Baculovirus Vector-Based Varicella-Zoster Virus Vaccine as a Promising Alternative with Enhanced Safety and Therapeutic Functions.

Varicella-zoster virus (VZV) poses lifelong risks, causing varicella and herpes zoster (HZ, shingles). Currently, varicella and HZ vaccines are predominantly live attenuated vaccines or adjuvanted subunit vaccines utilizing VZV glycoprotein E (gE). Here, we propose our vaccine candidates involving a comparative analysis between recombinant baculoviral vector vaccines (AcHERV) and a live attenuated vaccine strain, vOka. AcHERV vaccine candidates were categorized into groups encoding gE only, VZV glycoprotein B (gB) only, or both gE and gB (gE-gB) as AcHERV-gE, AcHERV-gB, and AcHERV-gE-gB, respectively. Humoral immune responses were evaluated by analyzing total IgG, IgG1, IgG2a, and neutralizing antibodies. Cell-mediated immunity (CMI) responses were evaluated by enzyme-linked immunospot (ELISPOT) assay and Th1/Th2/Th17 cytokine profiling. In the mouse model, AcHERV-gE-gB elicited similar or higher total IgG, IgG2a, and neutralizing antibody levels than vOka and showed robust VZV-specific CMI responses. From the perspective of antigens encoded in vaccines and their relationship with CMI response, both AcHERV-gB and AcHERV-gE-gB demonstrated results equal to or superior to AcHERV-gE, encoding only gE. Taken together, these results suggest that AcHERV-gE-gB can be a novel candidate for alleviating risks of live attenuated vaccine-induced latency and effectively preventing varicella during early stages of life while providing strong CMI for effective resistance against HZ and therapeutic potential in later stages of life.

IFN-α armed gE elicits superior immunogenicity compared to unmodified antigens and flagellin armed gE in mice.

Herpes zoster (HZ) induces significant pain and discomfort, which can seriously affect the quality of life of patients. At present, there is no specific treatment for HZ, and the mosteffective HZ control is vaccination. The main obstacle to developing an effective HZ vaccine is poorly induced cellular immune response. In this study, the IFN-α-gE-Fc fusion protein induced higher levels of humoral and cellular immunity compared to the unengineered gE antigen and higher levels of cellular immunity compared to the flagellin-gE-Fc fusion protein in a murine model. Compared with the marketed recombinant herpes zoster vaccine (Shingrix), IFN-α-gE-Fc can replace current used MPL adjuvant. At the same time, the immunogenicity of the IFN-α-gE-Fc + AQ was not weaker than that of the marketed recombinant zoster vaccine. The novel fusion protein provides a candidate entity for the development of a safe and effective novel HZ vaccine.

Pre-Clinical Development of an Adenovirus Vector Based RSV and Shingles Vaccine Candidate.

Respiratory syncytial virus (RSV) infection and shingles are two viral diseases that affect older adults, and a combined vaccine to protect against both could be beneficial. RSV infection causes hospitalisations and significant morbidity in both children and adults and can be fatal in the elderly. The RSV fusion (F) envelope glycoprotein induces a strong RSV-neutralising antibody response and is the target of protective immunity in the first RSV vaccine for older adults, recently approved by the FDA. An initial childhood infection with the varicella zoster virus (VZV) results in chickenpox disease, but reactivation in older adults can cause shingles. This reactivation in sensory and autonomic neurons is characterized by a skin-blistering rash that can be accompanied by prolonged pain. The approved protein-in-adjuvant shingles vaccine induces VZV glycoprotein E (gE)-fspecific antibody and CD4+ T cell responses and is highly effective. Here we report the evaluation of RSV/shingles combination vaccine candidates based on non-replicating chimpanzee adenovirus (ChAd) vectors. We confirmed the cellular and humoral immunogenicity of the vaccine vectors in mice using T cell and antibody assays. We also carried out an RSV challenge study in cotton rats which demonstrated protective efficacy following a homologous prime-boost regimen with our preferred vaccine candidate.

Herpes Virus Infections in Kidney Transplant Patients (HINT) - a prospective observational cohort study.

