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method by Langenberg et al., 1988
1. Samples obtain from lesion by using cotton swab
2.sample swab slowly at glass slide and allow to dry
3.apply biotinylated HSV DNA probe .
4.specimen heated to 92 degree celcius.
5.Incubate and wash step
6. Addition of another chemicals ex: chromogen-substrate and fast green counterstain.
7.view at 100x & 400x magnification (wet mount technique).

ADVANTAGE
- High sensitivity compared to tissue culture technique
-whole process require 2 hour 15 minutes.
-Less cost.

DISADVANTAGE
-Only applicable to detect large amount of samples / more than 20 HSV cells.
-Lack type sensitivity

REFERENCE
Langenberg, A., Smith, D., Brakel, C. L., Pollice, M., Remington, M., Winter, C., … Corey, L. (1988). Detection of 🤬 simplex virus DNA from genital lesions by in situ hybridization. Journal of Clinical Microbiology, 26(5), 933–7. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/2838505

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Genital 🤬 virus is passed from one person to another through sexual contact. This happens even if the person with the virus doesn't have symptoms or signs of infection.

Once the virus enters through the skin it travels along nerve paths. It may become dormant (inactive) in the nerves and remain there indefinitely.

Blister on infected area

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Genital 🤬 appears as one or more blisters on or around the genitals or 🤬. When these blisters burst they leave the tender sores known as ulcers. The first time a person has the 🤬 outbreak, the ulcers may take two to four weeks to heal. The next outbreaks may not occur for weeks, months, or even later. When they do, they usually are less severe than the first outbreak. 🤬 infection doesn't go away, but the outbreaks tend to become less frequent over time.

There are a few symptoms feel after being infected with 🤬 virus such as:
:: numbness, tingling, or burning in the genital region
:: burning sensation while urinating or having intercourse
:: painful urination, difficulty urinating, or a frequent need to urinate
:: Watery blisters in the genital area

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HSV virion and its two major modes of entry into cells. Structural components of a typical HSV virion are shown (box). HSV virions can enter into cells via a pH-independent fusion of viral envelope with the plasma membrane (I) or alternatively, via an endocytic pathway that may be phagocytosis-like (II) in terms of the viral uptake. In both pathways HSV particles may initially associate with filopodia-like membrane protrusions via heparan sulfate proteoglycan (HSPG). Unidirectional transport of extracellular particles bound to filopodia (HSV surfing) then brings the particles closer to the cell body for entry via interactions with the cellular receptors including gD receptor and possibly gB receptor. Fusion at the plasma membrane results in the release of the naked viral nucleocapsid in the cytoplasm for transport to the nucleus. Similarly, endocytosis also requires fusion of the enveloped particles with the vesicular membrane for the release of the viral nucleocapsid proximal to the nucleus.

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🤬 viruses establish lifelong infections, and the virus cannot yet be eradicated from the body. Treatment usually involves general-purpose antiviral 🤬 that interfere with viral replication, reduce the physical severity of outbreak-associated lesions, and lower the chance of transmission to others. Studies of vulnerable patient populations have indicated that daily use of antivirals such as aciclovir and valaciclovir can reduce reactivation rates.

Aciclovir

Valacyclovir

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Method by Ramaswamy et al., 2004.
- Obtain genital swab from patient
- Transfer swab in viral transport medium
-Maintain cell
DNA extraction
Perform real time PCR (amplify region of viral DNA polymerase)
-DNA sequence
Analysis result.

ADVANTAGE
-High detection rate than virus culture method and rapid result.
-convenient for clinical diagnostic

DISADVANTAGE
-Exposed to contamination.

REFERENCE
Ramaswamy, M., McDonald, C., Smith, M., Thomas, D., Maxwell, S., Tenant-Flowers, M., & Geretti, A. M. (2004). Diagnosis of genital herpes by real time PCR in routine clinical practice. Sexually Transmitted Infections, 80(5), 406–10. https://doi.org/10.1136/sti.2003.008201

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Abstract

Background: Virus isolation in cell culture is the recognised diagnostic gold standard for genital herpes. Although increasing evidence indicates that polymerase chain reaction (PCR) provides a more rapid and sensitive diagnostic method, its implementation in routine diagnostic settings has been limited by concerns over contamination and cost.

Objective: To evaluate the feasibility of replacing virus culture with PCR for the diagnosis of genital herpes in settings serving large populations of genitourinary medicine (GUM) attendees.

Methods: Genital swabs collected from 233 consecutive GUM attendees with suspected genital herpes were tested in parallel by virus culture and automated real time PCR. Three specimen preparation methods were evaluated and the assay reliability was assessed by repeat testing, comparison with a commercially available assay, and herpes simplex virus (HSV) sequence analysis. Probe melting temperatures (Tm) were used to differentiate between HSV types without additional post-PCR steps.

Results: HSV was detected in 79/233 (34%) samples by virus culture and 132/233 (57%) samples by PCR. PCR significantly increased HSV detection in both early (<5 days) and late (⩾5 days) presentations and in both first and recurrent episodes. HSV detection and typing by PCR was achieved within less than 4 hours leading to a significant reduction in labour compared to virus culture. Most specimens (120/132, 91%) were typed as HSV-2. Results were highly reproducible.

Conclusions: Real time PCR is a highly reproducible, rapid, and labour efficient method for HSV detection in genital swabs. Its implementation is feasible in routine diagnostic settings.

