247732, OK247733, OK247734, OK247735] (Additional files 3, 4).

Abbreviations

CHIKV:

Chikungunya virus

ELISA:

Enzyme-Linked ImmunoSorbent Assay

IGHV:

Immunoglobulin heavy-chain variable

IGKV:

Immunoglobulin kappa-chain variable

HCDR3:

Third complementary determining region of heavy-chain variable

Acknowledgements

We would like to thank Lev Kotler and Jan Kieleczawa at Wyzer Biosciences for their assistance in designing the NGS amplicons and generating the NGS data. To Dra. Gabriela Mellado for initial and encouraging discussions. Technical assistance by Guadalupe Zavala and the Unidad de Microscopía Electrónica IBT-UNAM is acknowledged. Also, we would like to thank Said Vázquez-Leyva, Luis Vallejo-Castillo and David Jáuregui-Zúñiga for assistance with purification and characterization of the antibodies.

Funding

This work was supported in part by the Grant 3661 from CONACYT to RCS (Rommel). CONACyT (Consejo Nacional para la Ciencia y Technology) is a Mexican government agency providing funding to research projects. This grant was approved within the call for Problemas Nacionales (National Problems) as part of an effort to address urgent and unmet health and medical needs in Mexico. The grant funded CHIKV isolation and characterization including whole genome sequencing and TEM, as well as panning of ALTHEA Gold Libraries™ with viral particles, screening and characterization of the antibodies.

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Authors and Affiliations

1. CONACyT-Unidad de Desarrollo e Investigación en Bioprocesos (UDIBI), Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City, México

M. Pedraza-Escalona

2. Unidad de Desarrollo e Investigación en Bioprocesos (UDIBI), Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City, México

O. Guzmán-Bringas, H. I. Arrieta-Oliva, K. Gómez-Castellano, J. Salinas-Trujano, J. Torres-Flores, J. C. Muñoz-Herrera, R. Camacho-Sandoval, P. Contreras-Pineda, R. Chacón-Salinas, S. M. Pérez-Tapia & J. C. Almagro

3. Laboratorio Nacional para Servicios Especializados de Investigación, Desarrollo e Innovación (I+D+i) para Farmoquímicos y Biotecnológicos, LANSEIDI-FarBiotec-CONACyT, Mexico City, México

M. Pedraza-Escalona, O. Guzmán-Bringas, H. I. Arrieta-Oliva, K. Gómez-Castellano, J. Salinas-Trujano, J. C. Muñoz-Herrera, R. Camacho-Sandoval, P. Contreras-Pineda, R. Chacón-Salinas, S. M. Pérez-Tapia & J. C. Almagro

4. Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional (ENCB-IPN), México City, México

R. Chacón-Salinas & S. M. Pérez-Tapia

5. GlobalBio, Inc, 320 Concord Ave., 02138, Cambridge, MA, USA

J. C. Almagro

Contributions

MPE contributed with planning and executing most of the experimental strategy of this work, coordinated TEM and CHIKV genome sequencing, participated in the manuscript writing, preparation of the figures, and manuscript editing processes. RCS2, SMPT and JCA devised the concept of the manuscript and contributed to the overall direction of the work, discussion of the results and general writing, preparation of the figures, and manuscript editing processes. OGB and RCS1 designed and executed ELISAs including the direct and sandwich assays, participated in the discussion of the results, and manuscript review. IAO, KGC, PCP and JCMH planned and executed the panning strategy using ALTHEA Gold Libraries™, screening, characterization of the positive clones, and IgG conversion and characterization. JST designed and implemented the protocols for CHIKV isolation and viral inactivation and participated in the discussion of the results. JTF provided valuable expert guidance to manipulate the virus, executed together with JSL, the protocols to isolate and characterize CHIKV and contributed with the manuscript concept, organization, writing and review of the text. All authors read and approved the final manuscript.

Corresponding author

Correspondence to J. C. Almagro.

Ethics declarations

Ethics approval and consent to participate

The serum sample used to obtain the CHIKV-033 isolate was collected under a written informed consent by the patient. All procedures for handling the virus and use of the ALTHEA Gold Libraries™ were approved by Research Ethic Committee at Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México (CEI-ENCB-SH-002-2018). All the studies presented here were performed under good laboratory practices.

Consent for publication

All the authors reviewed the final version of the manuscript and gave their approval to publish the work.

Competing interests

JC-A is involved in the development and commercialization of Althea Gold Libraries™. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be understood as a potential conflict of interest.

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Pedraza-Escalona, M., Guzmán-Bringas, O., Arrieta-Oliva, .I. et al. Isolation and characterization of high affinity and highly stable anti-Chikungunya virus antibodies using ALTHEA Gold Libraries™. BMC Infect Dis 21, 1121 (2021). https://doi.org/10.1186/s12879-021-06717-0
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DOI: https://doi.org/10.1186/s12879-021-06717-0

Keywords

Chikungunya virus

Venezuelan equine encephalitis Virus

Diagnostic

Human Antibodies

Next-Generation Sequencing

Phage display

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Isolation and characterization of high affinity and highly stable anti-Chikungunya virus antibodies using ALTHEA Gold Libraries™ - BMC Infectious Diseases

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Background More than 3 million infections were attributed to Chikungunya virus (CHIKV) in the 2014-2016 outbreak in Mexico, Central and South America, with over 500 deaths directly or indirectly related to this viral disease. CHIKV outbreaks are recurrent and no vaccine nor approved therapeutics exist to prevent or treat CHIKV infection.

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Tomatidine is a steroid that is being studied to help kill Chikungunya cells. It has been proven effective although it doesn't completely rid of the virus.

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Introduction

Chikungunya virus (CHIKV) was first identified in Tanzania between 1952 and 1953, where it caused an outbreak of chikungunya fever described as febrile polyarthralgia1. Within the last decade, CHIKV re-emerged and caused various large outbreaks in Africa, Asia and Central and South America2,3. Among those was an explosive epidemic on the Indian Ocean island of La Reunion, where over 266.000 people were affected4. Furthermore, since its introduction in the Americas in 2013, CHIKV has spread to over 45 countries in Central and South America causing more than 1.7 million infections5.

CHIKV is transmitted to humans via the mosquito vectors Aedes aegypti and Aedes albopictus 6. Important reasons for the drastic re-emergence of CHIKV is the expansion of the mosquito vector to urban areas with poor hygiene conditions, progressing climate change as well as the continuous increase in global transportation systems7. While other mosquito-borne arboviruses, such as dengue virus (DENV), only cause symptoms in a small fraction of infected individuals, CHIKV infection causes clinical manifestations in approximately 85% of infected individuals8. Acute chikungunya fever typically manifests as a rapid-onset illness characterized by fever, (poly)arthralgia, myalgia, maculopapular rashes, headaches and nausea9. Notably, 30 to 40% of patients suffer from persistent (poly)arthralgia that develops after clearance of the virus10. Appropriately, chikungunya in the African Makonde language translates as "that which bends up", referring to the debilitating joint pain patients often experience upon CHIKV infection6.

To date, the development of an effective treatment for CHIKV infection has not been successful. While various studies reported the development of CHIKV vaccine candidates and antiviral compounds in vitro and in animal models, there is no licensed vaccine or therapeutic available to prevent or treat CHIKV infection6,11,12,13. To combat CHIKV, we therefore currently rely on personal protective measures and vector control. The limited resources to control CHIKV infection and the rapid re-emergence emphasize the importance of identifying new compounds that effectively prevent or control CHIKV infection.

Tomatidine is a steroidal alkaloid derived from the stem and leaves of unripe, green tomatoes. It has been described to exhibit a variety of health-beneficial biological activities, including anti-metastatic activity14, anti-inflammatory activity15, anti-microbial activity16,17,18, and was shown to have a protective effect against age-related muscle atrophy19. Tomatidine was also found to exhibit antiviral activity towards the plant viruses Sunnhemp Rosette and Tobacco mosaic virus20. We recently identified tomatidine as a novel antiviral compound towards two re-emerging mosquito-borne flaviviruses: dengue virus (DENV) and zika virus (ZIKV)21. Potent antiviral activity was seen for all four DENV serotypes and a recent isolate of ZIKV. The most potent effect was seen for DENV serotype 2, with a half maximal effective concentration (EC50) of 0.82 µM. Tomatidine was shown to interfere with various stages of the viral replication cycle of DENV, yet predominantly after virus cell binding and internalization. No antiviral activity was observed for West Nile virus (WNV), a closely related mosquito-borne flavivirus.

Here, we evaluated the antiviral potential of tomatidine towards three different lineages of CHIKV, the East/Central/South African lineage, the original African isolate from 1953 as well as Asian lineage. We observed potent antiviral activity of tomatidine towards the three different CHIKV strains in Huh7 cells, with EC50 and EC90 values between 1.2 µM and 3.8 µM, respectively. Antiviral activity was also observed in Vero-WHO, HFF-1 and U2OS cells. In contrast to DENV, antiviral activity towards CHIKV was specifically seen at post-infection conditions. Tomatidine drastically reduced the number of infected cells and lead to an overall reduction in the number of produced progeny virions. Importantly, its antiviral activity was still observed at 24 hours post-infection, indicating that tomatidine effectively controls at least three rounds of CHIKV replication and highlighting its potential as an antiviral compound to treat CHIKV.

Results

Tomatidine inhibits CHIKV infection in various cell lines

First, we tested the antiviral effect of tomatidine on CHIKV in Vero-WHO cells, as these cells are highly permissive to infection and are often used in related studies6,22,23,24. Prior to the infection experiments, the cytotoxic profile of tomatidine in Vero-WHO cells was determined via an ATPLite assay. As shown in Supplementary Fig. S1a, tomatidine induced a dose-dependent reduction in ATP level with a CC50 value of 149 µM. The CC50 value represents the concentration of tomatidine needed to decrease the ATP level of the cells by 50%. The highest non-toxic tomatidine concentration (defined by survival rates above 75%) was 10 µM (Supplementary Fig. S1a) and was therefore used in subsequent experiments. Vero-WHO cells were incubated with 10 µM tomatidine or the equivalent volume of EtOH and infected with CHIKV-LR at MOI 0.5. Under standard infection conditions, 4.9 ± 0.0 Log infectious virus particles (8.1 × 10 ⁴ PFU/mL) were produced at 9 hpi (Fig. 1a). A comparable titer was observed for the 0.1% EtOH control (5.0 ± 0.1 Log), indicating that the solvent does not influence infectious virus particle production. In line with previous results on DENV and ZIKV21, tomatidine was found to exert significant antiviral activity towards CHIKV. In the presence of 10 µM tomatidine, the infectious virus titer was reduced to 4.4 ± 0.3 Log, which corresponds to a reduction by 76.8% when compared to the EtOH control (Fig. 1a).

Tomatidine reduces the production of infectious CHIKV particles. ( a) Antiviral effect of tomatidine on Vero-WHO cells infected with CHIKV-LR at MOI 0.5. Supernatants were collected 9 hpi. ( b-f) Huh7 cells were infected with distinct CHIKV strains at MOI 1 in presence of increasing concentrations of tomatidine or naringenin or the equivalent volume of EtOH. Supernatants were collected at 9 hpi. ( b,c) CHIKV-LR OPY1, ( d) CHIKV-LR OPY1 and naringenin ( e) CHIKV strain 99659, ( f) CHIKV strain S27. In all experiments data is represented as mean ± SEM from three independent experiments and differences were assessed with Student's t-test.