BACKGROUND: Kidney transplant recipients receive maintenance immunosuppressive therapy to avoid allograft rejection resulting in increased risk of infections and infection-related morbidity and mortality. Approximately 98% of adults are infected with varicella zoster virus, which upon reactivation causes herpes zoster. The incidence of herpes zoster is higher in kidney transplant recipients than in immunocompetent individuals, and kidney transplant recipients are at increased risk of severe herpes zoster-associated disease. Vaccination with adjuvanted recombinant glycoprotein E subunit herpes zoster vaccine (RZV) prevents herpes zoster in older adults with excellent efficacy (90%), and vaccination of kidney transplant candidates is recommended in Danish and international guidelines. However, the robustness and duration of immune responses after RZV vaccination, as well as the optimal timing of vaccination in relation to transplantation remain unanswered questions. Thus, the aim of this study is to characterize the immune response to RZV vaccination in kidney transplant candidates and recipients at different timepoints before and after transplantation. METHODS: The Herpes Virus Infections in Kidney Transplant Patients (HINT) study is a prospective observational cohort study. The study will include kidney transplant candidates on the waiting list for transplantation (n = 375) and kidney transplant recipients transplanted since January 1, 2019 (n = 500) from all Danish kidney transplant centers who are offered a RZV vaccine as routine care. Participants are followed with repeated blood sampling until 12 months after inclusion. In the case of transplantation or herpes zoster disease, additional blood samples will be collected until 12 months after transplantation. The immune response will be characterized by immunophenotyping and functional characterization of varicella zoster virus-specific T cells, by detection of anti-glycoprotein E antibodies, and by measuring cytokine profiles. DISCUSSION: The study will provide new knowledge on the immune response to RZV vaccination in kidney transplant candidates and recipients and the robustness and duration of the response, potentially enhancing preventive strategies against herpes zoster in a population at increased risk. TRIAL REGISTRATION: ClinicalTrials.gov (NCT05604911).

Identification of neutralizing epitopes on the D/A domain of the E2 glycoprotein of classical swine fever virus.

Classical swine fever virus (CSFV) shares high antigenic homology with other members of the genus Pestivirus. Because several pestivirus species can also infect swine, eliciting cross-reactive antibodies, it is important to define CSFV-specific epitopes for the differential diagnosis of classical swine fever (CSF) by serology. For this purpose, epitope mapping of seven monoclonal antibodies (mAbs), recognizing sites on the D/A domain of glycoprotein E2, was performed using recombinant expressed antigenic domains and mutants of E2, as well as an overlapping peptide library. Three CSFV-specific epitopes, i.e., 780-IEEMGDDFGFGLCPF-794, 810-NGSAFYLVCPIGWTG-824, and 846-REKPF-850, were identified within the D/A domain of E2. Site-directed mutagenesis further confirmed that residues 783-MGD-785, 789-FGLCPF-794, 813-AFYLVCPIGWTG-824, and 846-REK-848 were critical residues in these regions. In addition, a F789S difference within the epitope 780-IEEMGDDFGFGLCPF-794 was responsible for the absence of binding of two mAbs to the E2 protein of the live attenuated CSFV vaccine strain Riems. Structural modeling revealed that, the three epitopes are located near each other, suggesting that they may form a more complex conformational epitope on the D/A domain in vivo. Six of the mAbs neutralized viruses of diverse genotypes, indicating that the target epitopes are involved in virus interaction with cells. The binding of CSFV to cells was significantly reduced after pre-incubation with either truncated E2 proteins comprising the D/A domain or with the CSFV-specific mAbs targeting the domain D/A. These epitopes identified on the D/A domain are important targets for virus neutralization that might be involved in the early steps of CSFV infection. These findings reveal potential candidates for improving the differential diagnosis of pestiviruses by serology.

Rapid detection of varicella-zoster virus based on an immunochromatographic strip.

Varicella-zoster virus (VZV) is a highly infectious DNA virus that can cause varicella (chickenpox) and herpes zoster (HZ). A simple, sensitive and specific detection method is desirable for the VZV infection. In this study, VZV gE protein, expressed in CHO cells, was used to immunize BALB/c mice for the generation of monoclonal antibodies (mAbs). For the first time, we developed a colloidal gold-based immunochromatographic strip for rapid detection of VZV using a pair of mAbs against gE protein. The limit of detection (LOD) of the strip was 30 ng mL-1 of purified VZV gE antigen, and it could specifically test VZV without cross-reactivity with Enterovirus 71 (EV-71), Herpes simplex virus 1 (HSV-1) and Herpes simplex virus 2 (HSV-2). The coincidence rate between the strip and commercial real-time PCR diagnostic kit was 100% using vesicle as the clinical sample. Our strip provided a technical support for rapid and specific detection of VZV.

Safety and immunogenicity of a TK/ gI/gE gene-deleted feline herpesvirus-1 mutant constructed via CRISPR/Cas9 in feline.