In 2002, over 18 000 new cases of genital herpes were reported from genitourinary medicine clinics in the United Kingdom, making genital herpes the fourth most common sexually transmitted disease. These figures underestimate the true prevalence of symptomatic infections as atypical presentations are common and often misdiagnosed. Underdiagnosis of genital herpes is an important obstacle to effective management and control of transmission. The clinical diagnosis of genital herpes is both insensitive and non-specific and requires laboratory confirmation. Virus isolation in cell culture has long been regarded as the diagnostic gold standard and is the diagnostic method of choice in the United Kingdom. Virus isolation is slow and labour intensive, requiring up to 7-10 days for the appearance of cytopathic changes in cultured cells or before a negative result can be issued. Specificity is virtually 100%, but levels of virus shedding, quality of specimen, and transport conditions influence sensitivity. The rate of virus recovery declines significantly with time since the onset of lesions, from 52-93% for vesicles to 41-72% for ulcers and 19-27% for crusted lesions. Furthermore, lack of specimen refrigeration after collection and during transport markedly reduces virus viability.

Recent studies have shown that polymerase chain reaction (PCR) increases the rate of HSV detection in mucocutaneous swabs by 11-41% compared to virus culture. Although earlier PCR techniques were laborious, expensive and prone to contamination, newly developed real time PCR assays are fully automated and allow virus specific detection within a closed system, with fast turn around times and low risk of contamination. Whereas published protocols for herpes simplex virus (HSV) PCR do not always differentiate between HSV types, new technologies allow simultaneous detection and typing of HSV in a single reaction tube, based on software that analyses the different Tm values of HSV-1 and HSV-2 specific probes. This is a valuable feature for the use of PCR in routine clinical practice, as differentiating between HSV types provides important prognostic information in genital herpes.

The aim of this study was to compare the performance of virus culture and real time PCR for the diagnosis and typing of genital herpes in a clinical setting serving a large population of genitourinary medicine (GUM) clinic attendees. Three different specimen preparation methods were evaluated for their performance, cost, and labour intensity. Sequence variations within the target DNA polymerase gene region were investigated to assess their potential impact on the assay's performance.

MATERIALS AND METHODS

Patient populations

With ethics committee approval, genital swabs were collected from 233 consecutive GUM clinic attendees who presented with clinical features suggestive of genital herpes at the Caldecot Centre for Sexual Health at Kings College Hospital, London, between June 2002 and August 2002. The first part of the study recruited 145 consecutive patients. Their demographic and clinical data were recorded, including swab site, type of presentation (first or recurrent episode), duration of onset (<5 days or ⩾5 days), presence of ulceration, administration of antiviral drugs before disease onset (aciclovir, valaciclovir, or famciclovir), and HIV status. In the second part of the study, 88 additional genital swabs from consecutive patients presenting with symptoms consistent with genital herpes in the same clinical setting were tested, to provide a validation group for the larger subset.

Sampling

One swab was taken from each patient and placed in 2.5 ml viral transport medium (VTM) containing minimal essential medium (MEM) balanced with 25 mM Hepes (BioWhittaker, Europe) supplemented with 10% bovine serum albumin (BSA), 7.5% sodium bicarbonate, penicillin (500 U/ml), streptomycin (1 mg/ml) and amphotericin B (5 μg/ml). In the laboratory, samples were vortexed for 30 seconds and separated into five aliquots of 500 μl to be used for parallel virus culture and PCR.

Virus culture and typing

For virus isolation, 200 μl of the specimen in VTM were inoculated into two tubes containing a monolayer of Vero monkey kidney cells. Cultures were maintained in MEM-Hepes growth medium (BioWhittaker) supplemented with 25% fetal calf serum (FCS), and incubated at 37°C for 10 days. Cell cultures were examined daily for the appearance of a cytopathic effect (CPE). When a CPE was observed, the cells were scraped off the tube, dried on a slide, fixed in acetone and stained with fluorescein labelled murine monoclonal antibodies against HSV-1 and HSV-2 (Syva Microtak, Palo Alto, CA, USA) according to manufacturer's instructions.

Specimen preparation for PCR

With a subset of 140 samples, three different methods were used to prepare DNA from 200 μl of specimen in VTM: (1) manual DNA extraction with the QIAamp DNA Mini Kit (Qiagen, UK); (2) automated DNA extraction with the MagNA Pure LC (Roche Diagnostics, Germany), and (3) DNA precipitation with polyethylene glycol (PEG) and sodium chloride (NaCl). The QIAamp DNA Mini Kit was used in accordance with manufacturer's instructions with the following modifications: an aqueous solution of poly dA (5 μg/ml) (Amersham Biosciences, UK) was added during lysis and 230 μl of 99% ethanol was used at the first wash step to improve extraction in all samples. For the MagNA Pure LC instrument, the sample material was placed into the wells of the sample cartridge, with binding/lysis buffer, proteinase K and extraction carried out according to the manufacturer's instructions. DNA was eluted in 60 μl of sterile PCR grade deionised water (Sigma, UK) for the QIAamp DNA Mini kit and in 100 μl of elution buffer (Total Nucleic Acid Isolation kit, Roche Diagnostics) for the MagNA Pure LC system. PEG precipitation was performed by adding 200 μl of the specimen in VTM to a mixture of 2.5M NaCl (25 μl) and 20% PEG 8000 (25 μl). The mixture was vortexed, incubated on ice for 30 minutes, and centrifuged at 11 000 g for 20 minutes at 4°C. The supernatant was aspirated, pellet recentrifuged at 4°C for 5 minutes before the pellet was re-suspended in 20 μl of deionised water.

Real time PCR was performed with the LightCycler (Roche Diagnostics) using a method previously described, that amplifies a 215 bp region of the viral DNA polymerase gene. A total volume of 20 μl was obtained by adding 5 μl of DNA or precipitated virus to a PCR reaction mix in glass capillaries. DNA amplification was achieved using the Roche LC Fast Start DNA Master Hybridisation Probe kit (Roche Diagnostics) as described by the manufacturer. Forward (HSV POL F 5′-GCTCGAGTGCGAAAAAACGTTC-3′) and reverse primers (HSV POL A 5′-TGCGGTTGATAAACGCGCAGT-3′) and two fluorescently labelled hybridisation probes HSV-2 FLU (5′-gCgCACCAgATCCACgCCCTTgATgAgC-FAM-3′) and HSV-2 LCR (5′-LC Red 640-CTTgCCCCCgCAgATgACgCC phosphate-3′) (TIB MolBiol, Germany) were used at 5 μM and 1 μM respectively.