In order to validate these findings in a more relevant cell line for human infection, we next investigated the cytotoxic and antiviral potential of tomatidine in Huh7 cells (human hepatic cell line) as hepatocytes are targets during natural CHIKV infection25. Furthermore, like with Vero-WHO, these cells are also commonly used in antiviral CHIKV studies13,26,27,28. In our previous study on DENV, the cytotoxicity profile of tomatidine in Huh7 cells has been determined via the MTT assay, measuring the metabolic activity of the cell via mitochondrial activity21. Since mitochondrial activity is only one of many factors that determine cell viability, we here performed two additional cytotoxicity assays, the ATPLite assay, which measures the cellular ATP level and a trypan blue staining to detect the number of viable cells after tomatidine treatment. A dose-dependent decrease in ATP levels with increasing tomatidine concentrations was seen. The highest non-toxic tomatidine concentration was 20 µM and the CC50 value was defined as 156 µM (Supplementary Fig. S1b). The highest non-toxic concentration is slightly lower compared to the previously reported results for the MTT assay on Huh7 cells, where the highest non-toxic tomatidine concentration was defined as 30 µM21. The trypan blue staining revealed a significant reduction in total cell number at tomatidine concentrations >10 µM (Supplementary Fig. S1c). Based on the above described results, we defined 10 µM as the maximal non-toxic dose of tomatidine, in which metabolic activity, ATP levels and total cell numbers were not significantly changed. To investigate the potential additional cytopathic effect of CHIKV on the viability of tomatidine-treated-Huh7 cells, we next assessed the viability of Huh7 cells in the presence of 10 µM tomatidine under infection conditions via the MTS assay. As displayed in Supplementary Fig. S1d, where the results were normalized to a non-infected, non-treated control, infection with CHIKV at MOI 1 did not have an additional cytopathic effect on the Huh7 cells. Compared to the non-infected, non-treated control, no changes in cell viability were observed upon infection in the presence or absence of 10 µM tomatidine (Supplementary Fig. S1d). Hence, we decided to use 10 µM tomatidine as the highest concentration for all subsequent experiments in Huh7 cells.

To test the antiviral activity of tomatidine in Huh7 cells, cells were incubated with increasing concentrations of tomatidine (0.1 to 10 µM) at the time of infection with CHIKV-LR (MOI 1). On average 4.8 ± 0.2 and 4.9 ± 0.2 Log progeny infectious particles were produced for non-treated and EtOH-treated infection samples, respectively (Fig. 1b). Hence, the presence of EtOH did not influence the production of progeny infectious virus particles. Importantly, a dose-dependent decrease in the production of infectious CHIKV particles was observed in the presence of increasing tomatidine concentrations (Fig. 1b,c). In the presence of 10 µM tomatidine, 3.6 ± 0.2 Log infectious particles were produced, which corresponds to a 95.5% reduction in virus titer when compared to the EtOH control (Fig. 1b,c). Next, the EC50 and EC90 values (i.e. corresponding to 50% and 90% reduction in viral titer, respectively) were calculated by non-linear regression analysis. An EC50 and EC90 value of 1.3 µM and 3.5 µM was found, respectively (Fig. 1c). In order to calculate the SI, the CC50 value determined via ATPLite assay (156 µM) was divided by the EC50 value. Hence, the SI of tomatidine towards CHIKV infection in Huh7 cells is 120.

In order to further validate the antiviral effect of tomatidine towards CHIKV, we next evaluated its antiviral potential in two other human cell lines relevant during natural CHIKV infection, namely, skin fibroblast HFF-1 cells and bone osteosarcoma U2OS cells28,29,30. Similar to above, we again first determined the cytotoxic potential of tomatidine in both cell lines via the ATPLite assay. In both cell lines, a dose-dependent cytotoxic effect was observed, with a CC50 value of 239 µM in HFF-1 cells and 255 µM in U2OS cells (Supplementary Fig. S2a,b). Thereafter, we performed infection experiments at MOI 5 for HFF-1 cells and MOI 1 for U2OS cells in the presence of increasing tomatidine concentrations (0.5 to 10 µM) as described for the Huh7 cells. A higher MOI was used for HFF-1 cells given the relative lower permissiveness of these cells to CHIKV. In HFF-1 cells, a dose-dependent decrease in the number of infectious CHIKV particles was seen with increasing concentrations of tomatidine, with an EC50 value of 2.3 µM and a SI of 104 (Supplementary Fig. S2c). A similar pattern was observed in U2OS cells and led to an EC50 value of 3.9 µM and a SI of 65 (Supplementary Fig. S2d). Altogether, tomatidine exerts a significant antiviral effect towards CHIKV in all cell lines tested, the strongest effect being measured in Huh7 cells.

To compare the antiviral efficacy of tomatidine to another antiviral compound under our experimental settings, we next performed an antiviral study with naringenin, a natural flavonoid that has been reported to have potent antiviral activity towards CHIKV by Ahmadi et al. in 201624. To this end, infection experiments were performed in Huh7 cells using four different naringenin concentrations (20-150 µM) to determine the approximate EC50 value. At these concentrations, no cytotoxic effect was measured via the ATPLite assay (Supplementary Fig. S3). At the highest naringenin concentration tested, virus particle production was reduced by 77.6% (4.7 ± 0.3 Log for the EtOH control to 4.1 ± 0.2 Log in treated cells) (Fig. 1d). The approximate EC50 value for naringenin surrounds 40 µM. At this concentration, the CHIKV titer is reduced from 4.9 Log PFU (EtOH control) to 4.6 Log PFU, which corresponds to a reduction by 47%. Hence, in our experimental setup using Huh7 cells, tomatidine is more efficacious at lower concentrations than naringenin.

Next, we tested the antiviral effect of tomatidine towards CHIKV strains from other genotypes, namely the originally isolated African (S27) strain from 1953 and a recently isolated Asian CHIKV genotype (99659) in Huh7 cells31,32. For both strains, a strong dose-dependent decrease in virus particle production was observed (Fig. 1e,f). For the Asian strain, the EC50 and EC90 values were 2.0 µM and 3.7 µM, respectively (Fig. 1e). A slightly lower potency was observed towards the African strain, with EC50 and EC90 values of 3.8 µM and 13.8 µM, respectively (Fig. 1f). The respective SI was 78 for the Asian strain and 41.1 for the African strain. Altogether, the above described results demonstrate a potent, dose-dependent antiviral activity of tomatidine towards three distinct CHIKV genotypes in Huh7 cells with comparable EC50 and EC90 values.

Tomatidine has no direct virucidal effect on the CHIKV particle

To test whether the potent antiviral activity of tomatidine towards CHIKV is due to a direct virucidal effect of the compound on the CHIKV particle, 10 µM tomatidine was incubated with 2.5×10 ⁵ PFU of CHIKV either at room temperature or 37 °C for 2 h. Subsequently, the number of residual infectious CHIKV particles were determined via plaque assay on Vero-WHO cells. The lowest sample dilution used in the plaque assay was 1:10, which corresponds to final tomatidine concentration of 1 µM. At this concentration, we did not detect an antiviral effect of tomatidine in Vero-WHO cells (Supplementary Fig. S4) and therefore we can use the plaque assay on Vero-WHO cells as a readout for virucidal activity. Importantly, incubation of tomatidine with CHIKV alone had no effect on the infectious titer in comparison to the EtOH control (Fig. 2). Thus, the antiviral effect of tomatidine is not owed to a direct virucidal effect on the CHIKV particle.

Tomatidine has no direct virucidal effect on CHIKV particles. 2.5 × 10 ⁵ PFU of CHIKV-LR was incubated with 10 μM tomatidine for 2 h at room temperature (RT) or 37 °C. The infectious titer was determined by plaque assay on Vero-WHO cells. Data is represented as mean ± SEM from three independent experiments and differences were assessed with Student's t-test.

Tomatidine reduces CHIKV infection when added up to 6 h post-infection

To gain a better understanding of the kinetics of the antiviral activity of tomatidine, we next performed a time-of-drug-addition experiment. To this end, Huh7 cells were treated with 10 µM tomatidine pre-, during or post-infection (Fig. 3a). For the pre-treatment conditions, Huh7 cells were incubated with tomatidine for 2 or 1 h before infection. Thereafter, cells were washed to remove tomatidine and infection was initiated. In the during condition, tomatidine was added to the cells at the time of infection. At 2 hpi, inoculum was removed, cells were washed extensively and fresh medium without tomatidine was added. In case of the post-treatment conditions, tomatidine was added to the infected Huh7 cells at 2, 3, 4, 6, 7 or 8 hpi. For all conditions, cells were infected at MOI 1 and supernatants were collected at 9 hpi. Each time-point included a corresponding EtOH control. Whereas for the pre- and during conditions tomatidine did not have any inhibitory effect on progeny infectious virus titers, a significant reduction in infectious particle production was seen when the compound was added at post-infection conditions and up to 6 hpi (Fig. 3b). The strongest effect was observed at the 2 hpi treatment, with a reduction in infectious virus titer by 93.7% when compared to the corresponding EtOH control.

Tomatidine reduces CHIKV infection when added up to 6 hpi. ( a) Outline of the experimental set-up. ( b) Huh7 cells were infected with CHIKV-LR at MOI 1 and treated with 10 µM tomatidine or the equivalent volume of EtOH prior to infection (−2 or −1 h), during infection or at different times post-infection (2 to 8 hpi). Supernatants were collected 9 hpi. Data is represented as mean ± SEM from four independent experiments and differences were assessed with Student's t-test.

We next investigated whether tomatidine also has an effect on the production of genome-equivalent CHIKV copies (GEC) secreted by infected Huh7 cells. Hereto, Huh7 cells were infected with CHIKV-LR at MOI 1 and treated with 10 µM tomatidine or EtOH at the time of infection. At 9 hpi, the number of GEC was determined. For the non-treated and EtOH control, 6.8 ± 0.2 and 6.9 ± 0.1 Log GEC were detected, respectively (Fig. 4a). In the presence of tomatidine, the number of secreted GEC was reduced to 5.5 ± 0.2 Log GEC (corresponding to a 95.5% reduction) when compared to the EtOH control (Fig. 4a). Subsequent determination of the infectious titer and calculation of the GEC to PFU ratio revealed that there are no significant differences between the ratios in the absence and presence of tomatidine. In presence of EtOH, the ratio was determined as 356 ± 76 and for tomatidine a ratio of 334 ± 72 was calculated (Fig. 4b). Thus, the antiviral effect of tomatidine is related to a reduction in the overall production of GEC rather than specifically interfering with the infectious properties of secreted viruses.

Tomatidine has no effect on the specific infectivity of CHIKV. ( a) Huh7 cells were infected with CHIKV-LR at MOI 1 and treated with 10 µM tomatidine or the equivalent volume of EtOH at the time of infection. Supernatants were collected at 9 hpi and RT-qPCR was performed to determine GEC/mL. ( b) Ratio of GEC presented in ( a) and corresponding PFU determined via plaque assay. Data is represented as mean ± SEM from three independent experiments and differences were assessed with Student's t-test.

Tomatidine reduces CHIKV infection in Huh7 cells

Based on the findings that tomatidine predominantly acts between 2 and 6 hpi, we next investigated whether tomatidine influences the expression of the CHIKV E2-protein at the cell surface of infected Huh7 cells via flow cytometry. Huh7 cells were infected with CHIKV-LR at MOI 1 and treated with tomatidine or EtOH at the time of infection. In infected cells, the CHIKV E2-protein is translocated to the endoplasmatic reticulum and processed during transit through the secretory pathway prior to expression at the cell surface. Thus, changes within the expression level of E2 at the cell surface in the presence of tomatidine may be indicative for an inhibition of the E2 processing and transport to the cell surface. At 9 hpi, 2.8 ± 0.3% E2-positive cells were observed following infection at MOI 1 (Fig. 5a). At 16 hpi, i.e. after approximately two rounds of CHIKV replication, the number of E2-positive cells increased to 25.4 ± 5.2% (Fig. 5a). Comparable numbers were seen for the corresponding EtOH controls. Importantly, in the presence of tomatidine, the number of E2-positive cells was reduced to 0.8 ± 0.3% and 9.1 ± 2.0% at 9 and 16 hpi, respectively, which corresponds to a reduction by 78.8% (9 hpi) and 67.2% (16 hpi) compared to the EtOH control. Intriguingly, even though a clear reduction in the number of E2-positive cells was seen, a minor effect on the mean fluorescent intensity (MFI) within the E2-positive population was observed (Fig. 5b). At MOI 5, the same pattern was observed (Supplementary Fig. S5). Here, the percentage of infection was reduced by 75.6% (9 hpi) and 53.8% (16 hpi) in the presence of tomatidine, whereas the effect on the MFI was again minor (Supplementary Fig. S5a,b). Comparable results were obtained when cells were permeabilized to allow both intracellular and extracellular staining of E2 (Supplementary Fig. S6a-c). Furthermore, based on the fluorescence intensity measured in both experimental conditions, the vast majority of E2 is expressed at the cell surface at 9 hpi. Taken together, the above results imply that once infection is established, the expression and transport of the E2 protein to the cell surface is mostly unaffected.