Feline herpesvirus-1 (FHV-1) is the aetiological agent of feline viral rhinotracheitis, which accounts for approximately 50 % of all viral upper respiratory diseases in cats. Commercially available modified live vaccines containing FHV-1 are generally safe and effective, but these FHV-1 vaccines retain full virulence genes and can establish latency and reactivate to cause infectious rhinotracheitis in vaccine recipients, raising safety concerns. To address this shortcoming, we constructed a novel TK/gI/gE -gene-deleted recombinant FHV-1 (WH2020-ΔTK/gI/gE) through CRISPR/Cas9-mediated homologous recombination. The growth kinetics of WH2020-ΔTK/gI/gE were slightly delayed compared to those of the parent strain WH2020. Recombinant FHV-1 had severely impaired pathogenicity in cats. Felines immunized with WH2020-ΔTK/gI/gE produced high levels of gB-specific antibodies, neutralizing antibodies and IFN-ß. Additionally, WH2020-ΔTK/gI/gE provided greater protection against challenge with FHV-1 field strain WH2020 than did the commercial modified live vaccine. After challenge, the cats vaccinated with WH2020-ΔTK/gI/gE showed significantly fewer clinical signs, pathological changes, viral shedding, and viral loads in the lung and trigeminal ganglia than those vaccinated with the commercial vaccine or unvaccinated. Our results suggest that WH2020-ΔTK/gI/gE is a promising candidate as a safer and more efficacious live FHV-1 vaccine, with a decreased risk of vaccine-related complications, and could inform the design of other herpesvirus vaccines.

Preclinical immunogenicity of an adenovirus-vectored vaccine for herpes zoster.

Herpes zoster (HZ) results from waning immunity following childhood infection with varicella zoster virus (VZV) but is preventable by vaccination with recombinant HZ vaccine or live HZ vaccine (two doses or one dose, respectively). Vaccine efficacy declines with age, live HZ vaccine is contraindicated in immunosuppressed individuals, and severe local reactogenicity of recombinant HZ vaccine is seen in up to 20% of older adults, indicating a potential need for new vaccines. Nonreplicating chimpanzee adenovirus (ChAd) vectors combine potent immunogenicity with well-established reactogenicity and safety profiles. We evaluated the cellular and humoral immunogenicity of ChAdOx1 encoding VZV envelope glycoprotein E (ChAdOx1-VZVgE) in mice using IFN-γ ELISpot, flow cytometry with intracellular cytokine staining, and ELISA. In outbred CD-1 mice, one dose of ChAdOx1-VZVgE (1 × 107 infectious units) elicited higher gE-specific T cell responses than two doses of recombinant HZ vaccine (1 µg) or one dose of live HZ vaccine (1.3 × 103 plaque-forming units). Antibody responses were higher with two doses of recombinant HZ vaccine than with two doses of ChAdOx1-VZVgE or one dose of live HZ vaccine. ChAdOx1-VZVgE boosted T cell and antibody responses following live HZ vaccine priming. The frequencies of polyfunctional CD4+ and CD8+ T cells expressing more than one cytokine (IFN-γ, TNF-α and IL-2) were higher with ChAdOx1-VZVgE than with the conventional vaccines. Results were similar in young and aged BALB/c mice. These findings support the clinical development of ChAdOx1-VZVgE for prevention of HZ in adults aged 50 years or over, including those who have already received conventional vaccines.

Truncated glycoprotein E of varicella-zoster virus is an ideal immunogen for Escherichia coli-based vaccine design.

Varicella-zoster virus (VZV) is a highly infectious agent responsible for both varicella and herpes zoster disease. Despite high efficacy, there remain safety and accessibility concerns with the licensed vaccines. Here, we sought to produce a VZV gE immunogen using an E. coli expression system. We found that the soluble expression and yield of gE protein could be enhanced via C-terminal truncations to the protein, thereby facilitating a robust and scalable purification process for the purpose of vaccine manufacturing. The lead truncated gE (aa 31-358), hereafter referred to as tgE, was a homogenous monomer in solution and showed excellent antigenicity. Finally, we assessed and compared the immunogenicity of tgE with commercial vOka LAV and Shingrix vaccine. We found that aluminum-adjuvanted tgE was immunogenic as compared with vOka LAV. When adjuvanted with AS01B, a two-dose immunization of tgE showed comparable or better potency in antibody responses and cell-mediated immunity with those of the Shingrix vaccine at the same dosage, especially in terms of the proportion of IFN-γ-expressing CD4+ T cells. In conclusion, this method of E. coli-mediate tgE expression offers a cost-effective and scalable strategy to generate an ideal VZV gE immunogen for the development of both varicella and zoster vaccines.

Live Triple Gene-Deleted Pseudorabies Virus-Vectored Subunit PCV2b and CSFV Vaccine Undergoes an Abortive Replication Cycle in the TG Neurons following Latency Reactivation.