PCR conditions were as follows: initial denaturation at 95°C for 10 minutes (temperature transition rate of 20°C/s) to activate DNA Taq polymerase, followed by 50 cycles of denaturation at 95°C for 5 seconds (20°C/s transition), annealing at 50°C for 5 seconds (20°C/s transition) with single fluorescence acquisition, and extension at 72°C for 5 seconds (20°C/s transition). Melting curve analysis was performed as follows: annealing at 40°C for 30 seconds (20°C/s transition) followed by an increase to 80°C for 0 second (0.1°C/s transition) with continuous fluorescence acquisition, and cooling to 40°C for 30 seconds (20°C/s transition). The Tm of DNA fragments and hybridisation probes was used to differentiate between HSV types. The probe Tm was 59°C for HSV-1 and 64°C and/or 71°C for HSV-2. The RealArt HSV1/2 LC PCR kit (Artus Biotech, Germany) was used according to the manufacturer's instructions to confirm the results of 15 HSV positive specimens.

DNA sequencing

Samples were prepared for LightCycler PCR as described earlier but in the absence of hybridisation probes HSV-2 FLU and LCR. The final volume of 20 μl was made up with deionised water. Following amplification, the DNA was purified using QIAquick PCR Purification kits (Qiagen) according to manufacturer's instructions and eluted in 50 μl of deionised water.

DNA sequencing was performed by the Sanger dideoxy chain termination method using 3 μl of extracted DNA, 1 μl of each A, C, G, T reagent mix from the ThermoSequenase DYEnamic Direct Cycle sequencing kit (Amersham Pharmacia Biotech Inc, UK), 1 μl of 0.5 μM HSV forward and reverse primers (as previously described) labelled with CY5.5 and CY5 respectively (Sigma-Genosys Ltd, UK). Reactions were performed on a GeneAmp 9700 thermocycler (PerkinElmer Applied Biosystems, Beaconsfield, UK) under the following conditions: 25 cycles of denaturation at 94°C for 5 minutes, followed by further denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 70°C for 1 minute. Formamide loading dye (7 μl) was added, and the DNA denatured at 80°C for 2 minutes. Automated DNA sequencing reactions (2 μl) were run on an Trugene HIV-1 Opengene sequencing system (Visible Genetics, Ontario, Canada) under standard conditions using version 3.16 software as described by the manufacturer. Sequence data were compared to other HSV sequences present in the GenBank database to confirm HSV type using BLAST. Sequences were aligned using ClustalW version 1.81.

Analysis of results

The HSV detection rates for virus culture versus PCR according to clinical characteristics were compared by the χ ² or Fisher's exact test as appropriate. In the comparison of specimen preparation methods, the "true positive" reference was specimens that were either positive in both virus culture and PCR, or were virus culture negative but PCR positive in ⩾2 separate assays and by ⩾2 methods. Kappa values were calculated to determine the level of agreement of the three specimen preparation methods when compared to the reference.

RESULTS

In the first part of the study genital swabs were collected from 145 consecutive patients (85 men and 60 women) with suspected genital herpes, and tested in parallel by virus culture and PCR. The most common swab sites were the penile skin in males (63/85, 74%) and the vulva in females (48/60, 80%). HSV was detected in 45/145 (31%) specimens by culture and 77/145 (53%) specimens by PCR. The 32 specimens that were negative by culture but positive by PCR underwent extensive retesting. They were all confirmed HSV DNA positive in ⩾2 independent replicate PCR assays (mean 2.75 assays per sample). In addition, with a random subset of 15 samples, the in-house PCR results were confirmed by a commercially available PCR assay. Thus real time PCR increased the HSV detection rate by 71% (32/45). High reproducibility indicated a low risk of false positive results. No patient with positive culture had a negative PCR. The average time for a positive virus culture was 3 days (range 2-10 days), whereas negative results required 10 days of incubation. PCR results required less than 4 hours including time for specimen preparation, amplification, detection, analysis, and typing.

Table 1 shows the results of virus culture and PCR in relation to clinical presentation. By either method the rates of HSV detection were similar in men and women, and PCR increased HSV detection by 79% (19/24) and 62% (13/21) in the two groups respectively. Culture was more frequently positive in patients presenting <5 days of onset than in those who presented later (27/64, 42% versus 18/81 22%, p = 0.01). The proportion of patients with a positive PCR did not differ significantly between the two groups (38/64, 59% versus 39/81, 48%, p = 0.2). As a result, PCR increased HSV detection by 41% (11/27) in early disease and 116% (21/18) in late disease. The proportion of patients with a positive culture was similar in first episode and recurrent disease. PCR increased HSV detection by 58% (15/26) and 89% (17/19) in the two groups respectively. Improved detection by PCR was seen regardless of HIV status.

We subsequently extended the study to compare virus culture with PCR in further 88 consecutive genital swabs obtained from symptomatic patients in the same clinical setting. Of these, 34/88 (39%) were positive by culture and 55/88 (63%) by PCR. Thus, in this additional subset of samples, PCR increased HSV detection by 62% (21/34), validating the data from the larger subset. In the whole cohort, HSV detection rate was 79/233 (34%) by virus culture and 132/233 (57%) by PCR.

