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Epstein–Barr virus (EBV) was the first human tumor virus being discovered and remains to date the only human pathogen that can transform cells in vitro. 55 years of EBV research have now brought us to the brink of an EBV vaccine. For this purpose, recombinant viral vectors and their heterologous prime-boost vaccinations, EBV-derived virus-like particles and viral envelope glycoprotein formulations are explored and are discussed in this review. Even so, cell-mediated immune control by cytotoxic lymphocytes protects healthy virus carriers from EBV-associated malignancies, antibodies might be able to prevent symptomatic primary infection, the most likely EBV-associated pathology against which EBV vaccines will be initially tested. Thus, the variety of EBV vaccines reflects the sophisticated life cycle of this human tumor virus and only vaccination in humans will finally be able to reveal the efficacy of these candidates. Nevertheless, the recently renewed efforts to develop an EBV vaccine and the long history of safe adoptive T cell transfer to treat EBV-associated malignancies suggest that this oncogenic γ-herpesvirus can be targeted by immunotherapies. Such vaccination should ideally implement the very same immune control that protects healthy EBV carriers.

Infectious laryngotracheitis (ILT) is a highly contagious upper respiratory tract disease of chicken caused by a Gallid herpesvirus 1 (GaHV-1) belonging to the genus Iltovirus, and subfamily Alphaherpesvirinae within Herpesviridae family. The disease is characterized by conjunctivitis, sinusitis, oculo-nasal discharge, respiratory distress, bloody mucus, swollen orbital sinuses, high morbidity, considerable mortality and decreased egg production. It is well established in highly dense poultry producing areas of the world due to characteristic latency and carrier status of the virus. Co-infections with other respiratory pathogens and environmental factors adversely affect the respiratory system and prolong the course of the disease. Latently infected chickens are the primary source of ILT virus (ILTV) outbreaks irrespective of vaccination. Apart from conventional diagnostic methods including isolation and identification of ILTV, serological detection, advanced biotechnological tools such as PCR, quantitative real-time PCR, next generation sequencing, and others are being used in accurate diagnosis and epidemiological studies of ILTV. Vaccination is followed with the use of conventional vaccines including modified live attenuated ILTV vaccines, and advanced recombinant vector vaccines expressing different ILTV glycoproteins, but still these candidates frequently fail to reduce challenge virus shedding. Some herbal components have proved to be beneficial in reducing the severity of the clinical disease. The present review discusses ILT with respect to its current status, virus characteristics, epidemiology, transmission, pathobiology, and advances in diagnosis, vaccination and control strategies to counter this important disease of poultry.

High-intensity focused ultrasound (HIFU) is mainly used in tumor ablation and tumor vaccinology study. It has been shown to induce immune sensitization and enhance tumor responsiveness to other therapies., High-intensity focused ultrasound (HIFU), a noninvasive ablation therapy that has been widely used clinically in ablation of solid tumors, induces immune sensitization. We therefore in this study investigated whether HIFU treatment could enhance the efficacy of a herpes simplex virus 2 (HSV-2) vaccine. First, we observed that in HSV-2-positive cervical intraepithelial neoplasia (CIN) II patients, HIFU treatment induced significantly higher anti-HSV-2 neutralization response than surgical removal. Next, we tested the efficacy of HIFU-treated, UV-inactivated HSV-2-infected cells as a proof-of-concept vaccine in mice. Our data showed that HIFU-treated formulation significantly enhanced HSV-2 antibody titers and neutralization titers, compared to UV-, microwave (MW)-, or freeze-thaw (FT)-treated formulations. HIFU treatment also promoted the Th1/2 cell-mediated response. A long-term full protection was observed in mice that received the HIFU-treated formulation, and no weight loss was detected. Our findings indicate that the novel application of HIFU in vaccine production may represent a rational way to improve vaccine efficacy. IMPORTANCE High-intensity focused ultrasound (HIFU) is mainly used in tumor ablation and tumor vaccinology study. It has been shown to induce immune sensitization and enhance tumor responsiveness to other therapies. Our study has shown enhanced anti-HSV-2 response in HIFU-treated CIN II patients. Furthermore, in a murine model, we have demonstrated that HIFU-treated HSV-2 vaccine induced long-term protective immunity against lethal challenge. Our findings indicate that the novel application of HIFU in vaccine production may represent a rational way to improve vaccine efficacy.