Like other alpha herpesviruses, pseudorabies virus (PRV) establishes lifelong latency in trigeminal ganglionic (TG) neurons. Upon stress, the latent viruses in the TG neurons reactivate and are transported anterograde from the neuron cell bodies to the nerve endings in the nasal mucosa, where they replicate and are discharged in the nasal and oral secretions. Consequently, the virus is transmitted to other naïve animals. This cycle of latency and reactivation continues until the animal dies or is slaughtered. We have constructed a PRV triple mutant virus (PRVtmv) and used it as a live subunit vaccine vector against porcine circovirus 2b (PCV2b) and classical swine fever virus (CSFV) (PRVtmv+). We compared the latency reactivation properties of PRVtmv+ with its parent wild-type (wt) Becker strain following intranasal infection. The results showed that PRV wt and PRVtmv+ established latency in the TG neurons. Based on nasal virus shedding, immediate early (infected cell protein 0; ICP0) and late genes, MCP (major capsid protein) and gC (glycoprotein C) transcriptions, and viral DNA copy numbers in the TGs of latently infected and dexamethasone (Dex)-treated pigs, both PRV wt and PRVtmv+ reactivated from latency. We noticed that PRV wt virus replicated productively in the terminally differentiated, postmitotic TG neurons, but PRVtmv+ failed to replicate and, therefore, there was no virus production in the TG. In addition, we found that only the PRV wt virus was shed in the nasal secretions following the Dex-induced reactivation. Our results demonstrated that the PRVtmv+ is safe as a live viral subunit vaccine vector without the possibility of productive replication in the TG upon reactivation from latency and without subsequent nasal virus shedding. This property of PRVtmv+ precludes the possibility of vaccine virus circulation in pigs and the risk of reversion to virulence.

Establishment and verification of double antibody sandwich ELISA for detection of content of varicella-zoster virus glycoprotein E / 中国生物制品学杂志