HSV typing by LightCycler PCR

Figure 1 shows the reference Tm for HSV-1 and HSV-2. Among the first set of HSV positive samples, 71/77 (92%) were typed as HSV-2 and 6/77 (8%) as HSV-1. All typing results were confirmed in the replicate experiments. The 15 samples tested by the commercial PCR assay also yielded typing results identical to those obtained by the in-house assay. Among the second set of HSV positive samples, 49/55 (89%) were typed as HSV-2 and 6/55 (11%) as HSV-1. In the whole cohort, 120/132 (91%) HSV positive samples were typed as HSV-2. Fourteen randomly selected HSV-2 positive samples with Tm >60°C were sequenced and all confirmed as HSV-2 after alignment to known HSV-2 sequences (table 2). Among the 14 samples sequenced, four showed sequence variation in the FLU probe binding site within the 100 bp region of the DNA polymerase gene (table 2). No variation was observed in the target HSV-2 sequence among the 10 remaining specimens.

Feasibility of PCR testing for large volumes of samples

As specimen preparation is the most time consuming step in PCR testing, we compared the performance, labour intensity, and cost of three specimen preparation methods using manual DNA extraction with the QIAamp DNA mini kit, automated DNA extraction with MagNA Pure LC, and virus precipitation with PEG. In a comparison of 140 specimens, 120/140 (86%) were either negative or positive by all three methods, 12/140 (8%) were positive by two methods, and 8/140 (6%) were positive by only one method. Of the latter eight samples, all were confirmed positive by repeated specimen preparation and PCR testing, three were also positive by virus culture. Regarding as "true positive" specimens that were either positive by both virus culture and PCR or, if virus culture negative, positive in ⩾2 separate PCR assays and by ⩾2 methods, the HSV detection rate was PEG precipitation >MagNA Pure LC >QIAamp DNA mini kit (table 3). Kappa values for the three specimen preparation methods were ⩾0.84, indicating a high level of agreement. PEG precipitation detected 65/72 (90%) positive samples, including three samples positive by virus culture but negative by the other two preparation methods, and was more rapid and less expensive than the other two methods (table 3).

DISCUSSION

A virological confirmation of genital herpes provides the basis for appropriate management, and informs prognosis and counselling. To date, the routine diagnosis of genital herpes has mainly relied on virus isolation in cell culture. However, numerous recent reports have highlighted the limitations of this method. On practical consideration, virus culture is time consuming and requires highly stringent conditions for specimen transport and storage to preserve virus viability. Improved diagnostic methods are needed to improve recognition of genital herpes. In this study we demonstrated that real time PCR significantly increased the rate of HSV detection in genital swabs compared to virus culture and that its implementation is feasible for routine diagnostic settings.

PCR was shown to increase the overall rate of HSV detection by 61-71%. Even in patients presenting with visible genital ulcerations PCR detected 88% more infections than virus culture. Thus, in the absence of PCR testing, 53 of the 233 symptomatic patients would have received a negative result, preventing appropriate counselling on prognosis and risk of transmission. Our result is striking considering that a similar study by Scoular showed a 24% increased detection rate by PCR over virus culture. This observation may in part be because our study was conducted during summer months. Warm weather may have affected HSV viability after specimen collection and further diminished the sensitivity of virus culture.

The increase in detection afforded by PCR was observed in different clinical presentations including both first and recurrent episodes. In contrast with previous observations, we did not observe a different yield of virus culture in patients with first episode compared with those with recurrent disease. Predictably, the increase in detection by PCR was especially high among patients presenting late (⩾5 days) after the onset of symptoms, as declining levels of virus shedding in older lesions would favour the more sensitive technique. None the less, detection also increased significantly in patients who presented within 5 days of onset. The absence of a significant difference between culture and PCR yields in patients without visible ulceration is interesting, but the small number of observations precludes speculations about its significance.

Unlike a previous study by Scoular et al, the PCR method we used offered the advantage of providing HSV typing without additional, time consuming post-PCR steps. Sequencing of a subset of HSV-2 positive samples confirmed the accuracy of typing. The low level of sequence variation observed indicated high reliability of the assay. Among 132 HSV positive specimens, 91% were HSV-2 and 9% were HSV-1. HSV-2 accounted for the majority of both first and recurrent episodes. These findings are in contrast with results from other centres in the United Kingdom, which report HSV-1 to be responsible for more than ⩾50% of newly diagnosed cases of genital herpes.

To investigate the feasibility of implementing HSV PCR for large volume testing, three specimen preparation methods were compared to evaluate performance, labour, and cost. A high level of agreement was observed between the three methods. Virus precipitation by PEG, although not previously used for LightCycler PCR, had the highest detection rates, was the least expensive, and required the shortest incubation period, thus providing a valid option for specimen processing before PCR. Even though PCR has demonstrated increased sensitivity over virus culture, its implementation into routine clinical practice has been limited by concerns of contamination. Extensive retesting of positive samples in our study demonstrated that real time PCR assays are reliable and highly reproducible. This is a result of amplification occurring within a closed system, with a minimal risk of carryover contamination, although reproducibility was also helped by the high prevalence of disease in our sample. Although the costs of consumables for PCR (£2.44-£9.27) are higher than those for virus culture (£1.00-£1.55), real time PCR proved cost efficient by significantly reducing labour cost per sample.

With high levels of asymptomatic shedding and high prevalence of non-classic presentations, laboratory investigations provide guidance for patient management and controlling the spread of HSV infection. The sensitivity of nucleic acid amplification tests exceeds conventional laboratory methods to detect HSV, thus enabling greater diagnostic accuracy. Although virus culture continues to be the standard diagnostic test used in routine practice in the United Kingdom (D Brown and AM Geretti, unpublished observation), its ability to detect HSV in a large proportion of infected individuals is clearly deficient compared to PCR. Our data strongly support the implementation of PCR for the routine diagnosis of genital herpes.