Tomatidine reduces the cell surface expression of the CHIKV E2 protein. Huh7 cells were infected with CHIKV-LR at MOI 1 and treated with 10 µM tomatidine or the equivalent amount of EtOH at the time of infection. ( a) Cells were collected, fixed and stained for CHIKV E2 protein on the cell surface at 9 and 16 hpi. ( b) Relative fold changes in MFI in the presence of tomatidine compared to the EtOH control at 9 and 16 hpi. Data is represented as mean ± SEM from three independent experiments and differences were assessed with Student's t-test.

Antiviral activity of tomatidine is sustained for multiple rounds of CHIKV replication

During natural infection, the virus replicates continuously within the body. Therefore, we aimed to investigate for how long tomatidine is able to exert its antiviral activity. To this end, we infected cells with CHIKV-LR at MOI 0.01 in the presence of tomatidine and determined progeny virus particle production at 24 and 48 hpi. These time-points correspond to approximately three and six rounds of CHIKV replication, respectively. For samples collected at 48 hpi, the medium remained unchanged or was replaced with fresh medium containing 10 µM tomatidine or EtOH at 24 hpi. Figure 6 reveals that tomatidine reduces CHIKV particle production for at least three rounds of replication. The viral titer of the non-treated samples reached 5.3 ± 0.2 Log progeny infectious particles at 24 hpi (Fig. 6). A similar titer was seen for the EtOH control. Tomatidine treatment lead to a reduction in CHIKV titer by 87.8% to 4.3 ± 0.3 Log compared to the EtOH control (Fig. 6) at 24 hpi. At 48 hpi, no significant difference in infectious virus particle production could be observed between cells treated with EtOH (7.2 ± 0.3 Log) or tomatidine (6.4 ± 0.3 Log) (Fig. 6). Nevertheless, there was still a trend towards a reduction in virus titer in the presence of tomatidine. Importantly, replenishment of tomatidine at 24 hpi did lead to a significant reduction in infectious titer (95.8%) at 48 h (Fig. 6). Similar results were obtained at MOI 0.1, hence at a ten-times higher virus concentration (Supplementary Fig. S7). Here, tomatidine reduced the CHIKV titer by 85.7% at 24 hpi and by 90.6% at 48 hpi with tomatidine replenishment. Altogether, this suggests that tomatidine is able to effectively control CHIKV replication for at least three replication cycles independent of the viral load. To achieve protection at 48 hpi, a replenishment of tomatidine is necessary.

Anti-CHIKV activity of tomatidine is observed for multiple replication cycles. Huh7 cells were infected with CHIKV MOI 0.01 and treated with 10 µM tomatidine at the time of infection. Supernatants were collected 24 and 48 hpi. Alternatively, the medium was replaced by fresh medium containing 10 µM tomatidine or EtOH after 24 h. Data is represented as mean ± SEM from three independent experiments and differences were assessed with Student's t-test.

Tomatidine derivatives solasodine and sarsasapogenin inhibit CHIKV infection

Testing of structural derivatives of antiviral compounds is a common strategy to enhance their antiviral activity and/or can identify the structural regions of the compound that are relevant for the antiviral activity. We tested three commercially available tomatidine derivatives: tomatine, solasodine and sarsasapogenin for their antiviral effect towards CHIKV-LR in Huh7 cells. The structure of tomatidine and the above derivatives is depicted in Fig. 7a. Based on the cytotoxicity profile (Supplementary Fig. S8a-c), we used a concentration of 5, 5 and 20 µM for tomatine, solasodine and sarsasapogenin in the infectivity assays, respectively. Figure 7b shows that the infectious titer of the non-treated control is 5.02 Log PFU. The EtOH control for each compound showed comparable titers. Unexpectedly however, in presence of CHIKV, tomatine concentrations of 5, 2 and 1 µM lead to a strong cytotoxic effect with extensive cell death through which we were not able to analyze its true antiviral effect. Even at 2 and 1 µM tomatine extensive cell death was observed and therefore we excluded tomatine from further analysis. Solasodine and sarsasapogenin treatment lead to a significant, yet less potent reduction in infectious particle production when compared to the tomatidine control. While for tomatidine-treated cells a reduction to 3.8 Log PFU was observed (92.5% reduction compared to EtOH control), solasodine- and sarsasapogenin-treated cells reduced virus progeny production to 4.5 and 4.2 Log (Fig. 7b). This corresponds to a reduction by 82.5% and 76.4% compared to the EtOH control, respectively. Thus, within the group of tested tomatidine derivatives, tomatidine remains the most potent antiviral compound towards CHIKV.

Solasodine and sarsasapogenin show potent antiviral activity towards CHIKV. ( a) Structures of tomatidine, tomatine, solasodine and sarsasapogenin. ( b) Antiviral effect of tomatidine and its derivatives on infectious particle production following CHIKV-LR infection at MOI 1. Supernatants were collected 9 hpi. Corresponding treatment concentrations of different compounds: Tomatidine 10 µM, solasodine 5 µM, sarsasapogenin 20 µM. Data is represented as mean ± SEM from three independent experiments except for sarsasapogenin, where four independent experiments were performed, and the mean ± SEM from all four experiments is displayed. Differences were assessed with Student's t-test.

Discussion

We have identified the natural steroidal alkaloid tomatidine as a novel antiviral compound towards CHIKV. Tomatidine potently inhibited infection of three different CHIKV genotypes in Huh7 cells. The most potent antiviral effect can be seen towards the CHIKV La Reunion strain, which belongs to the CHIKV East/Central/South African genotype with an EC50 value of 1.3 µM and an SI of 120. For the other tested genotypes, an ancient African genotype and a more recent Asian genotype, EC50 values of 3.8 µM and 2.0 µM were determined, respectively. The corresponding SI are 41.1 (African strain) and 78 (Asian genotype). A direct virucidal effect of tomatidine on the CHIKV particle was excluded. Subsequent time-of-addition experiments demonstrate that the antiviral effect is caused at post-infection conditions and is maintained upon addition of the compound until 6 hpi. Tomatidine did not alter the specific infectivity of CHIKV. Moreover, we showed that tomatidine is able to control CHIKV replication for at least 3 rounds of replication. When testing commercially available structural derivatives of tomatidine, i.e. solasodine and sarsasapogenin, consistent yet slightly less potent antiviral effects towards CHIKV were seen.

The SI is a commonly used parameter in antiviral research to evaluate the specificity of antiviral compounds. The SI index is an adequate general parameter to define the specificity of newly discovered antivirals, however it only gives limited information as it is dependent on the experimental setup, i.e. the MOI and the cell type used. Compared to other reported antiviral compounds towards CHIKV, the SI of tomatidine shows high specificity and potency11,13,33,34. Therefore, tomatidine is a promising antiviral compound to treat CHIKV infection.

At the highest non-toxic concentration of tomatidine (10 µM), a 20-fold reduction in viral progeny was observed. Tomatidine was found to act at post-infection conditions as no antiviral effect was observed for the pre- and during infection treatments. Furthermore, a clear (~4-fold) reduction in the number of E2-positive cells in the presence of tomatidine was seen. However, this cannot completely explain the 20-fold reduction observed in the secretion of virus particles, especially since the cells that were infected with CHIKV in presence of tomatidine had substantial expression levels of viral E2. We therefore hypothesize that tomatidine interferes with multiple processes in the replicative cycle of CHIKV. First, infection is aborted after entry and membrane fusion but prior to E2 protein translation and transportation to the cell surface. Second, tomatidine may act on nucleocapsid formation, virion assembly and/or budding of progeny virions. The mode of action of tomatidine might be dependent on the concentration of the compound within the cells. Future studies should reveal the precise mode of action of tomatidine and whether it acts as a direct or host-directed antiviral compound in controlling CHIKV infection.

Recently, we have also demonstrated that tomatidine has a potent antiviral activity towards all four DENV serotypes and ZIKV but not WNV. Intriguingly, all three viruses belong to the flavivirus genus of the family of flaviviridae, and CHIKV, which is a member of the alphavirus genus of the family togaviridae, is much more distantly related to DENV than DENV to WNV. Interestingly, however, by comparing the results for DENV and CHIKV, similarities can be found. First, for both viruses the most potent antiviral effect is seen when tomatidine is added at 2 hpi. This suggests that for both viruses, an early but post-binding and entry step of the virus replication cycle is targeted by tomatidine. For CHIKV, tomatidine only showed effective protection for the post-treatment condition, whereas for DENV the pre and during treatment also showed a clear, albeit less potent, antiviral effect compared to the post-treatment. Therefore, tomatidine may target an additional, early step of the virus replication cycle in DENV infection. Alternatively, the difference between pre- and during treatment condition may also be explained by the differences in the replication time of DENV (24 hours) and CHIKV (8 hours). In this context, tomatidine may be internalized too slowly to exert its antiviral effect towards CHIKV, but not towards DENV. Furthermore, for both viruses the number of cells expressing the viral envelope protein revealed a potent, but much less pronounced antiviral effect compared to the effect seen on the viral particle production again pointing towards a shared mechanism. The question why we do not see an antiviral effect towards WNV, a virus that is much more closely related to DENV and ZIKV, however, remains to be elucidated.

The two out of three commercially available derivatives of tomatidine, solasodine and sarsasapogenin exhibited a constant but less potent antiviral activity compared to tomatidine. These results imply that structural groups altered in the derivatives may be in fact important determinants of tomatidine activity. Solasodine has an additional double bond within the steroidal ring structure, whereas sarsasapogenin is missing the nitrogen of the spiroaminoketal group. Previous studies on the antibacterial properties of tomatidine show that the two extremities of tomatidine, namely the beta-hydroxyl group and the spiroaminoketal group including the basic nitrogen, are responsible for its antibacterial activity35. The remaining steroidal rings serve as a structural scaffold. Since sarsasapogenin, which misses the basic nitrogen of tomatidine, shows less potent antiviral activity compared to solasodine and tomatidne, the basic nitrogen in the aminoketal group may be important for the antiviral activity of tomatidine towards CHIKV. Furthermore, and in line with Chagnon et al., the double bond within the steroid ring scaffold does not seem to change the antiviral potential of tomatidine. Altogether, these findings suggests that the basic nitrogen may be partly responsible for the antiviral activity of tomatidine. Whether the beta-hydroxyl group also relevant for tomatidine to exert its antiviral effect remains to be evaluated.

In order to further evaluate the potential of tomatidine as an antiviral drug, other important factors including the pharmacokinetic profile, as well as the protein-binding properties of tomatidine have to be taken into account. Unfortunately, to date literature on those aspects is scarce. Tomatidine has been used in several in vivo mouse studies and no toxicity was observed up to a concentration of 50 mg/kg19,36,37,38,39,40. Only one study measured the steady-state tomatidine plasma levels and revealed a plasma concentration of 287 ng tomatidine per mL after 2 month of oral treatment with 0.05% (w/w) tomatidine added to standard chow36. Whereas this study gives some insight into the distribution of tomatidine, further studies are needed to give an in-depth insight into the stability and biodistribution of tomatidine in vivo. With regard to protein-binding properties of tomatidine, there is no literature available that directly demonstrates binding of tomatidine to viral or cellular proteins. Nevertheless, numerous papers have demonstrated the ability of tomatidine to modulate different bacterial or host-cell pathways14,15,40,41. As an example, a study by Boulet et al. in 2017, demonstrated that tomatidine inhibits the Staphylococcus aureus ATP Synthase subunit C to exert its antibacterial properties17. Moreover, tomatidine has been shown to inhibit cellular ATF4 expression, which leads to a reduction in age-related muscle weakness and atrophy36. The ability of tomatidine to control ATF4 expression has also been shown by our recent publication from 2019, though this did not explain the antiviral activity of tomatidine towards DENV21. Collectively, despite the numerous functions of tomatidine further studies characterizing the pharmacokinetic profile as well as the protein binding properties of tomatidine are needed to further evaluate tomatidine as a potent antiviral drug.