@#ObjectiveTo develop and verify a double antibody sandwich ELISA for the quantitative detection of varicellazoster virus(VZV)glycoprotein E(gE).MethodsHybridoma cell lines secreting antibody against VZV-gE stably were screened by mouse hybridoma fusion technology,using VZV whole virus antigen as immunogen.The antibody titer in mouse ascites was detected by indirect ELISA.After purified by Hi Trap2=0.995 6,P=0.000 1)。结论 建立的双抗体夹心ELISA法具有良好的准确性、精密性和特异性，可用于VZV疫苗中g E抗原含量的快速检测。 ObjectiveTo develop and verify a double antibody sandwich ELISA for the quantitative detection of varicellazoster virus(VZV)glycoprotein E(gE).MethodsHybridoma cell lines secreting antibody against VZV-gE stably were screened by mouse hybridoma fusion technology,using VZV whole virus antigen as immunogen.The antibody titer in mouse ascites was detected by indirect ELISA.After purified by Hi Trap(TM)Mabselect(TM)Mabselect(TM)Su Re and Hi Trap(TM)Su Re and Hi Trap(TM)Desalting,monoclonal antibodies(m Abs)were analyzed for the purity by 12%SDS-PAGE,detected for the specificity by Western blot,and identified for the subtype by mouse monoclonal antibody typing kit.Capture antibody and enzyme-labeled antibody were screened by epitope superposition test,which were determined for the working concentrations by chessboard titration(capture antibody concentrations were 0.25,0.5,1,2,5 and 10μg/m L,enzyme-labeled antibody dilutions were 1∶500,1∶1 000,1∶2 000,1∶5 000 and 1∶10 000),and then a double antibody sandwich ELISA(DAS-ELISA)was developed for the detection of VZV-gE content.In addition,the linear range,accuracy,precision and specificity of the method were verified.The gE content in the supernatant of 3 batches of CHO-VZV-gE cells cultured in 7 L bioreactor for 1～14 d were detected by the developed method.ResultsFour positive hybridoma cell lines secreting specific antibodies against VZV-gE stably were obtained and named as m Ab-B2,m Ab-11,K9C7 and K9F4.The antibodies in mouse ascites showed titers of10(TM)Desalting,monoclonal antibodies(m Abs)were analyzed for the purity by 12%SDS-PAGE,detected for the specificity by Western blot,and identified for the subtype by mouse monoclonal antibody typing kit.Capture antibody and enzyme-labeled antibody were screened by epitope superposition test,which were determined for the working concentrations by chessboard titration(capture antibody concentrations were 0.25,0.5,1,2,5 and 10μg/m L,enzyme-labeled antibody dilutions were 1∶500,1∶1 000,1∶2 000,1∶5 000 and 1∶10 000),and then a double antibody sandwich ELISA(DAS-ELISA)was developed for the detection of VZV-gE content.In addition,the linear range,accuracy,precision and specificity of the method were verified.The gE content in the supernatant of 3 batches of CHO-VZV-gE cells cultured in 7 L bioreactor for 1～14 d were detected by the developed method.ResultsFour positive hybridoma cell lines secreting specific antibodies against VZV-gE stably were obtained and named as m Ab-B2,m Ab-11,K9C7 and K9F4.The antibodies in mouse ascites showed titers of106～106～107with purities of about 97%after purification,which all specifically bound to VZV whole virus protein with light chains ofκchain and heavy chains of Ig G_(2b),Ig G_1,Ig G_(2b)and Ig G_(2a)respectively.m Ab-B2 was determined as capture antibody and HPR-labeled m Ab-11 as enzyme-labeled antibody with the optimum working concentrations of 1.5μg/m L and 1∶5 000respectively.The internal reference concentration of gE antigen was in the range of 1.95～1 000 ng/m L,which showed a good linear relationship with A_(450).The four-parameter equation was Y=(0.15-3.99)/[1+(X/67.4)7with purities of about 97%after purification,which all specifically bound to VZV whole virus protein with light chains ofκchain and heavy chains of Ig G_(2b),Ig G_1,Ig G_(2b)and Ig G_(2a)respectively.m Ab-B2 was determined as capture antibody and HPR-labeled m Ab-11 as enzyme-labeled antibody with the optimum working concentrations of 1.5μg/m L and 1∶5 000respectively.The internal reference concentration of gE antigen was in the range of 1.95～1 000 ng/m L,which showed a good linear relationship with A_(450).The four-parameter equation was Y=(0.15-3.99)/[1+(X/67.4)(1.49)]+3.99,and R(1.49)]+3.99,and R2value was 0.999.The recoveries of accuracy verification were 94.9%～114.0%;The coefficients of variation(CVs)of precision verification were all less than 15%.Except CHO-VZV-gE cell culture supernatant,attenuated live varicella vaccine and attenuated live herpes zoster vaccine,the A_(450)of other samples were all less than 0.15 with no cross reaction.The content of gE in the supernatant of three batches CHO-VZV-gE cells increased gradually with the culture time,and was positively related with culture time within 14 days(R2value was 0.999.The recoveries of accuracy verification were 94.9%～114.0%;The coefficients of variation(CVs)of precision verification were all less than 15%.Except CHO-VZV-gE cell culture supernatant,attenuated live varicella vaccine and attenuated live herpes zoster vaccine,the A_(450)of other samples were all less than 0.15 with no cross reaction.The content of gE in the supernatant of three batches CHO-VZV-gE cells increased gradually with the culture time,and was positively related with culture time within 14 days(R2=0.995 6,P=0.000 1).ConclusionThe developed double antibody sandwich ELISA had good accuracy,precision and specificity,which might be used for rapid detection of gE antigen content in VZV vaccine.

Novel BC02 Compound Adjuvant Enhances Adaptive and Innate Immunity Induced by Recombinant Glycoprotein E of Varicella-Zoster Virus.

Both adaptive and innate immunity responses are necessary for the efficient elimination of different pathogens. However, the magnitude, quality and desired type of immune response specific to the co-administered antigen is largely determined by adjuvants. BC02 (BCG CpG DNA compound adjuvants system 02) is a novel compound adjuvant with independent intellectual properties, which is composed of BCG CpG DNA biological adjuvant with Al(OH)3 inorganic salt adjuvant acting as a delivery system. Its safety and strong adjuvant efficacy have been effectively verified in preclinical and clinical trials (Phase Ib, ClinicalTrials.gov Identifier: NCT04239313 and Phase II, ClinicalTrials.gov Identifier: NCT05284812). In this study, we report the level of cell-mediated immunity (CMI) and humoral immune response induced by the BC02 novel adjuvant combined with different doses of varicella-zoster virus (VZV) glycoprotein E (gE) in a mouse model. In addition, we conducted preliminary in vitro experiments to explore the enhancement of RAW264.7 cell immune activity by BC02 adjuvanted-gE experimental vaccine to activate innate immune response. The results showed that the BC02-adjuvanted low, medium or high dose of gE were highly effective in eliciting both CMI and humoral immune responses to the immunized mice, respectively. The production of gE-specific IFN-γ and IL-2-specific T cells was established within 28 days after booster immunization. In particular, the effect of BC02-adjuvanted medium dose of gE has been shown to be more prominent. Meanwhile, fluorescent antibody to membrane antigen (FAMA) and serum antibody plaque reduction tests have also shown that the BC02 adjuvanted-medium dose of gE antigen could induce the secretion of neutralizing antibodies against clinically isolated VZV strains in mice. In addition, our findings have shown that 1/25 dose of gE+BC02 medium dose experimental vaccine can significantly induce the secretion of innate immune cytokines TNF-A, MCP-1, IL-6 and GM-CSF and up-regulate the costimulatory molecules CD40, CD80 and I-A/I-E on RAW264.7 cells; and it has also been activated to form M2 macrophages. At the same time, RAW264.7 cells were stimulated for 12 h, and their phagocytosis was significantly enhanced. Taken together, these results suggest that the BC02 compound adjuvant offers a strategy to induce an appropriate innate and adaptive immunity against the different doses of the VZV gE protein to improve subunit vaccine efficacy, and BC02 may be a promising adjuvant candidate for subunit HZ vaccines.