Infectious laryngotracheitis virus (ILTV, Gallid alphaherpesvirus 1) causes severe respiratory disease in chickens and has a major impact on the poultry industry worldwide. Live attenuated vaccines are widely available and are administered early in the life of commercial birds, often followed by one or more rounds of revaccination, generating conditions that can favour recombination between vaccines. Better understanding of the factors that contribute to the generation of recombinant ILTVs will inform the safer use of live attenuated herpesvirus vaccines. This study aimed to examine the parameters of infection that allow superinfection and may enable the generation of recombinant progeny in the natural host. In this study, 120 specific-pathogen free (SPF) chickens in 8 groups were inoculated with two genetically distinct live-attenuated ILTV vaccine strains with 1-4 days interval between the first and second vaccinations. After inoculation, viral genomes were detected in tracheal swabs in all groups, with lowest copies detected in swabs collected from the groups where the interval between inoculations was 4 days. Superinfection of the host was defined as the detection of the virus that was inoculated last, and this was detected in tracheal swabs from all groups. Virus could be isolated from swabs at a limited number of timepoints, and these further illustrated superinfection of the birds as recombinant viruses were detected among the progeny. This study has demonstrated superinfection at host level and shows recombination events occur under a very broad range of infection conditions. The occurrence of superinfection after unsynchronised infection with multiple viruses, and subsequent genomic recombination, highlight the importance of using only one type of vaccine per flock as the most effective way to limit recombination.

Herpes simplex virus 1 (HSV-1) is a leading cause of infectious blindness, highlighting the need for effective vaccines. A single-cycle HSV-2 strain with the deletion of glycoprotein D, ΔgD-2, completely protected mice from HSV-1 and HSV-2 skin or vaginal disease and prevented latency following active or passive immunization in preclinical studies. The antibodies functioned primarily by activating Fc receptors to mediate antibody-dependent cellular cytotoxicity (ADCC). The ability of ADCC to protect the immune-privileged eye, however, may differ from skin or vaginal infections. Thus, the current studies were designed to compare active and passive immunization with ΔgD-2 versus an adjuvanted gD subunit vaccine (rgD-2) in a primary lethal ocular murine model. ΔgD-2 provided significantly greater protection than rgD-2 following a two-dose vaccine regimen, although both vaccines were protective compared to an uninfected cell lysate. However, only immune serum from ΔgD-2-vaccinated, but not rgD-2-vaccinated, mice provided significant protection against lethality in passive transfer studies. The significantly greater passive protection afforded by ΔgD-2 persisted after controlling for the total amount of HSV-specific IgG in the transferred serum. The antibodies elicited by rgD-2 had significantly higher neutralizing titers, whereas those elicited by ΔgD-2 had significantly more C1q binding and Fc gamma receptor activation, a surrogate for ADCC function. Together, the findings suggest ADCC is protective in the eye and that nonneutralizing antibodies elicited by ΔgD-2 provide greater protection than neutralizing antibodies elicited by rgD-2 against primary ocular HSV disease. The findings support advancement of vaccines, including ΔgD-2, that elicit polyfunctional antibody responses. IMPORTANCE Herpes simplex virus 1 is the leading cause of infectious corneal blindness in the United States and Europe. Developing vaccines to prevent ocular disease is challenging because the eye is a relatively immune-privileged site. In this study, we compared a single-cycle viral vaccine candidate, which is unique in that it elicits predominantly nonneutralizing antibodies that activate Fc receptors and bind complement, and a glycoprotein D subunit vaccine that elicits neutralizing but not Fc receptor-activating or complement-binding responses. Only the single-cycle vaccine provided both active and passive protection against a lethal ocular challenge. These findings greatly expand our understanding of the types of immune responses needed to protect the eye and will inform future prophylactic and therapeutic strategies.