Acknowledgments

The authors thank Dr Benedict Murdin (University of Surrey in Guildford, UK), for statistical support.

CONTRIBUTORS Study design: AMG, MS, MT-F; experimental work: MR, DT, SMl, MS; coordination of sampling and clinical data collection: CMcD; analysis of results: MR, AMG, MS; preparation of manuscript: MR, AMG.

View Abstract

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Diagnosis of genital herpes by real time PCR in routine clinical practice

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Background: Virus isolation in cell culture is the recognised diagnostic gold standard for genital herpes. Although increasing evidence indicates that polymerase chain reaction (PCR) provides a more rapid and sensitive diagnostic method, its implementation in routine diagnostic settings has been limited by concerns over contamination and cost.

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185278 ATHIFAH BINTI ISMAIL
185290 AYU NURSURIA BINTI ISFAK AHMAD
185593 NOR SYAFIQAH BINTI SHUKOR
185606 HALIMAHTUN SAADIAH BINTI ZAKARIA
185719 NUR FARRAH NABILAH BINTI KHAIR YUSOF

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According to the article release by WHO in 2017, they stated that the infection with HSV-2 is lifelong and incurable. It also one of the disease with global issue since 417 million people worldwide were living with the infection in 2012.

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Herpes simplex virus fact sheet with key facts and information on type 1 (HSV-1) and type 2 (HSV-2): signs and symptoms, transmission, challenges, treatment, global impact and WHO response.

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Vaccine development

To date no efficient vaccine for HSV-2 is available. But researcher are still finding the suitable vaccine that would be able to inhibit or 🤬 the virus.

1. Blocking 🤬 Simplex Virus 2 Glycoprotein E Immune Evasion as an Approach To Enhance Efficacy of a Trivalent Subunit Antigen Vaccine for Genital 🤬

-to improve the immunogenicity and efficacy of a gD2-based subunit antigen vaccine by inducing potent immunity to three antigens, two of which prevent HSV immune evasion from antibody and complement.

- gE2 is a candidate antigen for inclusion in a multivalent subunit vaccine that attempts to block HSV-2 immune evasion.
-when gE2 was added to gC2/gD2 to form a trivalent vaccine, neutralizing antibody titers with and without complement were significantly higher than those produced by gD2 alone.
-Our results indicate that gE2 functions in synergy with gC2 to protect the virus from antibody and complement neutralization. The addition of gE2 to a gC2/gD2 subunit antigen vaccine provides strong protection against HSV-2 vaginal challenge, preventing death and genital disease, and a high level of protection to lumbosacral dorsal root ganglia (DRG), which serve as the site of HSV-2 latency.

2. Vaccines and Microbicides Preventing HIV-1, HSV-2, and HPV Mucosal Transmission

-HIV-1 and HSV-2 coinfections are very common. HSV-2, as the main source of genital ulcers, can facilitate HIV-1 transmission (reviewed in Freeman et al., 2006)
-HSV-2 reactivation at the genital level can stimulate HIV-1 replication (Schacker et al., 1998)
-Treatment of HSV-2 infection with valacyclovir dramatically reduces genital and plasma HIV-1 mRNA levels (Nagot et al., 2007).
-Currently, the two established prevention methods for HSV-2 transmission are condoms (Wald et al., 2005) and HSV-2 antiviral treatment (Corey et al., 2004).
- At present, only two HSV-2 vaccines have successfully completed phase III clinical trials(Figure 1).
-The first one contains HSV-2 surface glycoproteins gB and gD, combined with the adjuvant MF59, known to induce T helper cell type 2 (Th2) responses.
-However, this trial showed no protection against HSV-2 acquisition and occurrence of genital ulcers, despite the elicitation of relatively high titers of neutralizing antibodies (Corey et al., 1999).
-The second candidate vaccine combined the truncated glycoprotein gD2 with the adjuvant aluminum hydroxide linked to MPL (alu-MPL), a known inducer of Th1 responses.
-A good level of protection was achieved with this vaccine, but only in HSV-2-seronegative women (Stanberry et al., 2002).
-In conclusion, the HSV-2 vaccine field still faces some major immunological issues.
-Another type of microbicide against HSV-2 aims at blocking viral entry into host cells, keratinocytes, and neurons. PRO 2000 and cellulose sulfate are sulfated polymers that interact with HIV-1 gp120 and with HSV-2 gB, thereby preventing HIV-1 as well as HSV-2 entry into target cells (Cheshenko et al., 2004).
-A combination of Carraguard and zinc was investigated by the Population Council. This compound, named PC-710, showed greater efficiency of HSV-2 protection in animal models when compared with HIV-1 protection. It may therefore serve as a microbicide against HSV-2, but its lack of activity against HIV-1 in clinical trials is a matter of concern (Skoler-Karpoff et al., 2008).

3. Topical microbicides for the prevention of genital 🤬 infection

-  compounds that prevent entry into target cells may be most advantageous. PRO 2000, Carraguard, polystyrene sulphonate and cellulose sulphate are sulphated or sulphonated polymers that interact directly with viral envelope glycoproteins (including glycoprotein B of HSV-2 and gp120 of HIV) and competitively inhibit viral attachment and prevent entry.²⁰ These products exhibit little or no cytotoxicity even at high doses, do not disrupt normal vaginal flora and do not induce a significant inflammatory response. However, the concentration of SLPI, which may be important in protecting the host from HIV and HSV, is substantially reduced following acute or chronic exposure of human epithelial cells to sulphonated/sulphated polymers in vitro.²⁰ It remains to be determined whether the reduction of SLPI observed in vitro would increase susceptibility to HSV, HIV or other STI. A potential limitation of this class of compounds is that they do not actually 🤬 the pathogen, and thus optimally might be used in combination with other candidate agents. Several of these compounds are in Phase II or III clinical trials.