The first study on analysis of the codon usage bias and evolutionary analysis of the glycoprotein envelope E2 gene of seven Pestiviruses.

Background and Aim: Pestivirus, a genus of the Flaviviridae family, comprises viruses that affect bovines, sheep, and pigs. Symptoms, including hemorrhagic syndromes, abortion, respiratory complications, and deadly mucosal diseases, are produced in infected animals, which cause huge economic losses to the farmers. Bovine viral diarrhea virus-1, bovine viral diarrhea virus-2, classical swine fever virus, border disease virus, Bungowannah, Hobi-like, and atypical porcine pestivirus belonging to the Pestivirus genus were selected for the study. This study aimed to estimate the codon usage bias and the rate of evolution using the glycoprotein E2 gene. Furthermore, codon usage bias analysis was performed using publicly available nucleotide sequences of the E2 gene of all seven Pestiviruses. These nucleotide sequences might elucidate the disease epidemiology and facilitate the development of designing better vaccines. Materials and Methods: Coding sequences of the E2 gene of Pestiviruses A (n = 89), B (n = 60), C (n = 75), D (n = 10), F (n = 07), H (n = 52), and K (n = 85) were included in this study. They were analyzed using different methods to estimate the codon usage bias and evolution. In addition, the maximum likelihood and Bayesian methodologies were employed to analyze a molecular dataset of seven Pestiviruses using a complete E2 gene region. Results: The combined analysis of codon usage bias and evolutionary rate analysis revealed that the Pestiviruses A, B, C, D, F, H, and K have a codon usage bias in which mutation and natural selection have played vital roles. Furthermore, while the effective number of codons values revealed a moderate bias, neutrality plots indicated the natural selection in A, B, F, and H Pestiviruses and mutational pressure in C, D, and K Pestiviruses. The correspondence analysis revealed that axis-1 significantly contributes to the synonymous codon usage pattern. In this study, the evolutionary rate of Pestiviruses B, H, and K was very high. The most recent common ancestors of all Pestivirus lineages are 1997, 1975, 1946, 1990, 2004, 1990, and 1990 for Pestiviruses A, B, C, D, F, H, and K, respectively. This study confirms that both mutational pressure and natural selection have played a significant role in codon usage bias and evolutionary studies. Conclusion: This study provides insight into the codon usage bias and evolutionary lineages of pestiviruses. It is arguably the first report of such kind. The information provided by the study can be further used to elucidate the respective host adaptation strategies of the viruses. In turn, this information helps study the epidemiology and control methods of pestiviruses.

Construction and characterisation of glycoprotein E and glycoprotein I deficient mutants of Australian strains of infectious laryngotracheitis virus using traditional and CRISPR/Cas9-assisted homologous recombination techniques.

In alphaherpesviruses, glycoproteins E and I (gE and gI, respectively) form a heterodimer that facilitates cell-to-cell spread of virus. Using traditional homologous recombination techniques, as well as CRISPR/Cas9-assisted homologous recombination, we separately deleted gE and gI coding sequences from an Australian field strain (CSW-1) and a vaccine strain (A20) of infectious laryngotracheitis virus (ILTV) and replaced each coding sequence with sequence encoding green fluorescent protein (GFP). Virus mutants in which gE and gI gene sequences had been replaced with GFP were identified by fluorescence microscopy but were unable to be propagated separately from the wildtype virus in either primary chicken cells or the LMH continuous chicken cell line. These findings build on findings from a previous study of CSW-1 ILTV in which a double deletion mutant of gE and gI could not be propagated separately from wildtype virus and produced an in vivo phenotype of single-infected cells with no cell-to-cell spread observed. Taken together these studies suggest that both the gE and gI genes have a significant role in cell-to-cell spread in both CSW-1 and A20 strains of ILTV. The CRISPR/Cas9-assisted deletion of genes from the ILTV genome described in this study adds this virus to a growing list of viruses to which this approach has been used to study viral gene function.