Objectives Cost-effectiveness analyses (CEA) are based on the value judgment that health outcomes (eg, quantified in quality-adjusted life-years; QALYs) are all equally valuable irrespective of their context. Whereas most published CEAs perform extensive sensitivity analysis on various parameters and assumptions, only rarely is the influence of the QALY-equivalence assumption on cost-effectiveness results investigated. We illustrate how the integration of alternative social value judgments in CEA can be a useful form of sensitivity analysis. Methods Because varicella-zoster virus (VZV) vaccination affects 2 distinct diseases (varicella zoster and herpes zoster) and likely redistributes infections across different age groups, the program has an important equity dimension. We used a cost-effectiveness model and disentangled the share of direct protection and herd immunity within the total projected QALYs resulting from a 50-year childhood VZV program in the UK. We use the UK population’s preferences for QALYs in the vaccine context to revalue QALYs accordingly. Results Revaluing different types of QALYs for different age groups in line with public preferences leads to a 98% change in the projected net impact of the program. The QALYs gained among children through direct varicella protection become more important, whereas the QALYs lost indirectly through zoster in adults diminish in value. Weighting of vaccine-related side effects made a large difference. Conclusions Our study shows that a sensitivity analysis in which alternative social value judgments about the value of health outcomes are integrated into CEA of vaccines is relatively straightforward and provides important additional information for decision makers to interpret cost-effectiveness results.

Live attenuated vaccine strains have been developed for Varicella-Zoster virus (VZV). Compared to clinically isolated strains, the vaccine strains contain several non-synonymous mutations in open reading frames (ORFs) 0, 6, 31, 39, 55, 62, and 64. In particular, ORF62, encoding an immediate-early (IE) 62 protein that acts as a transactivator for viral gene expression, contains six non-synonymous mutations, but whether these mutations affect transactivation activity of IE62 is not understood. In this study, we investigated the role of non-synonymous vaccine-type mutations (M99T, S628G, R958G, V1197A, I1260V, and L1275S) of IE62 in Suduvax, a vaccine strain isolated in Korea, for transactivation activity. In reporter assays, Suduvax IE62 showed 2- to 4-fold lower transactivation activity toward ORF4, ORF28, ORF29, and ORF68 promoters than wild-type IE62. Introduction of individual M99T, S628G, R958G, or V1197A/I1260V/L1275S mutations into wild-type IE62 did not affect transactivation activity. However, the combination of M99T within the N-terminal Sp transcription factor binding region and V1197A/I1260V/L1275S within the C-terminal serine-enriched acidic domain (SEAD) significantly reduced the transactivation activity of IE62. The M99T/V1197A/I1260V/L1275S mutant IE62 did not show considerable alterations in intracellular distribution and Sp3 binding compared to wild-type IE62, suggesting that other alteration(s) may be responsible for the reduced transactivation activity. Collectively, our results suggest that acquisition of mutations in both Met 99 and the SEAD of IE62 is responsible for the reduced transactivation activity found in IE62 of the VZV vaccine strains and contributes to attenuation of the virus.