Our current in vitro findings identify tomatidine as a promising antiviral compound to treat CHIKV infection. Toxicity profiles, time-of-addition studies and durability experiments demonstrate a potent and robust antiviral activity. Tomatidine shows a potent antiviral effect when added up to 6 hpi, which is rare among the currently identified potential antiviral compounds towards CHIKV. Nevertheless, further studies regarding the efficacy in vivo and the pharmacokinetics of tomatidine are essential to further evaluate its potential as an antiviral compound. Aside from the ability of tomatidine to inhibit CHKV infection, its reported anti-inflammatory activities as well as interferon-stimulating effects may also be of importance as this may alleviate the symptoms associated with CHIKV fever15,38.

Methods

Cell culture

The human hepatocarcinoma cell line Huh7 (JCRB0403), kindly provided by Tonya Colpitts (University of South Carolina) and the human bone osteosarcoma cell line U2OS (ATCC HTB-96) were maintained in Dulbecco's minimal essential medium (DMEM) Glutamax (Gibco, The Netherlands) supplemented with 10% FBS (Lonza, Basel, Switzerland), 100 U/mL penicillin and 100 mg/mL streptomycin (PAA Laboratories, Pasching, Austria). The human skin fibroblast cell line HFF-1 (ATCC SCRC-1041) was maintained as described above, with the exception that the medium was supplemented with 15% FBS. Vero-WHO cells, renal cells derived from the African Green monkey, (WHO Reference Cell Bank 10-87, ATCC CCL-81) were maintained in DMEM containing 5% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. All cell lines were cultured at 37 °C and 5% CO ₂.

Virus stocks and titration

CHIKV (La Reunion OPY1) was a kind gift from A. Merits (University of Tartu, Estonia). CHIKV strain S27 was kindly provided by S. Guenther, Bernhard-Nocht-Institute for Tropical Medicine and was isolated in 1953 in Tanzania. CHIKV strain 99659, an Asian genotype isolated in 2014 from the British Virgin Islands in the Caribbean was a kind gift from M. Diamond (Washington University, School of Medicine). For virus production, Vero-WHO cells were infected at a multiplicity of infection (MOI) of 0.01 and progeny virions were harvested at 46 h post-infection (hpi). Subsequently, the virus-containing supernatants were centrifuged to clarify from cell debris, aliquoted and stored at −80 °C. Infectious virus titers were determined by plaque assay on Vero-WHO cells and defined as the number of plaque forming units (PFU) per mL. Briefly, Vero-WHO cells were seeded at a density of 13×10 ⁴ cells/well in a 12-well plate. At 24 h post-seeding, cells were infected with 10-fold serial dilutions of the sample in duplo. At 2 hpi, wells were overlaid with 1% seaplaque agarose (Lonza, Swiss) prepared in 2x MEM. Plaques were counted at 44 hpi. The number of genome-equivalent copies (GEC) per mL was determined via Q-RT-PCR using a StepOne Real-Time PCR instrument (Applied Biosystems, Foster City, CA, USA), as described previously42. RNA extraction was performed using a QIAmp viral RNA mini kit (QIAGEN, Venlo, The Netherlands). cDNA synthesis from viral RNA was performed using Omniscript (QIAGEN) and the primers 5′AGCTCCGCGTCCTTTACCA-3′ (forward) and 5′-GCCAAATTGTCCTGGTCTTCCT-3′ (reverse). For qPCR the TaqMan probe 5′-FAMCACTGTAACTGCCTATGCAAACGGCGAC-TAMRA-3′ (Eurogentec, Maastricht, The Netherlands) was used. DNA amplification was performed for 40 cycles of 15 s at 95 °C and 60 s at 60 °C. The number of GEC was determined using a standard curve (correlation coefficient >0.995) of a quantified cDNA plasmid containing the CHIKV E1 sequences (pCHIKV-LS3 1B).

Chemicals

Tomatidine hydrochloride, solasodine, and sarsasapogenin were purchased from Sigma Aldrich (St. Louis, Missouri, USA) and dissolved in absolute ethanol (EtOH) to a final concentration of 5 mM. Tomatine (Santa Cruz Biotechnology) was prepared in absolute EtOH to a final concentration of 2.5 mM. Naringenin was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA) and dissolved in absolute EtOH to a final concentration of 50 mM. All stock solutions were stored at −20 °C and used for a maximum of 3 months. The final EtOH concentration was below 0.1% in all infection experiments.

Cytotoxicity assay

The cytotoxicity of tomatine, tomatidine, solasodine and sarsasapogenin was determined in vitro via an ATPLite Luminescence Detection assay system. Huh7, U2OS, HFF-1 or Vero-WHO cells were seeded in a white polystyrene 96-well plate at a density of 8.0×10 ³. At 24 h post-seeding, cells were treated with increasing concentrations of the different compounds ranging from 1 to 500 µM. At 24 h post-treatment, 50 µL mammalian cell lysis solution was added per well and the plate was incubated for 5 min at room temperature on an orbital shaker. Subsequently, 50 µL substrate solution was added to wells and the plate was incubated for 5 min, as before. Then, the plate was incubated for 10 min in the dark. Luminescence was measured with a microplate reader (Biotek, Sinergy, HT, Vermont, USA). Cytotoxicity was expressed as follows:

$$ \% \,{Cytotoxicity}=\frac{({\rm{Abs}}\,{\rm{sample}})-({\rm{Abs}}\,{\rm{blank}})}{({\rm{Abs}}\,{\rm{negative}}\,{\rm{control}})-({\rm{Abs}}\,{\rm{blank}})}\ast 100$$

For tomatidine, cytotoxicity in Huh7 cells was also measured via determining the total cell number. To this end, Huh7 cells were seeded in a 12-well plate at a density of 1.2 × 10 ⁵ cells/well. At 24 h post-treatment, cells were trypsinized with 1x Trypsin/EDTA (Gibco), stained with trypan blue (Sigma) and counted manually using a Buerker counting chamber depth 0.100 mm (Marienfeld, Lauda-Koenigshofen, Germany).

Additionally, the cytotoxic effect at the highest non-toxic concentration of tomatidine (10 µM) was evaluated in Huh7 cells in the presence of CHIKV via the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (MTS assay). To this end, Huh7 cells were seeded in a 96-well plate as described above and infected with CHIKV-LR at MOI 1. At the time of infection, cells were treated with 10 µM tomatidine or the equivalent volume of EtOH. At 24 h post infection, 20 µL MTS/PMS solution was added per well and the plate was further incubated for 2 h at 37 °C. Subsequently, 10% formaldehyde was added to inactivate the virus and the absorbance was measured at 490 nm with a microplate reader. Cytotoxicity was expressed as described for the ATPLite assay. Values were then displayed as percentage compared to a non-infected, non-treated control.

Antiviral assays

Vero-WHO, Huh7, U2OS and HFF-1 cells were infected with CHIKV at MOI 0.5, 1 or 5. Unless indicated otherwise, tomatidine, solasodine, sarsasapogenin or naringenin (or the equivalent volume of ethanol) were added at the time of infection. The virus inoculum was removed at 2 hpi, after which the cells were washed three times, and compound-containing medium was added until sample collection. For the time-of-addition experiments, Huh7 cells were treated with tomatidine either pre-, during or post-infection. For the pre-treatment, 10 µM tomatidine was added 1 or 2 h before infection, as indicated. At the time of infection, cells were washed three times before the addition of the virus inoculum. For the during condition, tomatidine was added together with the virus inoculum and was present for only for 2 h. For the post-treatment condition, tomatidine was added at 1, 2, 4, 6, 7 and 8 hpi. In all experiments, the virus inoculum was removed after two hours and the supernatant was collected at 9 or 16 hpi.

Virucidal effect

CHIKV-LR (2.5×10 ⁵ PFU) was incubated in the presence or absence of 10 µM tomatidine or the equivalent volume of ethanol at room temperature or 37 °C for 2 h in a total volume of 250 µL. Subsequently, the infectious virus titer was determined via plaque assay.

Flow cytometry

Huh7 cells were trypsinized with 1x Trypsin/EDTA (Gibco) and fixed with 2% paraformaldehyde. After fixation, the cells were stained with a rabbit anti-E2-stem antibody (obtained from G. Pijlman, Wageningen University, Wageningen, The Netherlands) diluted 1:1000 and a AF647 conjugated chicken anti-rabbit antibody diluted 1:300 (Life Technologies, Carlsbad, California, USA). Flow cytometry analysis was performed with a FACSCalibur cytometer (BD Biosciences) and analysis was performed via Kaluza 1.1.

Durability study

Huh7 cells were infected with CHIKV-LR OPY1 at MOI 0.01 or MOI 0.1 and treated with 10 µM tomatidine or EtOH, as described in the antiviral assay section. Supernatants were collected at 24 or 48 hpi. For the samples collected at 48 hpi, the medium remained unchanged after infection or was replaced by fresh complete medium containing 10 µM tomatidine or EtOH at 24 hpi. Virus titer was determined via plaque assay.

Statistical analysis

The EC50 and EC90 values represent the tomatidine concentration that reduces virus particle production by 50 or 90%, respectively. The CC50 and CC90 values, represent the tomatidine concentration that causes 50 and 90% cytotoxicity, respectively. The corresponding dose-response curves were fitted by non-linear regression analysis using a sigmoidal model. The calculated selectivity index (SI) represents the ratio of CC50 to EC50. All data was analyzed with GraphPad Prism (La Jolla, CA, USA) and data are presented as mean ± SEM. Statistical differences were evaluated via Student's t-test, a value of p ≤ 0.05 was considered significant, with *p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.

Acknowledgements

B.T. was supported by the Graduate School of Medical Sciences of the University of Groningen and a grant from the de Cock Hadders Stichting. L.M.M., M.D.T., I.A.R.Z. and J.M.S. were supported by the University Medical Center Groningen. The corresponding funders had no involvement in the design of the study, collection and analysis of data, publication decisions or manuscript preparation. The University Medical Center Groningen has filed a patent application involving this research. Inventors on this application are B.T., M.D.T. and J.M.S.

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Author notes

Mayra Diosa-Toro

Present address: Programme in Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, 169857, Singapore

Authors and Affiliations

Department of Medical Microbiology and Infection Prevention, University of Groningen; University Medical Center Groningen, Groningen, the Netherlands

Berit Troost, Lianne M. Mulder, Mayra Diosa-Toro, Denise van de Pol, Izabela A. Rodenhuis-Zybert & Jolanda M. Smit

Contributions

The project was supervised by J.M.S. and I.A.R.Z. J.M.S., B.T. and M.D.T. designed the study. B.T. prepared all figures and wrote the manuscript. Experimental work of Figures 2 and 3 was performed by L.M.M. ATPLite assays were performed by M.D.T., L.M.M. and D.V.P. All authors reviewed the manuscript.

Corresponding author

Correspondence to Jolanda M. Smit.

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Troost, B., Mulder, L.M., Diosa-Toro, M. et al. Tomatidine, a natural steroidal alkaloid shows antiviral activity towards chikungunya virus in vitro. Sci Rep 10, 6364 (2020). https://doi.org/10.1038/s41598-020-63397-7
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Tomatidine, a natural steroidal alkaloid shows antiviral activity towards chikungunya virus in vitro - Scientific Reports

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In recent decades, chikungunya virus (CHIKV) has re-emerged, leading to outbreaks of chikungunya fever in Africa, Asia and Central and South America. The disease is characterized by a rapid onset febrile illness with (poly)arthralgia, myalgia, rashes, headaches and nausea. In 30 to 40% of the cases, CHIKV infection causes persistent (poly)arthralgia, lasting for months or even years after initial infection.

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This article talks about strong correlations between disease incidence and climatic variables, socio-economic indicators, and highlights the complex interactions among arboviruses.