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Our mind map link for reference

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Herpes Simplex Virus : A Coggle Diagram about PATHOGEN (EPIDEMIOLOGY, TRANSMISSION, MECHANISM OF INFECTION, SYMPTOMS and TREATMENT), IMMUNOLOGY (INNATE IMMUNE RESPONSE, References, DETECTION USING IMMUNOLOGY METHOD and ADAPTIVE IMMUNE RESPONSE) and GENETIC (MOLECULAR DETECTION and VACCINE DEVELOPMENT)

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HSV can be a major risk factor for HIV infection.  There are studies that shows the anti HSV therapy can slow the rate and reduce the number of copies of HIV virus in the woman genital tracts. Currently the antiviral treatment can help to minimize the severity of the infection but the researcher still finding the new vaccine that could prevent or limiting the infection of HSV, There are a few of vaccine that are being tested but do not successful in the clinical trials because they are tested by using the mouse models which all the immune response that had been observed is mediated by a murine cell. So, to produce a vaccine that is suitable for human, the researcher has develop a humanized mouse strain that has the human umbilical cord that resulting to provide the mouse  the abilities to produce both innate and adaptive human immune cells.

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ABSTRACT

Genital herpes, caused by herpes simplex virus type 2 (HSV-2), is one of the most prevalent sexually transmitted diseases worldwide and a risk factor for acquiring human immunodeficiency virus. Although many vaccine candidates have shown promising results in animal models, they have failed to be effective in human trials. In this study, a humanized mouse strain was evaluated as a potential preclinical model for studying human immune responses to HSV-2 infection and vaccination. Immunodeficient mouse strains were examined for their abilities to develop human innate and adaptive immune cells after transplantation of human umbilical cord stem cells. A RAG2 −/− γc −/− mouse strain with a BALB/c background was chosen as the most appropriate model and was then examined for its ability to mount innate and adaptive immune responses to intravaginal HSV-2 infection and immunization. After primary infection, human cells in the lymph nodes were able to generate a protective innate immune response and produce gamma interferon (IFN-γ). After intravaginal immunization and infection, human T cells and NK cells were found in the genital tract and iliac lymph nodes. In addition, human T cells in the spleen, lymph nodes, and vaginal tract were able to respond to stimulation with HSV-2 antigens by replicating and producing IFN-γ. Human B cells were also able to produce HSV-2-specific immunoglobulin G. These adaptive responses were also shown to be protective and reduce local viral replication in the genital tract. This approach provides a means for studying human immune responses in vivo using a small-animal model and may become an important preclinical tool.

Genital herpes, caused primarily by herpes simplex virus type 2 (HSV-2), is one of the most prevalent sexually transmitted diseases in the world and is associated with substantial morbidity ( 13). After initial infection of the genital tract, the virus establishes latency within the nervous system and thus maintains lifelong infection in humans. Latent virus can reactivate and cause recurrent symptoms, including genital lesions; however, subclinical infection and asymptomatic viral shedding also occur ( 11, 35, 40, 53). HSV-2 has gained increasing interest in the light of evidence that it is a major risk factor for human immunodeficiency virus type 1 (HIV-1) acquisition and transmission and for the progression of HIV-1 infection ( 8, 9, 17, 25, 37, 55, 56). In addition, there is evidence that anti-HSV therapy can reduce the amount of infectious HIV-1 in the genital tracts of women ( 9, 45). Although antiviral treatment is available and can reduce the severity of the infection, compliance problems, as well as difficulty in diagnosing infection in patients, have hampered efforts to control the disease. A vaccine would provide a more effective way of preventing or limiting infection and would therefore greatly reduce the social and economic burdens caused by HSV-2 infection.

Several vaccine candidates exist; however, they have proven to be less successful in clinical trials than anticipated, and new strategies may need to be developed ( 24, 61). A key concern is that preclinical vaccine strategies have been evaluated largely by using studies performed with mouse models of HSV-2 infection and, thus, the immune responses observed were mediated by murine cells. As a consequence, the results of these studies may not accurately represent the human immune response to infection. In order to develop an effective vaccine and/or treatment, it is necessary to understand which immune mechanisms provide protection against infection at the site of viral entry, the vaginal tract, and how these immune responses can be induced in humans.

Innate and adaptive immune responses are both important for controlling HSV-2 infection. Innate immune cells such as NK and NKT cells are required for protection against genital HSV-2 infection in mice ( 1) and in humans; NK cells accumulate at sites of HSV-2 infection and can lyse HSV-infected cells ( 30, 67). Adaptive immune responses to HSV-2 include the cellular response mediated by CD4⁺ and CD8⁺ T cells and the humoral response mediated by B cells and antibodies. There is much evidence that T cells play a crucial role in protection against HSV-2 in mice and humans ( 28). T cells are present in herpes lesions, and depletion of T cells in mice greatly reduces protection ( 16, 27, 29, 30, 44, 51, 70). Gamma interferon (IFN-γ), which is produced early after infection by NK cells and later by CD4⁺ T cells, has been shown to be a crucial cytokine for the control of HSV ( 43, 52, 58, 63). Although HSV-2-specific antibodies are produced in response to infection and vaccination, a correlation with protection in humans has not been established ( 2, 3, 7, 10, 11, 48). In mice, a role for antibodies early after infection has been shown; however, if B cells are knocked out, mice are still able to eventually clear the virus ( 16, 50). Although we do not have a complete understanding of the components that are necessary for protection, it appears that both innate and adaptive immune responses will be required and that it will be important to elicit these responses at the site of infection in the genital tract.