Baculovirus Display of Varicella-Zoster Virus Glycoprotein E Induces Robust Humoral and Cellular Immune Responses in Mice.

Varicella-zoster virus (VZV) is the causative agent of varicella and herpes zoster (HZ) and can pose a significant challenge to human health globally. The initial VZV infection-more common in children-causes a self-limiting chicken pox. However, in later life, the latent VZV can become reactivated in these patients, causing HZ and postherpetic neuralgia (PHN), a serious and painful complication. VZV glycoprotein E (gE) has been developed into a licensed subunit vaccine against HZ (Shingrix). However, its efficacy relies on the concomitant delivery of a robust adjuvant (AS01B). Here, we sought to create a new immunogen for vaccine design by displaying the VZV-gE on the baculovirus surface (Bac-gE). Correct localization and display of gE on the engineered baculovirus was verified by flow cytometry and immune electron microscopy. We show that Bac-gE provides excellent antigenicity against VZV and induces not only stronger gE-specific CD4+ and CD8+ T cell responses but also higher levels of VZV-specific neutralizing antibodies as compared with other vaccine strategies in mice. Collectively, we show that the baculovirus display of VZV-gE confers ideal humoral and cellular immune responses required for HZ vaccine development, paving the way for a baculovirus-based vaccine design.

Alphaherpesvirus glycoprotein E: A review of its interactions with other proteins of the virus and its application in vaccinology.

The viral envelope glycoprotein E (gE) is required for cell-to-cell transmission, anterograde and retrograde neurotransmission, and immune evasion of alphaherpesviruses. gE can also interact with other proteins of the virus and perform various functions in the virus life cycle. In addition, the gE gene is often the target gene for the construction of gene-deleted attenuated marker vaccines. In recent years, new progress has been made in the research and vaccine application of gE with other proteins of the virus. This article reviews the structure of gE, the relationship between gE and other proteins of the virus, and the application of gE in vaccinology, which provides useful information for further research on gE.

Functional Analysis of a Frontal miRNA Cluster Located in the Large Latency Transcript of Pseudorabies Virus.

MicroRNAs (miRNAs) have been identified as a class of crucial regulators of virus-host crosstalk, modulating such processes as viral replication, antiviral immune response, viral latency, and pathogenesis. Pseudorabies virus (PRV), a model for the study of alphaherpesvirus biology, codes for 11 distinct miRNAs mapped to the ~4.6 kb intron of Large Latency Transcript (LLT). Recent studies have revealed the role of clusters consisting of nine and eleven miRNA genes in the replication and virulence of PRV. The function of separate miRNA species in regulating PRV biology has not been thoroughly investigated. To analyze the regulatory potential of three PRV miRNAs located in the frontal cluster of the LLT intron, we generated a research model based on the constitutive expression of viral miRNAs in swine testis cells (ST_LLT [1-3] cell line). Using a cell culture system providing a stable production of individual miRNAs at high levels, we demonstrated that the LLT [1-3] miRNA cluster significantly downregulated IE180, EP0, and gE at the early stages of PRV infection. It was further determined that LLT [1-3] miRNAs could regulate the infection process, leading to a slight distortion in transmission and proliferation ability. Collectively, our findings indicate the potential of LLT [1-3] miRNAs to retard the host responses by reducing viral antigenic load and suppressing the expansion of progeny viruses at the early stages of infection.

Immunogenicity of Varicella Zoster Virus DNA Vaccines Encoding Glycoprotein E and Immediate Early Protein 63 in Mice.

Herpes zoster (HZ) is caused by the reactivation of latent varicella-zoster virus (VZV) from the sensory ganglia due to aging or immunosuppression. Glycoprotein E (gE) is a widely used vaccine antigen for specific humoral and cellular immune responses. Immediate early protein 63 (IE63) is expressed during latency, suggesting that it is a potential antigen against HZ reactivation. In this study, HZ DNA vaccines encoding gE, IE63, IE63-2A-gE (where 2A is a self-cleaving sequence), or IE63-linker-gE were developed and investigated for immunogenicity in mice. The results showed that each HZ DNA vaccine induced VZV-specific antibody production. The neutralizing antibody titer elicited by IE63-2A-gE was comparable to that elicited by gE or live attenuated HZ vaccine (LAV). IE63-2A-gE-induced gE or IE63-specific INF-γ+ T cell frequencies in splenocytes were comparable to those of LAV. Furthermore, IE63-2A-gE, gE, or IE63 led to a significant increase in IFN-γ (IE63 stimulation) and IL-2 (gE stimulation) secretion compared to LAV, showing a Th1-biased immune response. Moreover, IE63-2A-gE and gE induced cytotoxic activity of CD8+ T cells compared to that of LAV. This study elucidates that the IE63-2A-gE DNA vaccine can induce both humoral and cell-mediated immune responses, which provides a candidate for the development of an HZ vaccine.