Live herpesvirus-vectored vaccines are widely used in veterinary medicine to protect against many infectious diseases. In poultry, three strains of herpesvirus vaccines are used against Marek’s disease (MD). However, of these, only the herpesvirus of turkeys (HVT) has been successfully developed and used as a recombinant vaccine vector to induce protection against other avian viral diseases such as infectious bursal disease (IBD), Newcastle disease (ND) or avian influenza (AI). Although effective when administered individually, recombinant HVT vectors have limitations when combined in multivalent vaccines. Thus there is a need for developing additional viral vectors that could be combined with HVT in inducing protection against multiple avian diseases in multivalent vaccines. Gallid herpesvirus 3 (GaHV3) strain SB-1 is widely used by the poultry industry as bivalent vaccine in combination with HVT to exploit synergistic effects against MD. Here, we report the development and application of SB-1 as a vaccine vector to express the VP2 capsid antigen of IBD virus. A VP2 expression cassette was introduced into the SB-1 genome at three intergenic locations (UL3/UL4, UL10/UL11 and UL21/UL22) using recombineering methods on the full-length pSB-1 infectious clone of the virus. We show that the recombinant SB-1 vectors expressing VP2 induced neutralising antibody responses at levels comparable to that of commercial HVT-based VAXXITEKHVT+IBD vaccine. Birds vaccinated with the experimental recombinant SB-1 vaccine were protected against clinical disease after challenge with the very virulent UK661 IBDV isolate, demonstrating its value as an efficient viral vector for developing multivalent vaccines against avian diseases., Veterinary medicine: an effective vaccine platform to overcome multiviral interference Gallid herpesvirus 3 (GaHV3) strain SB-1 is effective in delivering vaccine material to cells, overcoming a major roadblock in poultry vaccination. UK researchers, led by the Prof. Venugopal Nair OBE at the Pirbright Institute, have constructed a vaccine against Marek's disease virus (MDV) and infectious bursal disease viruses (IBDV). Both MDV and IBDV are deadly viruses of poultry industry. For this purpose, GaHV3 strain SB-1 (a vaccine strain of virus against MDV) was used and genetic materials for VP2 (one of the surface glycoproteins of IBDV) was incorporated into the genome of SB-1. Cells inoculated with the viral vaccine produce the VP2 proteins of IBDV which then primes a host’s immune system response against infection. Birds immunized with the team’s vaccine remained free of clinical symptoms after IBDV infection, highlighting the potential of SB-1 to be used as a vector against other avian diseases. The use of SB-1 also sidesteps a constraint of previous vaccines, based on herpesvirus of turkeys (HVT), which is recommended not to be combined with other HVT based vaccines. SB-1 vaccines can be administered alongside HVT vaccines and they can provide a better immunity against MDV, highlighting the potential of SB-1 to be used as a vector against other avian vidal diseases.

Exercise (with appropriate intensity and duration) is a natural modulator of immune responses and may be useful to increase the vaccine response towards antigen. According to the fact that rural area responding butter than urbon area to vaccine protocol, this study was undertaken to test the hypothesis that short term exercise training as an adjuvant for antigen such as herpes simplex virus type 1 (HSV-1) in animal models. Mice with/without access to short term exercise training were immunized intramuscularly with inactivated KOS strain of HSV-1. Immune responses was investigated with regards of both cell-mediated and humoral immunity. In this study by using short term exercise training as an adjuvant enhanced Th1 response while it did not show significant effect on Th2 responses towards HSV-1 immunization. Also, immunoglobulin G (IgG) 2a/IgG1-ratios increased in vaccine with short term exercise training group. These results suggested that coupling short term moderate exercise training as a mild adjuvant with vaccination may enhance cell-mediated immune responses especially Th1 responses.