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Jasmine Morgan,

Clare Strode,

J. Enrique Salcedo-Sora

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Abstract

Dengue, Zika and chikungunya are diseases of global health significance caused by arboviruses and transmitted by the mosquito Aedes aegypti, which is of worldwide circulation. The arrival of the Zika and chikungunya viruses to South America increased the complexity of transmission and morbidity caused by these viruses co-circulating in the same vector mosquito species. Here we present an integrated analysis of the reported arbovirus cases between 2007 and 2017 and local climate and socio-economic profiles of three distinct Colombian municipalities (Bello, Cúcuta and Moniquirá). These locations were confirmed as three different ecosystems given their contrasted geographic, climatic and socio-economic profiles. Correlational analyses were conducted with both generalised linear models and generalised additive models for the geographical data. Average temperature, minimum temperature and wind speed were strongly correlated with disease incidence. The transmission of Zika during the 2016 epidemic appeared to decrease circulation of dengue in Cúcuta, an area of sustained high incidence of dengue. Socio-economic factors such as barriers to health and childhood services, inadequate sanitation and poor water supply suggested an unfavourable impact on the transmission of dengue, Zika and chikungunya in all three ecosystems. Socio-demographic influencers were also discussed including the influx of people to Cúcuta, fleeing political and economic instability from neighbouring Venezuela. Aedes aegypti is expanding its range and increasing the global threat of these diseases. It is therefore vital that we learn from the epidemiology of these arboviruses and translate it into an actionable local knowledge base. This is even more acute given the recent historical high of dengue cases in the Americas in 2019, preceding the COVID-19 pandemic, which is itself hampering mosquito control efforts.

Author summary

Viruses transmitted by Ae. aegypti mosquitoes (dengue, Zika, chikungunya) are amongst the most significant public health concerns of recent years due to the increase in global cases and the rapid spread of the vectors. The primary method of controlling the spread of these arboviruses is through mosquito control. Understanding factors associated with risk of these viruses is key for informing control programmes and predicting when outbreaks may occur. Climate is an important driver in mosquito development and virus reproduction and hence the association of climate with disease risk. Socio-economic factors contribute to perpetuate disease risk. Areas of high poverty have abundance of suitable habitat for Ae. aegypti (e.g. due to poor housing and sanitation). This study investigated the factors affecting arbovirus incidence in three distinct regions of Colombia: Bello, Cúcuta and Moniquirá. The results show significant relationships between disease incidence and temperature, precipitation and wind speed. A decline in dengue following outbreaks of Zika (2016) is also reported. Measures of poverty, including critical overcrowding and no access to improved water source were also found to be higher in areas of higher disease incidence. The results of this study highlight the importance of using a multifactorial approach when designing vector control programs in order to effectively distribute local health care resources.

Citation: Morgan J, Strode C, Salcedo-Sora JE (2021) Climatic and socio-economic factors supporting the co-circulation of dengue, Zika and chikungunya in three different ecosystems in Colombia. PLoS Negl Trop Dis 15(3): e0009259. https://doi.org/10.1371/journal.pntd.0009259

Editor: Rebecca C. Christofferson, Louisiana State University, UNITED STATES

Received: September 4, 2020; Accepted: February 20, 2021; Published: March 11, 2021

Copyright: © 2021 Morgan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: Funding for this study was provided by Edge Hill University's Graduate Teaching Assistant scheme (JM) ( https://www.edgehill.ac.uk/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Vector-borne diseases are one of the most significant public health burdens globally, with 80% of the total world population at risk [ 1]. Arboviruses, including dengue, Zika and chikungunya, are of particular concern due to the recent increase in global cases promoted by the rapid spread of both their primary mosquito vector Aedes aegypti as well as their secondary vector Aedes albopictus [ 2]. Dengue infection can be asymptomatic but clinical presentations range from mild dengue fever (DF), a febrile illness similar to influenza, to the severe forms of dengue; dengue shock syndrome (DSS) and dengue haemorrhagic fever (DHF) [ 3]. Most Zika (ZIKV) infections are asymptomatic, with only approximately 20% of infections causing symptoms [ 4, 5]. The clinical presentations of symptomatic ZIKV can include Zika fever, congenital Zika syndrome and Guillain-Barré syndrome. Congenital Zika syndrome refers to a group of birth defects, notably Microcephaly, which have been associated with ZIKV infection during pregnancy [ 6, 7]. Infection with the chikungunya virus (CHIKV) is characterised by sudden onset fever, rash and arthralgia [ 8]. Joint pain associated with CHIKV is debilitating and whilst typically lasting a few days can last for many months or even years [ 9].

Dengue causes an estimated 390 million infections per year and has a distribution that covers every continent of the world with the exception of Antarctica [ 10]. The number of global dengue cases reported to the WHO has increased 15 fold over the last 20 years, with deaths also seeing a significant increase (4-fold) [ 11]. The first epidemics of ZIKV were reported in Yap, Micronesia (2007) and French Polynesia (2013); outbreaks were reported in Brazil in 2015 and 2016 which then led to a rapid spread of ZIKV to 48 countries within the Americas and the Caribbean [ 12]. ZIKV epidemics have also been reported in Singapore [ 13], Vietnam [ 14], Thailand [ 15] and Cape Verde [ 16]. CHIKV was first reported in Tanzania in 1952 and has since rapidly spread across the globe causing sporadic and significant epidemics in Asia, India, Europe and The Americas [ 17]. The epidemiology of CHIKV is notable due the sporadic patterns of outbreaks, likely caused by introduction of the virus into urban environments from the sylvatic cycle with nonhuman primates the most likely major reservoir host [ 18- 20].

Spreading from its ancestral home in West Africa 400-500 years ago via the slave trade Ae. aegypti is found in tropical and sub-tropical regions [ 21]. Meteorological conditions directly influence the incidence of arboviruses by modulating vector mosquito populations. Conditions favourable to Ae. aegypti include an ideal temperature range between 20-35°C for mosquito development, fertilisation and vector competence [ 22]. Increases in temperature also increase viral replication rates within Ae. aegypti, increasing viral load and hence reducing extrinsic incubation periods and increasing transmission [ 23]. Non-climatic factors promoting Ae. aegypti populations include vegetation index, urbanisation and accessibility to human populations [ 24, 25]. Latin America is significantly affected by Ae. aegypti borne viruses due to its habitat suitability for the vector, tropical climate and often limited medical resources and vector control programmes [ 26, 27]. Colombia, located in the north-west corner of South America, has 140,612 km ² of suitable habitat for Ae. aegypti throughout the country based on the presence of climatic characteristics [ 28]. Ae. aegypti in Colombia has been found at altitudes up to 2,300 m above sea level [ 29]. The presence of Ae. aegypti across Colombia is mirrored by a high nationwide incidence of dengue, Zika and chikungunya. Dengue has been consistently reported in Colombia over the past two decades causing an average of 84,926 cases each year (1980-2019). Zika was first reported in Colombia in 2015 and was followed by a significant outbreak of 91,711 cases in 2016. Chikungunya was first detected in Colombia in 2013, causing 275,907 cases in that single year [ 30]. Colombia is now hyperendemic for dengue [ 31] as well as endemic for both Zika and chikungunya [ 32].

In addition to climatic variables, socio-economic (SE) factors can contribute to the spread of mosquito borne diseases. This is particularly acute with Ae. aegypti, a highly anthropophilic species which lives within or in close proximity to human dwellings breeding in domestic water storage containers. Poor housing construction together with high population density and inadequate sanitation with little to no access to clean running water are key SE factors promoting Ae. aegypti populations [ 33]. In the absence of suitable vaccines for dengue, Zika or chikungunya, disease prevention is currently based on Ae. aegypti control. This is challenging with a diurnal biting mosquito. A compound effect is the development of insecticide resistance in populations of Ae. aegypti reported in areas of Colombia [ 34].

This study aims to investigate the epidemiology of these three arboviruses (dengue, Zika and chikungunya) co-circulating in a single vector species ( Ae. aegypti) in three distinct eco-systems in Colombia between 2007-2017. In addition to having differing climatic and socio-economic profiles the three locations selected as our study domain-Bello, Cúcuta and Moniquirá - have contrasting levels of Ae. aegypti circulation [ 35- 38]. There is also a higher prevalence of insecticide resistance in Ae. aegypti from Cúcuta, specifically to common larvicides (i.e. temephos) used in vector control interventions for disease prevention [ 37]. The national vector interventions monitored by the National Insecticide Resistance Surveillance Network (Colombia's National Institute of Health: INS) [ 39], following WHO recommendations (WHO) [ 40], continue to yield mixed local outcomes in control of mosquito populations and arboviral diseases [ 41- 43]. We sought to assemble and model comprehensive sets of data for this recent 11-year period to investigate the climatological as well as the socio-demographic traits in these three locations that might correlate with disease prevalence-a proxy for levels of vector circulation. This is with the aim to elucidate the influencers from local ecosystems that could ultimately dictate the efficacy of vector and vector borne disease interventions. We used multifactorial approaches of several meteorological and socio-economic factors with disease incidence. We show that specific climatological factors are strong drivers for these arboviral diseases to which contextual socio-economical characteristics can act as modifiers. Importantly, we find a discriminatory pattern between these three diseases highlighting unexpected dynamics of transmission between Zika and dengue particularly in an area of high dengue circulation.

Methods

Study locations

Three study municipalities: Bello, Cúcuta and Moniquirá ( Fig 1) were chosen due to their geographical separation, and distinct climate characteristics, demographics and burden of Ae. aegypti borne diseases ( Table 1).

Disease incidence data

Cases of dengue, severe dengue (dengue shock syndrome and dengue haemorrhagic fever), chikungunya and Zika reported in each epidemiological week were obtained for the period of 2007 to 2017 from SIVIGILIA (National Public Health Surveillance System, Colombian National Institute of Health) [ 46]. This period of study (2007-2017) had consistent reporting of dengue along with the epidemics of both Zika and chikungunya. Chikungunya was first reported in 2014 and Zika in 2015. For the purpose of analysis week 53 was removed from any years in which there were 53 epidemiological weeks (2008 and 2014) this ensured continuity across the data set with all years comprising of 52 weeks when analysed. Only cases confirmed by SIVIGILIA were used in this analysis, confirmations are made based on laboratory tests and epidemiological links. The disease incidence data used in this study is provided in detail in S1 Table.

Climate data

Daily weather data for Bello, Cúcuta and Moniquirá were obtained from the NASA Langley Research Center (LaRC) POWER Project [ 47]. The meteorological data has a resolution of 0.5 o latitude and 0.5 o longitude, with data taken from 6.3367 o latitude and -75.5596 o long for Bello, 7.8891 o latitude and -72.4967 o for Cúcuta and 5.879 o latitude and -73.5736 o longitude for Moniquirá. The weather data was obtained for the same time period as the disease incidence data (2007-2017) and converted to epidemiological weeks to correspond with the dates of the disease incidence data. The weather variables included for each municipality were: maximum temperature (Tmax), minimum temperature (Tmin), average temperature (Tavg), maximum wind speed (WSmax), minimum wind speed (WSmin), average wind speed (WSavg), total precipitation and average humidity (Havg). The climate data used in this study is provided in detail in S1 Table.

Population and socio-economic data

Population data for each municipality were obtained using population projections by Colombia's National Administrative Department of Statistics (Departamento Administrativo Nacional de Estadística) (DANE) ( www.dane.gov.co) [ 45]. The multidimensional poverty index (MPI) was implemented by the Oxford Poverty and Human Development Initiative and the United Nations Development Program's Human Development Report Office as a direct method for measuring poverty [ 48]. The MPI at municipality level was obtained from DANE using data collected in the 2018 National Population and Housing Census and using the indicators and respective weightings listed in S2 Table [ 49]. The overall multidimensional poverty index for each study municipality is shown in Table 2 along with the values for each socio-economic indicator in each municipality. The socioeconomic variables included in the MPI calculation and their specific interpretation within Colombia are explained in S2 Table. The overall MPI calculations can be interpreted as higher values indicating higher proportion of the population in poverty. Households with values of > 33.3% in any indicators are classed as poor.

Statistical analysis

Patterns of disease incidence by location.

Differences in the total burden of all three Ae. aegypti borne viruses as well as the individual burden of dengue and severe dengue were investigated using the total number of cases from 2007-2017. Poisson Generalised Linear Models (GLMs) were initially carried out because they allow for examination of non-linear data with response variables that are not normally distributed [ 50], revealing overdispersion (data variance greater than expected for the given model) statistics of 545, 542 and 123 for total disease, dengue and severe dengue respectively. To correct for the large overdispersion the GLMs were recalculated with negative binomial errors [ 51] using the glm.nb function of the R package MASS [ 52]. Differences between all three municipalities were tested with Tukey pairwise comparisons using the glht function of the multcomp R package [ 53]. Incidence of chikungunya in 2015 was initially modelled using a Poisson GLM which revealed an overdispersion statistic of 2.9. The standard errors were therefore corrected using quasi-GLMs where the variance was theta x mu. Where mu was the mean of the dependant variable and theta the dispersion parameter of the quasi-GLMs [ 51]. Quasi-GLMs were conducted using the glm function from the R package stats [ 54]. Zika incidence was modelled for the year 2017 only, initially a Poisson GLM was used and an overdispersion statistic of 99 was detected. As the overdispersion statistic was above 20 it was corrected for using negative binomial errors [ 51]. Total Ae. aegypti borne disease and dengue incidence were also modelled for 2015 and 2016 using quasi-GLMs to account for low level overdispersion except for total disease in 2016 which had a dispersion statistic of 90 and was therefore modelled with a negative binomial GLM. Population was used as an offset in all models in order to standardise disease incidence by population size.