The lack of an effective vaccine and accurate translation of results obtained with mice to humans indicates a need for a more relevant preclinical model to study human immune responses and disease. Substantial improvements in the development of humanized mice have made them a novel tool for the study of human diseases ( 69). Human CD34⁺ stem cells have been injected into several immunodeficient mouse strains, such as NOD/SCID/γc −/− and RAG2 −/− γc −/− mice, in which superior engraftment has resulted in multilineage differentiation of the human cells ( 23, 64). These novel humanized mice have been shown to develop human immune responses to pathogens such as Epstein-Barr virus, dengue virus, and influenza virus and to immunization with cholera toxin ( 33, 64, 66, 68). In addition, humanized mice can support infection with HIV after systemic or mucosal challenge in the vaginal tract and rectum ( 4- 6, 62, 65). HSV-2 infection in humanized mice has not been examined, and mucosal immunization that can provide protection from infection with wild-type virus has also not been demonstrated. In addition, although it is clear that adaptive immune responses can be generated in humanized mice, innate responses to viral infection have not been extensively examined.

In this study, we evaluated three immunodeficient mouse strains for their abilities to engraft human umbilical cord-derived stem cells and support the differentiation of these cells into important innate and adaptive immune cells. The most appropriate model was then used to examine mucosal immune responses following primary HSV-2 infection, immunization, and secondary HSV-2 challenge. We show for the first time that the humanized mice can mount protective human NK cell-mediated innate immune responses to primary mucosal infection with HSV-2. In addition, mucosal immunization and infection can induce HSV-2-specific antibody production and, to a greater extent, T-cell-mediated responses both systemically and locally in the genital tracts of humanized mice. We further show that mucosal immunization can provide protection against a lethal intravaginal (IVAG) challenge with HSV-2.

MATERIALS AND METHODS

RESULTS

DISCUSSION

A novel mouse model in which human immune responses to HSV-2 can be observed may be an important tool for preclinical evaluation of antiviral therapies and vaccines. This study has demonstrated the ability of an immunodeficient mouse strain to engraft human stem cells and support the development of human immune cells that are essential for innate and adaptive immunity to HSV-2. Importantly, the human immune cells can migrate to the vaginal tract and respond to mucosal primary infection, immunization, and secondary infection. The human immune response is robust enough to provide protection against a lethal dose of HSV-2 and significantly impair local viral replication.

In an attempt to identify the most appropriate murine recipient for human stem cells, we investigated three immunodeficient mouse strains for their abilities to support engraftment. The NOD/SCID strain has been used frequently to evaluate the health and repopulating ability of human stem cells ( 21, 36, 57). NOD/SCID mice lack murine T and B cells and have impaired innate immune functions, which allows for consistent and relatively high levels of human immune cell engraftment (as measured by the presence of human CD45⁺ cells in the bone marrow and spleen tissues). Although human immune cells from multiple myeloid, erythroid, and B-cell lineages have been detected in these mice, this study and others have shown that human T and NK cells do not develop ( 12, 20, 22, 26, 46). This lack of differentiation makes this model inappropriate for studies involving innate and adaptive immune responses, and it was therefore not used any further in this work. As an alternative to the NOD/SCID mutations, the RAG2 −/− and γc −/− mutations confer a complete and permanent lack of T, B, and NK cells. RAG2 −/− γc −/− mice with two different backgrounds, C57BL/6 and BALB/c, were tested. We evaluated both strains for their abilities to support human stem cell engraftment and differentiation. Interestingly, there was a complete contrast between the two strains, as the C57BL/6 mice did not support any engraftment while the BALB/c mice supported complete engraftment. In addition, the BALB/c RAG2 −/− γc −/− mice developed a thymus which contained human T cells, as well as mesenteric lymph nodes containing human T, B, and NK cells. It is not clear why there is such a disparity between the reconstitution results for these strains. Since the RAG2 −/− and γc −/− mutations are the same, differences in the background strain may be responsible. As the focus of this research was determining the most appropriate strain to use for studying the human innate and adaptive immune responses, we used the BALB/c RAG2 −/− γc −/− mice for all future experiments.

Our knowledge of the immune responses generated against HSV-2 has been obtained primarily from in vivo studies using mouse models of HSV-2 and in vitro studies using human samples. This approach has limited our ability to apply the results to in vivo human responses and thus understand what components of the immune response are essential for protection in humans. The innate immune response to HSV-2 is not fully understood, and our knowledge of in vivo human NK cell functions, such as IFN-γ production, is lacking. In mice, NK and NKT cells are required for protection against mucosal HSV-2 infection ( 1) and IFN-γ is produced locally after infection ( 43, 63). In humans, NK cells accumulate at the site of HSV-2 infection ( 30), and in one study, patients with severe HSV infections were found to be lacking in NK cells ( 15). In a separate study, we have shown that human NK and NKT cells develop in the BALB/c RAG2 −/− γc −/− mouse model and that these cells are functional (Kwant-Mitchell et al., submitted). They can produce human IFN-γ in response to IL-15 and can kill NK-sensitive K562 cells both in vitro and in vivo. In the present study, human NK cells developed in the spleens, thymuses, lymph nodes, and vaginal tracts of mice with reconstituted human cell populations. NK cells isolated from the mesenteric lymph nodes of infected mice produced human IFN-γ in response to stimulation with IL-15 and infection with HSV-2. In addition, the human innate immune response was capable of protecting mice against a primary genital HSV-2 infection and could significantly limit vaginal viral replication.