Bacterins obtained from recombinant bacteria displaying heterologous antigens in its surface coded by prokaryotic rather than eukaryotic expression plasmids (here called “spiny” bacterins or spinycterins), have been used to increase fish immunogenicity of recombinant viral protein fragments. To explore their immunogenicity, five bacterial-specific membrane anchor-motifs characterized in the literature (Nmistic, Mistic, NTD, YAIN and YBEL) were genetically fused to the immunorelevant cystein-free 117 amino acid fragment II from the ORF149 of cyprinid herpes virus 3 (frgIICyHV3). The fusion of anchor-motifs to the N-terminus of frgIICyHV3 enriched expression in E.coli outer membranes as demonstrated by ELISA, immunofluorescence and flow cytometry of formaldehyde-fixed recombinant bacteria (spinycterins). Unconventional low-intensity ultrasound inducing mucosal micropores in a reversible non-harmful manner was used before carp or zebrafish immersion on spinycterin suspensions as a practical delivery alternative to fish-to-fish injection. After ELISA screening for anti-frgIICyHV3-specific antibodies of spinycterin-immunized fish plasma, the YBEL constructs were identified as the most immunogenic in both carp and zebrafish, correlating with one of the best expressed recombinant proteins as demonstrated by Western blot and surface enriched as demonstrated by ELISA and flow cytometry. The use of prokaryotic expression plasmids to express viral immunorelevant protein fragments in traditionally used fish vaccination bacterins should reduce the environmental concerns raised by DNA vaccination based on eukaryotic expression plasmids. Therefore, spinycterins may be a useful alternative to develop safer fish viral vaccines and mass vaccination methods.

Vaccination is a proven intervention against human viral diseases; however, success against Herpes Simplex Virus 2 (HSV-2) remains elusive. Most HSV-2 vaccines tested in humans to date contained just one or two immunogens, such as the virion attachment receptor glycoprotein D (gD) and/or the envelope fusion protein, glycoprotein B (gB). At least three factors may have contributed to the failures of subunit-based HSV-2 vaccines. First, immune responses directed against one or two viral antigens may lack sufficient antigenic breadth for efficacy. Second, the antibody responses elicited by these vaccines may have lacked necessary Fc-mediated effector functions. Third, these subunit vaccines may not have generated necessary protective cellular immune responses. We hypothesized that a polyvalent combination of HSV-2 antigens expressed from a DNA vaccine with an adjuvant that polarizes immune responses toward a T helper 1 (Th1) phenotype would compose a more effective vaccine. We demonstrate that delivery of DNA expressing full-length HSV-2 glycoprotein immunogens by electroporation with the adjuvant interleukin 12 (IL-12) generates substantially greater protection against a high-dose HSV-2 vaginal challenge than a recombinant gD subunit vaccine adjuvanted with alum and monophosphoryl lipid A (MPL). Our results further show that DNA vaccines targeting optimal combinations of surface glycoproteins provide better protection than gD alone and provide similar survival benefits and disease symptom reductions compared with a potent live attenuated HSV-2 0ΔNLS vaccine, but that mice vaccinated with HSV-2 0ΔNLS clear the virus much faster. Together, our data indicate that adjuvanted multivalent DNA vaccines hold promise for an effective HSV-2 vaccine, but that further improvements may be required.

The human herpes simplex virus type 1 (HSV-1) is an extremely rampant human pathogen, and its infection could cause life-long diseases, including the central nervous system disorders. The glycoproteins of HSV-1 such as glycoprotein B, glycoprotein C, glycoprotein D, glycoprotein H, and glycoprotein L are highly involved in mediating the viral attachment and infection of the host cell. Therefore, immunoinformatic approaches followed by molecular dynamics simulation and systems biology has been used to analyze these glycoproteins in order to propose effective peptide-based vaccine candidates against the HSV-1 infection. The ElliPro and NetCTL.1.2 online tools were employed to forecast the B- and T-lymphocyte (CTL) epitopes for gB, gC, gD, gH, and gL. The 3D coordinates of these epitopes were modeled and docked against the human major histocompatibility complex molecule-1. The outcomes obtained from postdocking analysis along with TAP (Transporter associated with antigen processing), MHC binding, and C-terminal cleavage score assisted in the selection of potential epitopes. These epitopes were further subjected to molecular dynamics simulation and systems biology approach which showed significant results. On the basis of these substantial outcomes, peptides are proposed that could be used to provoke immunity against the HSV-1 infection.