Development of an Indirect ELISA Kit for Rapid Detection of Varicella-Zoster Virus Antibody by Glycoprotein E.

Varicella-zoster virus (VZV), a highly infectious agent that causes varicella (chickenpox), can also cause zoster (shingles), a disorder that is frequently associated with severe neuralgia. A reliable serological VZV diagnostic assay would be useful for identifying unprotected individuals and for surveilling post-vaccination immunoprotection status. Toward this goal, VZV membrane glycoprotein E (gE), the immunodominant VZV protein, served as target antigen in an indirect ELISA kit developed here to detect anti-VZV antibodies in clinical samples. For target antigen preparation, Chinese hamster ovary (CHO) cells were modified to express and secrete the VZV gE ectodomain, which was subsequently purified and used as coating antigen in an indirect ELISA. Ultimately, the optimal purified gE coating antigen concentration was determined to be 2 µg.ml-1 and the OD450nm detection cutoff value was 0.286. The coefficient of variation (CV) of intra-assay and inter-assay were <10 and 15%, respectively. A comparative test of 66 clinical samples showed that the coincidence rate was 93.9% between the indirect ELISA and a commercial varicella-zoster virus IgG ELISA kit. Thus, the indirect ELISA kit developed here may be useful for achieving rapid, sensitive, and specific detection of anti-VZV antibodies.

The Attenuated Pseudorabies Virus Vaccine Strain Bartha Hyperactivates Plasmacytoid Dendritic Cells by Generating Large Amounts of Cell-Free Virus in Infected Epithelial Cells.

Pseudorabies virus (PRV) is a porcine alphaherpesvirus and the causative agent of Aujeszky's disease. Successful eradication campaigns against PRV have largely relied on the use of potent PRV vaccines. The live attenuated Bartha strain, which was produced by serial passaging in cell culture, represents one of the hallmark PRV vaccines. Despite the robust protection elicited by Bartha vaccination, very little is known about the immunogenicity of the Bartha strain. Previously, we showed that Bartha-infected epithelial cells trigger plasmacytoid dendritic cells (pDC) to produce much higher levels of type I interferons than cells infected with wild-type PRV. Here, we show that this Bartha-induced pDC hyperactivation extends to other important cytokines, including interleukin-12/23 (IL-12/23) and tumor necrosis factor alpha (TNF-α) but not IL-6. Moreover, Bartha-induced pDC hyperactivation was found to be due to the strongly increased production of extracellular infectious virus (heavy particles [H-particles]) early in infection of epithelial cells, which correlated with a reduced production of noninfectious light particles (L-particles). The Bartha genome is marked by a large deletion in the US region affecting the genes encoding US7 (gI), US8 (gE), US9, and US2. The deletion of the US2 and gE/gI genes was found to be responsible for the observed increase in extracellular virus production by infected epithelial cells and the resulting increased pDC activation. The deletion of gE/gI also suppressed L-particle production. In conclusion, the deletion of US2 and gE/gI in the genome of the PRV vaccine strain Bartha results in the enhanced production of extracellular infectious virus in infected epithelial cells and concomitantly leads to the hyperactivation of pDC. IMPORTANCE The pseudorabies virus (PRV) vaccine strain Bartha has been and still is critical in the eradication of PRV in numerous countries. However, little is known about how this vaccine strain interacts with host cells and the host immune system. Here, we report the surprising observation that Bartha-infected epithelial porcine cells rapidly produce increased amounts of extracellular infectious virus compared to wild-type PRV-infected cells, which in turn potently stimulate porcine plasmacytoid dendritic cells (pDC). We found that this phenotype depends on the deletion of the genes encoding US2 and gE/gI. We also found that Bartha-infected cells secrete fewer pDC-inhibiting light particles (L-particles), which appears to be caused mainly by the deletion of the genes encoding gE/gI. These data generate novel insights into the interaction of the successful Bartha vaccine with epithelial cells and pDC and may therefore contribute to the development of vaccines against other (alphaherpes)viruses.