Patterns of disease incidence over time.

For the pattern of disease over time we used total yearly incidence data. Poisson GLMs were initially used to model each disease in each municipality but overdispersion was detected in some models, hence error distributions were adjusted accordingly. For Bello incidence of both total disease (dengue, severe dengue, chikungunya and Zika) and dengue alone were modelled using quasipoisson GLMs, correcting for overdispersions of 3.69 and 3.63 respectively. For Cúcuta a negative binomial GLM was required for total disease incidence in order to correct for overdispersion of 32.52 and quasi-GLM was used for dengue incidence due to slight overdispersion of 4.87. Severe dengue incidence in Cúcuta was not found to be significantly overdispersed when modelled with a Poisson GLM (overdispersion statistic = 1.79), as the overdispersion statistic was <2. For Moniquirá both total disease incidence and dengue incidence alone were modelled using quasi-GLMs, correcting for respective overdispersion statistics of 2.39 and 2.34 [ 51]. Population was used as an offset in all models in order to standardise disease incidence by population size. All quasi-GLMs were conducted using the glm function from the R package stats [ 54] and negative binomial GLMs used the glm.nb function of the R package MASS [ 52]. Differences between the years were tested with Tukey pairwise comparisons using the glht function of the multcomp R package [ 53].

Patterns of climate over time and between municipalities.

Patterns in climate over time in each municipality were visualised by fitting the data with a non-parametric approach (locally weighted scatterplot smoothing) as implemented in R's ggplot2 loess (local polynomial regression fitting) method. Local fitting uses the distance of data in the neighbourhood of each dependent variable (time) to weight the least squares of the independent variable (climate variable quantitative measurement). The size of the neighbourhood is controlled by the ggplot's span parameter in geom_smooth or stat_smooth. The default span applied here uses tricubic weighting (proportional to (1 − (distance/max distance)^3)^3) [ 55].Tukey's Honest Significant difference method was used following an ANOVA to test for differences in each climate variable between the study municipalities using the R package stats [ 54].

Generalised additive models.

The correlations between climatic variables and the total disease incidence (dengue, severe dengue, chikungunya and Zika) across all three locations were investigated using a generalised additive model (GAM). The weekly disease incidence and weather data for each municipality was converted into 4-week data, matching the dates of epidemiological months. Combining the data into 4-week periods rather than individual weeks reduced zero inflation improving the reliability of the GAM outputs. All climate variables were lagged by plausible time lags for their effect on disease incidence, of 4 and 8 weeks. Square root transformations were used for total disease incidence and each weather variable due to non-normal distribution. Generalised additive models were chosen due to their ability to model non-linear relationships between a response variable (disease incidence) and multiple explanatory variables (climate variables) [ 56]. A quasi-maximum likelihood Poisson GAM was used in order to prevent possible overdispersion [ 51]. Population size was used as an offset to standardise disease incidence by population. Initially all climate variables with both 4 and 8-week time lags were assumed to have a non-linear relationship and were therefore modelled as smoothed terms. Subsequent analysis of the effective degrees of freedom ( edf) was used to identify variables with edf = 1.0, suggesting linearity. These variables were then included in the model as linear rather than smoothed terms. Generalised cross validation (GCV) was used to determine the most appropriate model. Generalised additive modelling and subsequent model validation was conducted using the R package mgcv [ 57]. Visualisation of GAM estimations were conducted using the mgcViz R package [ 58].

Socio-economic factors.

Principle components analysis (PCA) was used for dimensional reduction to allow the inclusion of socio-economic data with previously compiled geographic and climate data. The PCA was conducted using the R package stats [ 54] and visualisations of the PCA were created using the factoextra R package [ 59].

Results

General disease incidence between 2007-2017

Dengue was the most prevalent of the three diseases throughout Colombia ( Fig 2 and S3 Table). Cúcuta carried the highest disease burden of the three municipalities followed by Moniquirá, with Bello having the lowest disease incidence. The total number of confirmed cases of all Ae. aegypti borne diseases (dengue, severe dengue, chikungunya and Zika) during the period of 2007-2017 were 5,727, 32,328 and 1,005 in Bello, Cúcuta and Moniquirá respectively. The breakdown of cases per 100,000 people of all three diseases between 2007 and 2017 in these locations were 1,301, 6,094 and 4,669 in Bello, Cúcuta and Moniquirá, respectively. When discriminating by disease per 100,000 people, the incidence of dengue was 1,263 in Bello, 5,106 in Cúcuta and 4,566 in Moniquirá. Chikungunya had 27 cases in Bello, 154 in Cúcuta and 61 Moniquirá. Incidence of Zika was lowest in Bello with 11 cases compared to 834 in Cúcuta and 42 in Moniquirá.

For visualisation of the spread of disease data over the study period, 4-week smooth moving averages (SMA) were applied ( Fig 3). SMA were only used for data visualisation ( Fig 3) and not during the analysis of the disease data. Dengue and severe dengue were consistently reported throughout the period of 2007-2017. Dengue data showed at least three spikes: one in 2009 ( Fig 3), followed by two more by the end of 2014 and beginning of 2017.The latter of these peaks started approximately two years prior (2015) ( Fig 3). Chikungunya cases were only reported between 2014-2017, hence Fig 3 shows chikungunya cases for this time period only. This was similar for Zika, whose first cases were reported in 2015, with the highest number of cases reported in week 468 in 2016 ( Fig 3). There were no obvious seasonal patterns in disease incidence reported across the three municipalities ( Fig 3), due to the close location of the study municipalities to the Equator. However the major peaks depicting the chikungunya (2015) and Zika (2016) outbreaks in Cúcuta (2015) both occurred within the early part of the year, with cases beginning to increase from the last few months of the previous year, this is also true of the spike in severe dengue in Cúcuta in 2010. Whilst Colombia is an Equatorial country and does not experience distinctive seasonal patterns in climate incidence, the spike in severe dengue in Cúcuta in 2010 corresponds to higher than average temperatures as identified by analyses of meteorological characteristics conducted in this paper. The timings of the chikungunya and Zika peaks do not however correspond to any deviations in meteorological characteristic averages and are more likely due to the timing of importation of the viruses into the local areas.

Patterns of disease incidence per location

Total disease incidence over the 11-year period was significantly lower in Bello than in Cúcuta (p = < 0.001) and Moniquirá (p = 0.005) ( Fig 4A) for all three diseases. The number of dengue cases were similarly high between Moniquirá and Cúcuta ( p = 0.99). However, severe dengue incidence was significantly different across all three municipalities: Cúcuta had the highest burden of severe dengue when compared to both Bello (p = <0.001) and Moniquirá (p = 0.005), and Moniquirá had a significantly higher burden of severe dengue when compared to Bello (p = 0.047) ( Fig 4A).

The incidence data were also analysed separately for the years when the outbreaks of chikungunya and Zika were reported, 2015 and 2016, respectively ( Fig 4B and 4C). This allowed for a more directly and meaningful comparison of the burden represented by these three diseases. Cases of chikungunya in 2015 were not significantly different between any of the municipalities ( Fig 4B). However, Cúcuta had significantly higher incidence of Zika than both Bello (p = <0.001) and Moniquirá (p = <0.001) ( Fig 4C). Interestingly, in the same year of the Zika outbreak (2016) each municipality had a significantly different number of dengue cases. Cúcuta had the lowest incidence of dengue, and Moniquirá the highest ( Fig 4C). While Cúcuta had the highest incidence of dengue in previous years, in 2016 the same location experienced the lowest incidence of dengue accompanied by the highest incidence of Zika ( Fig 4C).

Patterns of disease incidence during 2007 to 2017

We compared the number of cases per 100,000 people in each year for dengue, severe dengue, chikungunya and Zika from 2007 to 2017 in Bello, Cúcuta and Moniquirá ( Fig 5). The initial cases of Zika were first confirmed in Colombia in 2015, with cases reported in both Bello and Cúcuta from that year onwards. The first Zika case in Moniquirá was not confirmed until 2016. Whilst cases of Zika were relatively low in Bello and Moniquirá, Cúcuta experienced large outbreaks ( Fig 5).

The data analysed per location ( Fig 4) and per year ( Fig 5) suggested that Zika displaced dengue in Cúcuta from 2015 to 2016. The number of Zika cases per 100,000 population in 2015 were 125 and 705 in 2016 whilst dengue was present in 313 and 150 cases, respectively ( Fig 5B). On the other hand, in Bello and Moniquirá, where the incidence of Zika was much lower (Bello; 1 in 2015 and 3 in 2016, Moniquirá; 0 in 2015 and 42 in 2016), there was an incremental trend for dengue during this same transition from 2015 to 2016 ( Fig 5A and 5C). Following the significantly high dengue incidence in Bello and Moniquirá in 2016, the incidence stabilised, and the incidence reported in 2017 were not statistically different to that of years prior to 2016. This was not the case however in Cúcuta where incidence of dengue continued to fall, with the lowest incidence of the study period observed in 2017 ( Fig 5B). In 2017 incidence of Zika was also much lower with only 3 cases per 100,000 people reported in Cúcuta in 2017 ( Fig 5A and 5C).

Effect of climate on disease incidence

The geographical settings for the three locations studied here Bello, Cúcuta and Moniquirá ( Table 1) determine three different climate systems-ecosystems. These three different ecosystems are expected to establish contrasting behavioural patterns for the mosquito vector species that transmit dengue, Zika and chikungunya. The results presented in this section related to the quantitative climate variables as explained in Methods and inferred here to be proxy determinants of disease transmission by Ae. aegypti. We initially summarised the climate variables of each municipality from 2007-2017 using LOESS to show the patterns over time ( Fig 6). All parameters used here for temperature (Tmax, Tmin, Tavg) and wind speed (WSmax, WSmin, WSavg) were highest in Cúcuta. Bello had the lowest wind speed and highest relative humidity (Havg) and precipitation. There was a large fluctuation of 8°C in Tmax and 5°C in Tavg in 2010 in all three locations ( Fig 6). Otherwise the recorded climatic variables fluctuate within a similar range throughout these 11 years. The variability between municipalities was greater than within and therefore granted the GAMs as applied in this study. We found that the climate experienced by the three municipalities was significantly different (p = <0.001) over this 11-year period for all the climate parameters included in this study ( S4 Table).

We explored potential relationships between the time series climate data and total disease incidence in Bello, Cúcuta and Moniquirá at shorter 4 and 8-week time lags using a generalised additive model (GAM) as explained in Methods. The estimates from the quasipoisson GAM explained 57.6% of the variance in total disease incidence over time ( Table 3). Effective degrees of freedom ( edf) close to 1 represent relation close to linearity while high edf values for the smooth terms suggest that the relationship between climatic variables and disease incidence is non-linear. The GAM identified significant relationships between disease incidence and precipitation at 4 and 8-week lags, average humidity (4-week lag), minimum temperature (4 and 8-week lags), average temperature (8-week lag), maximum wind speed (4 and 8-week lag) and average wind speed (4-week lag) ( Table 3).

The climate variables significantly correlated to total disease incidence as presented in Table 3 were further investigated by plotting the smoothed variance of the latter against the ranges covered for each climate variable ( Fig 7). The analysis of the three most significant climatic contributors to total disease incidence -temperature, wind speed, and precipitation-delineated clear trends on how climate affected disease transmission. Increasing Tmin from 8°C to 16°C, at either 4 or 8-week, reduced the total disease incidence while the average temperatures (Tavg) up to 24°C contributed to incremental levels of disease ( Fig 7A-7C). This could be an indication of the need for fluctuations at low temperatures in order to facilitate increased virus transmission.