Herpes simplex virus type-2 (HSV-2) is a common cause of genital infections throughout the world. Currently no prophylactic vaccine or therapeutic cure exists against the virus that establishes a latent infection for the life of the host. Intravaginal microbivac is a developing out-of-the-box strategy that combines instant microbicidal effects with future vaccine-like benefits. We have recently shown that our uniquely designed zinc oxide tetrapod nanoparticles (ZOTEN) show strong microbivac efficacy against HSV-2 infection in a murine model of genital infection. In our attempts to further understand the antiviral and immune bolstering effects of ZOTEN microbivac and to develop ZOTEN as a platform for future live virus vaccines, we tested a ZOTEN/HSV-2 cocktail and found that prior incubation of HSV-2 with ZOTEN inhibits the ability of the virus to infect vaginal tissue in female Balb/c mice and blocks virus shedding as judged by plaque assays. Quite interestingly, the ZOTEN-neutralized virions elicit a local immune response that is highly comparable with the HSV-2 infection alone with reduced inflammation and clinical manifestations of disease. Information provided by our study will pave the way for the further development of ZOTEN as a microbivac and a future platform for live virus vaccines.

Herpes Simplex Virus (HSV) is a highly prevalent sexually transmitted infection that aside from causing cold sores and genital lesions, causes complications in the immunocompromised and has facilitated a large proportion of HIV acquisition globally. Despite decades of research, there is no prophylactic HSV vaccine ready for use in humans, leaving many questioning whether a prophylactic vaccine is an achievable goal. A previous HSV vaccine trial did have partial success in decreasing acquisition of HSV2 - promising evidence that vaccines can prevent acquisition. However, there is still an incomplete understanding of the immune response pathways elicited by HSV after initial mucosal infection and how best to replicate these responses with a vaccine, such that acquisition and colonisation of the dorsal root ganglia could be prevented. Another factor to consider in the rational design of an HSV vaccine is adjuvant choice. Understanding the immune responses elicited by different adjuvants and whether lasting humoral and cell-mediated responses are induced is important, especially when studies of past trial vaccines found that a sufficiently protective cell-mediated response was lacking. In this review, we discuss what is known of the immune control involved in initial herpes lesions and reactivation, including the importance of CD4 and CD8 T cells, and the interplay between innate and adaptive immunity in response to primary infection, specifically focusing on the viral relay involved. Additionally, a summary of previous and current vaccine trials, including the components used, immune responses elicited and the feasibility of prophylactic vaccines looking forward, will also be discussed.

We are interested in developing a vaccine that prevents genital herpes. Adjuvants have a major impact on vaccine immunogenicity. We compared two adjuvants, an experimental Merck Sharp & Dohme lipid nanoparticle (LNP) adjuvant, LNP-2, with CpG oligonucleotide combined with alum for immunogenicity in mice when administered with herpes simplex virus type 2 (HSV-2) glycoproteins C, D and E (gC2, gD2, gE2). The immunogens are intended to produce neutralizing antibodies to gC2 and gD2, antibodies to gD2 and gE2 that block cell-to-cell spread, and antibodies to gE2 and gC2 that block immune evasion from antibody and complement, respectively. Overall, CpG/alum was better at producing serum and vaginal IgG binding antibodies, neutralizing antibodies, antibodies that block virus spread from cell-to-cell, and antibodies that block immune evasion domains on gC2. We used a novel high throughput biosensor assay to further assess differences in immunogenicity by mapping antibody responses to seven crucial epitopes on gD2 involved in virus entry or cell-to-cell spread. We found striking differences between CpG/alum and LNP-2. Mice immunized with gD2 CpG/alum produced higher titers of antibodies than LNP-2 to six of seven crucial epitopes and produced antibodies to more crucial epitopes than LNP-2. Measuring epitope-specific antibodies helped to define mechanisms by which CpG/alum outperformed LNP-2 and is a valuable technique to compare adjuvants.