The adaptive immune response has been studied more extensively in an effort to understand which components are most important for protection against infection, limitation of symptoms, and prevention of transmission. In mice, the best protection against vaginal HSV-2 infection is achieved by immunizing with attenuated virus HSV-2 Tk −, delivered via the genital mucosa ( 52). CD4⁺ and CD8⁺ T-cell responses, as well as IFN-γ production, have been shown to play crucial roles in this protection ( 39, 41, 43, 44, 51). In addition, IgG has been shown to be the primary HSV-2-specific antibody in vaginal secretions following IVAG infection, whereas the role of secretory IgA seems to be less important ( 38, 42, 47, 49). In humans, it is not clear whether protective responses are mediated by T cells, antibodies, or some combination of the two. HSV-2-infected patients have been examined, and both HSV-2-specific antibodies and T cells have been detected in genital lesions ( 2, 14, 27, 28, 30, 54). However, results from several vaccine trials have indicated that the production of antibodies may not be sufficient for protection, and thus, cellular immunity is now regarded as an essential component of a protective human anti-HSV-2 immune response. In addition to the type of immune response that is important for protection, the best route of administration for a vaccine has been debated. Since HSV-2 infection is a mucosal infection that is initiated at the vaginal mucosa, many studies have been aimed at inducing local, genital immune responses as well as systemic ones. In mice, both intranasal and IVAG vaccine deliveries have proven to be effective at inducing genital immune responses, and there is evidence that intranasal immunization also elicits genital immune responses in humans ( 18, 19, 31, 32, 34, 60). In this study, we have shown that human T cells migrate to the vaginal tract after immunization and infection. In addition, iliac lymph nodes, which drain the genital tract, develop only after vaginal infection and also contain T cells. The human T cells from the spleen, lymph nodes, and vaginal tract are functional and respond specifically to HSV-2 antigens by replicating and producing human IFN-γ. Human B cells are also functional, as they produce antibodies which can be detected in the serum and are also able to make HSV-2-specific IgG after immunization and infection. Since engraftment, expansion, and homing of human cells is observed in our model, there is likely some degree of cross-species reactivity between mice and humans; however, this issue is known to represent a potential problem with current humanized mouse models. Indeed, several strategies to improve humanized mouse models involve making human-derived molecules more available to the engrafted cells.

The ultimate test of a vaccine is whether it can provide protection against infection with wild-type virus. In this study, two immunization approaches were used. One group of mice with reconstituted human cell populations was immunized IVAG with an attenuated strain of HSV-2, and another group was infected IVAG with a low dose of wild-type HSV-2. Both of these immunization strategies induced a human immune response that was strong enough to protect against a secondary IVAG wild-type HSV-2 infection. Furthermore, local viral titers in the genital tracts of immunized mice were kept significantly lower than those in the genital tracts of unimmunized mice, and many immunized mice showed no detectable virus. It is therefore clear from these studies that the human immune cells in the mice are functional and are able to mount antigen-specific systemic and mucosal immune responses after immunization and infection.

HSV-2 is becoming more prevalent in the human population and is emerging as a significant risk factor for HIV infection. There is no effective vaccine for HSV-2, and many candidates that showed promise in murine studies showed little effect in humans. The present study outlines a model that may potentially be used for preclinical testing of vaccines, as well as the study of human immune responses in an in vivo setting. Humanized BALB/c RAG2 −/− γc −/− mice can mount both innate and adaptive immune responses to HSV-2, and human immune cells can migrate into the genital mucosa, where natural infection most often occurs.

ACKNOWLEDGMENTS

We thank M. Ito at the Central Institute for Experimental Animals, Kawasaki, Japan, for generously providing us with BALB/c RAG2 −/− γc −/− mice.

This work was supported by grants from the Canadian Institutes of Health Research (CIHR). K.L.R. is partially supported by a Career Scientist Award from the Ontario HIV Treatment Network (OHTN).

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Mucosal Innate and Adaptive Immune Responses against Herpes Simplex Virus Type 2 in a Humanized Mouse Model

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Genital herpes, caused by herpes simplex virus type 2 (HSV-2), is one of the most prevalent sexually transmitted diseases worldwide and a risk factor for acquiring human immunodeficiency virus. Although many vaccine candidates have shown promising results in animal models, they have failed to be effective in human trials.

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From the studies, the humanized mouse cell can produce lymph node and thymus. The innate immune response is able to protect the mice against primary genital HSV-2 infection and could significantly limit vaginal viral replication.It occur after the infection is the production of the NK cells that can lyse the HSV infected cell. Cytokine that produce from NK cells and also CD4⁺ is gamma interferon(IFN-ɣ) . NK cells found to be accumulate at the site of infection and produce by spleen, thymus and vaginal tract. NK produce gamma interferon after being induce by IL-15  and infection with HSV-2

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Then for the adaptive, responsible for the protection against infection,limitation of symptoms and preventation of transmission. there are cellular response that are mediated by CD4⁺ and CD8⁺ . Then the humoral response also produce mediated by B cell producing antibodies. Human T cells starts to multiply  after exposed to the HSV antigen. Lymph node and spleen start to produce gamma interferon(IFN-ɣ) locally after the infection occured. Human B cells also produce specific HSV atigent which is IgG

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Vaccine that are still in the research are the IVAG with attenuated strain of HSV-2 and the IVAG with a low dose of wild type HSV-2. Both shown the positive result in induced the human immune response and can be used for protection against secondary IVAG wild typeHSV-2 infection. Both of these trial vaccine may be potentially used in the preclinical testing of vaccine

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In vitro (Latin for within the glass) refers to the technique of performing a given procedure in a controlled environment outside of a living organism. Many experiments in cellular biology are conducted outside of organisms or cells. One of the abiding weaknesses of in vitro experiments is that they fail to replicate the precise cellular conditions of an organism, particularly a microbe. To cite one example among many, the lysates or extracts from culture-grown spirochetes do not reflect antigens expressed in the mammalian Borrelia:

In vivo (Latin for “within the living”) refers to experimentation using a whole, living organism as opposed to a partial or dead organism. Animal studies and clinical trials are two forms of in vivo research. In vivo testing is often employed over in vitro because it is better suited for observing the overall effects of an experiment on a living subject.