Increasing wind speed above 1 m/s was associated by an increase in disease incidence with a peak around 2 m/s after which disease incidence declined until around 3 m/s where a small rise in disease risk can also be seen. This relationship between maximum wind speed and disease incidence was observed after both 4 and 8-week time lags ( Fig 7D and 7E). The decisive influence on wind speed was substantiated by the negative effect on disease incidence at incremental average wind speeds ( Fig 7F). Precipitation showed a positive relationship with disease incidence above 90 mm at 4-week time lags and above 25 mm for the data with 8-week time lag ( Fig 7G-7H). The high level of non-linearity shown for the relationship between average humidity and disease incidence ( edf = 7.5) ( Table 3) is detailed in Fig 7I. Disease incidence increased as humidity increased between 55-60%. This was followed by a slight decrease and plateau between 60-70%, a more rapid increase was observed between 70 and 75% above which disease incidence begins to decline ( Fig 7I).

Socio-economic profiles

We followed a holistic approach by further including socio-economic data for these three locations in the investigation for modifiers to the disease transmission of dengue, Zika and chikungunya. The overall multidimensional poverty index (see Methods) was lowest in Bello at 14.2. Cúcuta and Moniquirá had similar MPIs at 25.7 and 27.1 respectively ( Table 2).

The overall MPIs in Cúcuta and Moniquirá were similar. However, there were differences in specific poverty measures relevant to the transmission of vector borne viral diseases. Cúcuta had higher rates of overcrowding (16.4%), barriers to both childhood and youth services (2.2%) and healthcare services (5.2%) and inappropriate exterior wall material (6.1%) ( Table 2). The findings also pointed to other socio-economic indexes that also affect general health and well-being in Moniquirá: inadequate excreta disposal (sanitation) (12.1%), no access to an improved water source (20.4%), illiteracy (15.3%) and low education achievement (59.4%) were all highest in Moniquirá ( Table 2).

In order to introduce the socio-economic data into the analyses undertaken with the epidemiological and climatic data we carried out a dimensionality reduction and correlation with a principal component analysis (PCA). Importantly there was a clear separation of the three municipalities along both dimensions PC1 and PC2 that together integrates 89.9% of the compiled parameters ( Fig 8). This approach made apparent a discriminatory set of factors both climatic and socio-economic for all three locations. Cúcuta had an extensive combination of climate factors (i.e. wind speed and temperature) that together with school absence, dependency, overcrowding, wall material and school failure are potential modifiers of dengue, Zika and chikungunya risk in this municipality. Interestingly, Moniquirá showed mainly socio-economic variables (i.e. water source, sanitation, illiteracy, low education, flooring material, child labour, high MPI, informal work) to be potential modifiers for disease risk. On the other hand, Bello had mainly climate variables as potential modifiers of disease transmission-average humidity, precipitation and elevation-with only health insurance as a socio-economic factor.

Discussion

This study set out to determine the longitudinal dynamics of three Aedes arboviral diseases co-circulating in three regions of Colombia over a 11-year period between 2007 and 2017. We found significant differences in the burden of viruses among the three municipalities studied. Bello had the lowest level of disease incidence across all diseases. Cúcuta had the highest incidence of severe dengue (2007-2017) and Zika (2016) and the highest overall disease incidence. In addition to climatic factors the burden of these vector borne diseases in Cúcuta can be compounded by local current socio-political dynamics. Cúcuta is on Colombia's border with Venezuela, a country which has faced an economic, political and health crisis in recent years [ 60, 61]. The humanitarian crisis has led to large migration of Venezuelan citizens and refugees to neighbouring countries, with Colombia receiving the highest number of Venezuelan migrants. The number of Venezuelan migrants in Colombia increased from 48,714 in 2015 to 600,000 in 2017 [ 62]. This has had a significant impact on public health in Colombia, with infectious and vector-borne diseases particularly effected [ 63- 68]. The recent COVID-19 pandemic has intensified the humanitarian crisis in Venezuela, with bordering countries including Colombia closing their borders. The International Rescue Committee have reported a crisis for Venezuelan migrants in Cúcuta due to reduced access to health and other services [ 69]. COVID-19 is also providing challenges for disease surveillance and control programs as well as public health systems in Colombia and this is likely to have long lasting impact on vector-borne diseases [ 70, 71]. It is therefore more imperative than ever that we more fully understand the dynamics of these important diseases which are likely to escalate over the coming years.

The arrival of chikungunya and Zika in 2015 established a co-transmission of three different arboviruses by Ae. aegypti. Unexpectedly, a reduction in dengue cases was found in parallel to the spike in Zika cases in the year of the Zika epidemic of 2016 in Cúcuta. The same location in the years prior to the Zika outbreak had consistently presented high incidences of dengue. Moreover, in Cúcuta the incidence of both dengue and severe cases of dengue were lower in 2017, the year following the Zika epidemic, than in any of the 10 years prior. The decline of dengue following Zika reported in this study agrees with the overall decline in dengue incidence across the whole of Colombia [ 72, 73] and has also been observed in other dengue endemic countries across the Americas [ 73, 74]. In 2017 the total number of dengue cases across the Americas was lower than any of the 10 previous years [ 75] with a 73% decline between 2016 and 2017 alone [ 74].

Changes in epidemiological surveillance systems can cause the identification of inaccurate patterns of disease incidence. However, we did not observe any indication of significant changes to the surveillance system used to report Aedes borne viruses to SIVIGILIA, the database used here. However, the circulation of multiple viruses in the same localities at the same time does provide challenges for surveillance systems. Clinical presentation of Zika is very similar to that of dengue [ 27, 76] and this can cause cases to be misidentified when laboratory testing is not conducted. We note that although the cases analysed in this study are all confirmed cases, confirmation is not always done by laboratory testing but also by epidemiological links. Misidentification could therefore explain the increase in dengue that was observed in Bello and Moniquirá in 2016, where low incidence of Zika was reported. Increased dengue incidence in 2015 and 2016 in other regions has also been reported and attributed to potential misidentification of Zika [ 74]. Misidentification could also be a factor in the observed decline of dengue in Cúcuta in 2016, with dengue cases being misidentified as Zika during the Zika outbreak.

Coinfection of the primary vector Ae. aegypti with multiple arboviruses (i.e. DENV, CHIKV, ZIKV) has been reported following laboratory exposure [ 77- 82], with an enhanced susceptibility to ZIKV (PMID 33214283). Aedes mosquitoes have also been shown capable of transmitting more than one arbovirus in a single biting event [ 78, 82, 83]. Although coinfection has yet to be found in wild Ae. aegypti [ 83]. Coinfection of multiple Aedes borne viruses has been reported in mammalian hosts including humans [ 84- 90]. In Colombia patients have been diagnosed with DENV-CHIKV [ 66, 91, 92], DENV-ZIKV [ 66, 92], CHIKV-ZIKV [ 66, 92] and DENV-CHIKV-ZIKV [ 27, 67] coinfections. However, the frequency of DENV-ZIKV co-infections seems low at 8.8% [ 92]. DENV and ZIKV co-transmission in mice through the bite of Ae. aegypti mosquitoes showed preferential transmission of ZIKV [ 82].

Host cross-immunity of ZIKV and DENV could have been a contributing factor in the dengue declines observed in this study. The observed decline in dengue cases following Zika outbreaks reported within this study and in others across the Americas suggest that there may be cross-immunity between ZIKV and DENV in humans [ 72- 74, 93- 119]. Flavivirus immunity involves a T cell response and studies have reported cross-reactivity of CD4+ and CD8+ T cells to both DENV and ZIKV [ 120- 124]. Cross-reactivity of antibodies and T cells and cross-immunity from Zika, although not necessarily conferring cross-protection, has been presented as the most probable reason explaining the decline of dengue across the Americas in 2017 [ 74].

Specific climatic factors associated here significantly affected disease incidence. We found significant co-relationship between average temperature and wind speed with disease transmission, with a peak at around 2m/s, consistent with findings of previous studies [ 125- 128]. Ae. aegypti has a small flight range of 200 m and high wind speeds reduce mosquito flight distances while low winds mean reduced dispersion of mosquitoes. We found the optimum wind speed to be around 2 m/s which is in line with current knowledge of mosquito flight [ 125, 129, 130]. A significant relationship was also found between increasing minimum temperature and decreasing disease incidence, contrasting to the findings of some other studies [ 130- 133]. Exposure of Ae. aegypti to fluctuations at low temperatures has been associated with shorter DENV extrinsic incubation periods (EIP) and increased virus dissemination from the midgut when compared to exposure to constant temperatures with the same mean [ 134].Reduced EIP and increased virus dissemination increase transmission potential [ 23]and could explain the relationship between increasing minimum temperature and decreasing disease incidence observed in this study. Climate variables can be used to build predictive models to anticipate when outbreaks of dengue, chikungunya and Zika are likely to occur [ 135- 138]. This is useful in the prioritisation of vector-control resources. Recent modelling studies have reported an increase in the incidence and geographical spread Ae. aegypti borne viruses when using climate change simulation models [ 139- 143]. This highlights the importance of consideration of environmental factors when assessing risk of vector-borne disease [ 144].

We report differences in measurements of socio-economic variables between Bello, Cúcuta and Moniquirá. Bello, the municipality with the lowest burden of Aedes borne viruses also had the lowest poverty index, whilst Cúcuta and Moniquirá were much higher in both disease incidence and multidimensional poverty. Higher incidence of Aedes borne disease has been associated with lower socio-economic status and higher poverty levels [ 145- 152]. Cúcuta had the highest rate of critical overcrowding. Overcrowding has been reported to be an important contributing factor to dengue incidence [ 144- 146, 153]. Inadequate sanitation, and no access to improved sources of water are both well-known contributing factors in increasing burden of Aedes borne disease due to the ecology of Aedes mosquitoes [ 33, 144, 148- 150, 154]. These socio-economic risk factors were highest in Moniquirá where there were also high levels of low educational achievement and illiteracy. Illiteracy and low educational level have previously been associated with increased vulnerability to dengue in Colombia and Brazil [ 155, 156].

This study has potential limitations, due to the nature and availability of the data used. Co-infections were unable to be analysed due to the inability to identify these within the data set, despite co-infections likely occurring during our study period. The investigation into the effects of socioeconomics on disease incidence are limited by the unavailability of temporal data, therefore changes in socioeconomics and their impact on disease burden over time could not be analysed.

Having different ecosystems Bello, Cúcuta and Moniquirá presented a valuable opportunity to explore longitudinal arboviral disease incidence over 11 years that encompassed a Zika epidemic. Chikungunya was the only disease for which incidence did not significantly differ between the three municipalities. Cúcuta had the greatest disease incidence having the most favourable climatic factors and greater poverty index but as it borders with Venezuela mass movement of people is also suggested to be a contributing factor. Climatic factors associated with disease incidence were precipitation, average humidity, temperature and wind speed. Co-transmission of dengue and Zika during the epidemic led to a significant reduction of dengue cases in Cúcuta where dengue had previously been high. This significant finding warrants further investigation. Where the poverty index was low, as in Bello, so was the disease incidence. Socio-economic factors such as barriers to health and childhood services, inadequate sanitation, poor housing and poor water supply were implicated as drivers of disease transmission. Aedes aegypti and Ae. albopictus are increasing their geographical range and climate change is predicted to alter the distribution of these vectors and hence disease risk. Arboviral epidemiology is further complicated by humanitarian crises (e.g. political and economic crises Venezuela leading to mass migration) and the COVID-19 pandemic which reinforces the urgency for understanding the dynamics of these global health problems. Context dependent and actionable understanding of the drivers for disease transmission that consider local dynamics, both climatic and socio-economic, should contribute to the design of more effective vector mosquito control programmes [ 157].

Acknowledgments

We acknowledge Dr Ashley Lyons from Liverpool Hope University for advice on statistical techniques.

References

1. 39. Colombia MdlPSRd Instituto Nacional de Salud RdC OP. Gestion para la vigilancia entomologica y control de la transmision del dengue. 2012.

2. 44. Instituto de Hidrología M y EA. Características Climatológicas (1981-2010). 2014 [cited 2 Oct 2019]. Available from: https://web.archive.org/web/20160815030620/ http://www.ideam.gov.co/web/tiempo-y-clima/clima

3. 50. Wood , Simon N. Generalized Additive Models: An Introduction with R. CRC Press; 2017.