The ubiquitous Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and is etiologically linked to the development of several malignancies and autoimmune diseases. EBV has a multifaceted life cycle that comprises virus lytic replication and latency programs. Considering EBV infection holistically, we rationalized that prophylactic EBV vaccines should ideally prime the immune system against lytic and latent proteins. To this end, we generated highly immunogenic particles that contain antigens from both these cycles. In addition to stimulating EBV-specific T cells that recognize lytic or latent proteins, we show that the immunogenic particles enable the ex vivo expansion of cytolytic EBV-specific T cells that efficiently control EBV-infected B cells, preventing their outgrowth. Lastly, we show that immunogenic particles containing the latent protein EBNA1 afford significant protection against wild-type EBV in a humanized mouse model. Vaccines that include antigens which predominate throughout the EBV life cycle are likely to enhance their ability to protect against EBV infection., Author summary Human herpesviruses are tremendously successful pathogens that establish lifelong infection in a substantial proportion of the population. The oncogenic γ-herpesvirus EBV, like other herpesviruses, expresses a plethora of open-reading frames throughout its multifaceted life cycle. We have developed a prophylactic vaccine candidate in the form of immunogenic particles that contain several EBV antigens. This is in stark contrast to the vast majority of EBV vaccines candidates that contain only one or two EBV antigens. Our immunogenic particles were shown capable of stimulating several EBV-specific T-cell clones in vitro. The immunogenic particles were also capable of expanding cytolytic EBV-specific T cells ex vivo and provided a protective benefit in vivo when used as a prophylactic vaccine.

A recent phase 3 trial with soluble herpes simplex virus 2 (HSV-2) glycoprotein D (gD2t) in adjuvant failed to show protection against genital herpes. We postulated that live attenuated HSV-2 would provide more HSV antigens for induction of virus-specific antibodies and cellular immunity than would gD2t. We previously reported an HSV-2 mutant, HSV2-gD27, in which the nectin-1 binding domain of gD2 is altered so that the virus is impaired for infecting neural cells, but not epithelial cells, in vitro and is impaired for infecting dorsal root ganglia in mice (K. Wang, J. D. Kappel, C. Canders, W. F. Davila, D. Sayre, M. Chavez, L. Pesnicak, and J. I. Cohen, J Virol 86:12891–12902, 2012, doi:10.1128/JVI.01055-12). Here we report that the mutations in HSV2-gD27 were stable when the virus was passaged in cell culture and during acute infection of mice. HSV2-gD27 was attenuated in mice when it was inoculated onto the cornea, intramuscularly (i.m.), intravaginally, and intracranially. Vaccination of mice i.m. with HSV2-gD27 provided better inhibition of challenge virus replication in the vagina than when the virus was used to vaccinate mice intranasally or subcutaneously. Comparison of i.m. vaccinations with HSV2-gD27 versus gD2t in adjuvant showed that HSV2-gD27 induced larger reductions of challenge virus replication in the vagina and reduced latent viral loads in dorsal root ganglia but induced lower serum neutralizing antibody titers than those obtained with gD2t in adjuvant. Taken together, our data indicate that a live attenuated HSV-2 vaccine impaired for infection of neurons provides better protection from vaginal challenge with HSV-2 than that obtained with a subunit vaccine, despite inducing lower titers of HSV-2 neutralizing antibodies in the serum. IMPORTANCE Genital herpes simplex is one of the most prevalent sexually transmitted diseases. Though HSV-2 disease is usually mild, it can be life threatening in neonates and immunocompromised persons. In addition, genital herpes increases the frequency of HIV infection and transmission. HSV-2 maintains a latent infection in sensory neurons and cannot be cleared with antiviral drugs. The virus frequently reactivates, resulting in virus shedding in the genital area, which serves as a source for transmission. A prophylactic vaccine is needed to prevent disease and to control the spread of the virus. Previous human trials of subunit vaccines have been unsuccessful. Here we report the results of vaccinating mice with a new type of live attenuated HSV-2 vaccine that is impaired for infection of neurons and provides better protection of mice than that obtained with a subunit vaccine. The strategy of altering the cell tropism of a virus is a new approach for a live attenuated vaccine.