4. 51. Thomas R. Data Analysis with R Statistical Software A Guidebook for Scientists. 2nd ed. Eco-explore; 2017.

5. 56. Zuur AF, Ieno EN, Walker NJ, Saveliev AA, Smith GM. Mixed Effects Models and Extensions in Ecology with R. 1st ed. New York: Springer; 2009.

6. 58. Fasiolo M, Nedellec R, Goude Y, Wood , Simon N. Scalable visualisation methods for modern Generalized Additive Models. 2018. Available from: https://arxiv.org/abs/1707.03307

7. 60. Nelson RM. Venezuela's Economic Crisis: Issues for Congress. Congr Res Serv Libr Congr. 2018. Available from: https://pdfs.semanticscholar.org/c60d/ab688b21f960f6ada089e1c3bce87395fb9d.pdf

8. 71. Wenham C, Lotta G, Pimenta N. Mosquitoes and Covid-19 are a ticking time bomb for Latin America. In: LSE Latin America and Caribbean [Internet]. 2020 [cited 11 Jun 2020]. Available from: http://eprints.lse.ac.uk/104150/

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Climatic and socio-economic factors supporting the co-circulation of dengue, Zika and chikungunya in three different ecosystems in Colombia

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Author summary Viruses transmitted by Ae. aegypti mosquitoes (dengue, Zika, chikungunya) are amongst the most significant public health concerns of recent years due to the increase in global cases and the rapid spread of the vectors. The primary method of controlling the spread of these arboviruses is through mosquito control.

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The Chikungunya virus has recently resurfaced in Paraguay and Brazil. Problems like misdiagnosis and limited laboratory testing have caused this resurgence.

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Chikungunya virus has recently begun infecting more and more in South America. Their healthcare system isn't the best so they aren't able to treat it as well.

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When it comes to public health crises and emergencies, it is hard to compete with the impact of the Covid pandemic, undeniably the most challenging period Latin America's weak and unequal health systems have faced in decades, resulting in more than 1.76 million deaths since 2020.

But Covid is not the sole instance of a virus that the region's limited health apparatus struggles to contain. The perennial examples are dengue fever and chikungunya, which have long afflicted poor communities in tropical and subtropical climates, and, unfortunately, have expanded their reach in recent years.

Dengue epidemics have been recorded in Brazil since the early 1990s, but figures are on the rise. According to the Oswaldo Cruz Foundation, a federal biological research organization, reported cases of the mosquito-borne disease increased by 162 percent from 2021 to 2022, approaching 1.5 million last year.

Last year was also the deadliest for the disease in recent Brazilian history, with 1,016 dengue deaths registered, close to the totals seen in the 1980s, when the virus wreaked havoc across the country. The number of deaths recorded last year in Brazil alone is close to the total number of fatalities counted in all of Latin America in 2019, during the 2019-2020 dengue fever epidemic.

The record broke two years of relatively low dengue fever incidence during the pandemic. Experts attribute the...

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Dengue has long plagued the continent, and the numbers have grown more alarming recently, but a new vaccine is also raising hopes.

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CEPI is working towards finding a vaccine for this virus. Unfortunately, this virus will most likely only be available for adults 18+.

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Licenced Chikungunya vaccines are on the horizon - CEPI

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Enabling access to them in endemic countries will be crucial to combatting this debilitating disease

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Valneva is going through the licensing process for a vaccine. This would be very monumental if this were approved since it would cause relief for many South American countries.

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Saint-Herblain (France), August 29, 2023 - Valneva SE (Nasdaq: VALN; Euronext Paris: VLA), a specialty vaccine company, today announced that Health Canada has completed screening validation of the Company's regulatory application for marketing approval of its single-shot chikungunya vaccine candidate VLA1553 in persons aged 18 years and above, and has determined that the New Drug Submission (NDS) application is sufficiently complete to permit a substantive review. Based on Health Canada's performance standard to process an NDS application, the Company believes the regulatory review could be completed by mid-2024.

This is the second regulatory application for VLA1553 filed by Valneva, and the Company intends to make additional regulatory submissions in 2023. A Biologic License Application (BLA) is currently under priority review[1] by the U.S. Food and Drug Administration (FDA)[2].

VLA1553 is currently the only chikungunya vaccine candidate worldwide for which regulatory review processes are underway and, if approved, it could become the first licensed chikungunya vaccine available to address this unmet medical need. It would also represent the third vaccine Valneva[3] has brought from early research and development to approval.

Juan Carlos Jaramillo, MD, Chief Medical Officer of Valneva, commented, "Chikungunya represents a major threat for people traveling to or living in areas where chikungunya virus and the mosquitos that transmit it are present, including popular destinations for U.S. and Canadian travelers. This threat continues to grow as shown by the recent epidemiological alert issued by the Pan American Health Organization (PAHO)[4]. No vaccine or specific treatments are currently available for this debilitating disease, and we will continue to work diligently to make VLA1553 available in different territories as quickly as possible."

The regulatory submissions with Health Canada and the FDA follow final pivotal Phase 3 data in March 2022[5], final lot-to-lot consistency results in May 2022 and positive twelve-month persistence data in December 2022. The Company's pivotal Phase 3 results were published in the Lancet in June 2023.

A clinical study of VLA1553 in adolescents aged 12 to 17 years is ongoing in Brazil, for which Valneva reported initial Phase 3 safety data in adolescents yesterday[9]. This study, conducted by Valneva's partner Instituto Butantan and funded by the Coalition for Epidemic Preparedness Innovations (CEPI), is intended to support label extension in this age group following a potential initial regulatory approval in adults from the FDA. The study is also expected to support licensure of the vaccine in Brazil, which would be the first potential approval for use in endemic populations, as well as regulatory submission in Europe.

The vaccine was granted PRIority MEdicine (PRIME) designation by the European Medicines Agency (EMA) in 2020 and also received FDA Fast Track, Breakthrough Therapy and Priority Review designations in 2018, 2021 and 2023 respectively.
About Chikungunya
Chikungunya is a mosquito-borne viral disease caused by the chikungunya virus (CHIKV), a Togaviridae virus, transmitted by Aedes mosquitoes. Infection leads to symptomatic disease in up to 97% of humans after four to seven days following the mosquito bite. While mortality with CHIKV is low, morbidity is high, and the global market for vaccines against chikungunya is estimated to exceed $500 million annually by 2032[10]. Clinical symptoms include acute onset of fever, debilitating joint and muscle pain, headache, nausea, rash and chronic arthralgia. Chikungunya virus often causes sudden large outbreaks with high attack rates, affecting one-third to three-quarters of the population in areas where the virus is circulating. The high-risk areas of infection for travelers are places where chikungunya virus-carrying mosquitos are endemic, including the Americas, parts of Africa, and Southeast Asia, and the virus has spread to more than 110 countries[11]. As of July 2022, more than three million cases have been reported in the Americas[12] and the economic impact is considered to be significant. The medical and economic burden is expected to grow as the CHIKV primary mosquito vectors continue to spread geographically. There are no preventive vaccines or effective treatments available and, as such, chikungunya is considered to be a major public health threat.
About VLA1553
VLA1553 is a live-attenuated, single dose investigational vaccine candidate targeting the chikungunya virus, which has spread to over 110 countries[13]. It has been designed by deleting a part of the chikungunya virus genome.

Valneva reported final data from the pivotal Phase 3 trial of VLA1553 in March 2022[14], final lot-to-lot consistency results in May 2022[15] and positive twelve-month persistence data in December 2022.

If approved, VLA1553 would expand Valneva's existing commercial vaccines portfolio and as such, Valneva intends to commercialize this vaccine, leveraging its existing manufacturing and commercial operations.

To make VLA1553 more accessible to Low- and Middle-Income Countries (LMIC), Valneva and Instituto Butantan in Brazil signed an agreement in January 2021 for the development, manufacturing and marketing of VLA1553[17]. The collaboration falls within the framework of the agreement signed between CEPI and Valneva in July 2019[18], which provides funding of up to $24.6 million with support from the European Union's Horizon 2020 program.

VLA1553 received FDA Fast Track, Breakthrough Therapy designations and Priority Review in 2018, 2021 and 2023, respectively. VLA1553 was also granted PRIority MEdicine (PRIME) designation by the European Medicines Agency (EMA) in 2020.
About Valneva SE
Valneva is a specialty vaccine company focused on the development, manufacturing and commercialization of prophylactic vaccines for infectious diseases with significant unmet medical need. The Company takes a highly specialized and targeted approach to vaccine development and then applies its deep understanding of vaccine science to develop prophylactic vaccines addressing these diseases. Valneva has leveraged its expertise and capabilities both to commercialize three vaccines and to rapidly advance a broad range of vaccine candidates into and through the clinic, including candidates against the chikungunya virus and Lyme disease.

Valneva Investor and Media Contacts

Laetitia Bachelot-Fontaine

VP Global Communications & European Investor Relations

M +33 (0)6 4516 7099

laetitia.bachelot-fontaine@valneva.com

Joshua Drumm, Ph.D.

VP Global Investor Relations

M +001 917 815 4520

joshua.drumm@valneva.com

Forward-Looking Statements
This press release contains certain forward-looking statements relating to the business of Valneva, including with respect to the progress, timing, results and completion of research, development and clinical trials for product candidates, to regulatory approval of product candidates and review of existing products. In addition, even if the actual results or development of Valneva are consistent with the forward-looking statements contained in this press release, those results or developments of Valneva may not be sustained in the future. In some cases, you can identify forward-looking statements by words such as "could," "should," "may," "expects," "anticipates," "believes," "intends," "estimates," "aims," "targets," or similar words. These forward-looking statements are based largely on the current expectations of Valneva as of the date of this press release and are subject to a number of known and unknown risks and uncertainties and other factors that may cause actual results, performance or achievements to be materially different from any future results, performance or achievement expressed or implied by these forward-looking statements. In particular, the expectations of Valneva could be affected by, among other things, uncertainties and delays involved in the development and manufacture of vaccines, unexpected clinical trial results, unexpected regulatory actions or delays, competition in general, currency fluctuations, the impact of the global and European credit crisis, and the ability to obtain or maintain patent or other proprietary intellectual property protection. Success in preclinical studies or earlier clinical trials may not be indicative of results in future clinical trials. In light of these risks and uncertainties, there can be no assurance that the forward-looking statements made during this presentation will in fact be realized. Valneva is providing the information in these materials as of this press release and disclaim any intention or obligation to publicly update or revise any forward-looking statements, whether as a result of new information, future events, or otherwise.

Valneva Announces PDUFA Date Extension for Chikungunya Virus Vaccine Candidate - Valneva
This statement refers to Valneva and its predecessor Intercell https://www.paho.org/en/documents/epidemiological-alert-chikungunya-increase-region-americas
[9] Valneva Reports Positive Initial Phase 3 Safety Data in Adolescents for its Single-Shot Chikungunya Vaccine Candidate - Valneva

[10] VacZine Analytics Chikungunya virus vaccines Global demand analysis. February 2020
PAHO/WHO data: Number of reported cases of chikungunya fever in the Americas (Cumulative Cases 2018-2022 and Cases per year 2013-2017). https://www.paho.org/data/index.php/en/mnu-topics/chikv-en/550-chikv-weekly-en.html. Last accessed 25 Jul 2022.
[14] Valneva Successfully Completes Pivotal Phase 3 Trial of Single-Shot Chikungunya Vaccine Candidate

[15] Valneva Successfully Completes Lot-to-Lot Consistency Trial for its Single-Shot Chikungunya Vaccine Candidate

[17] Valneva and Instituto Butantan Sign Final Agreement on Single-Shot Chikungunya Vaccine for Low and Middle Income Countries

[18] CEPI awards up to $23.4 million to Valneva for late-stage development of a single-dose Chikungunya vaccine

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Health Canada Accepts Valneva's Chikungunya Vaccine License Application for Review - Valneva

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Saint-Herblain (France), August 29, 2023 - Valneva SE (Nasdaq: VALN; Euronext Paris: VLA), a specialty vaccine company, today announced that Health Canada has completed screening validation of the Company's regulatory application for marketing approval of its single-shot chikungunya vaccine candidate VLA1553 in persons aged 18 years and above, and has determined that the New Drug [...]