Viral vaccines have traditionally protected against disease, but for viruses that establish latent infection, it is desirable for the vaccine to reduce infection to reduce latent infection and reactivation. While seroconversion has been used in clinical trials of herpes simplex virus (HSV) vaccines to measure protection from infection, this has not been modeled in animal infection systems. To measure the ability of a genital herpes vaccine candidate to protect against various aspects of infection, we established a non-lethal murine model of genital HSV-2 infection, an ELISA assay to measure antibodies specific for infected cell protein 8 (ICP8), and a very sensitive qPCR assay. Using these assays, we observed that immunization with HSV-2 dl5-29 virus reduced disease, viral shedding, seroconversion, and latent infection by the HSV-2 challenge virus. Therefore, it may be feasible to obtain protection against genital disease, seroconversion and latent infection by immunization, even if sterilizing immunity is not achieved.

We previously isolated a herpes simplex virus 1 (HSV-1) mutant, KOS-NA, that carries two nonsynonymous mutations in UL39, resulting in L393P and R950H amino acid substitutions in infected cell protein 6 (ICP6). Our published data studying KOS-NA pathogenesis strongly suggest that one of these ICP6 substitutions expressed from KOS-NA, R950H, severely impaired acute viral replication in the eyes and trigeminal ganglia of mice after inoculation onto the cornea and consequently impaired establishment and reactivation from latency. Because of its significant neuroattenuation, we tested KOS-NA as a potential prophylactic vaccine against HSV-1 in a mouse model of corneal infection. KOS-NA stimulated stronger antibody and T cell responses than a replication-competent ICP0-null mutant and a replication-incompetent ICP8-null mutant optimized for immunogenicity. Immunizations with the ICP0-, ICP8-, and KOS-NA viruses all reduced replication of wild-type HSV-1 challenge virus in the corneal epithelium to similar extents. Low immunizing doses of KOS-NA and the ICP8- virus, but not the ICP0- virus, protected mice against eyelid disease (blepharitis). Notably, only KOS-NA protected almost completely against corneal disease (keratitis) and greatly reduced latent infection by challenge virus. Thus, vaccination of mice with KOS-NA prior to corneal challenge provides significant protection against HSV-1-mediated disease of the eye, even at a very low immunizing dose. These results suggest that KOS-NA may be the foundation of an effective prophylactic vaccine to prevent or limit HSV-1 ocular diseases.IMPORTANCE HSV-1 is a ubiquitous human pathogen that infects the majority of the world's population. Although most infections are asymptomatic, HSV-1 establishes lifelong latency in infected sensory neurons, from which it can reactivate to cause deadly encephalitis or potentially blinding eye disease. No clinically effective vaccine is available. In this study, we tested the protective potential of a neuroattenuated HSV-1 mutant (KOS-NA) as a vaccine in mice. We compared the effects of immunization with KOS-NA to those of two other attenuated viruses, a replication-competent (ICP0-) virus and a replication-incompetent (ICP8-) virus. Our data show that KOS-NA proved superior to the ICP0- and ICP8-null mutants in protecting mice from corneal disease and latent infection. With its significant neuroattenuation, severe impairment in establishing latency, and excellent protective effect, KOS-NA represents a significant discovery in the field of HSV-1 vaccine development.

Herpes simplex viruses 1 and 2 are among the most ubiquitous human infections and persist lifelong in their host. Upon primary infection or reactivation from ganglia, the viruses spread by direct cell–cell contacts (cell-to-cell spread) and thus escape from the host immune response. We have developed a monoclonal antibody (mAb 2c), which inhibits the HSV cell-to-cell spread, thereby protecting from lethal genital infection and blindness in animal models. In the present study we have designed a nanoparticle-based vaccine to induce protective antibody responses exceeding the cell-to-cell spread inhibiting properties of mAb 2c. We used biodegradable calcium phosphate (CaP) nanoparticles coated with a synthetic peptide that represents the conformational epitope on HSV-1 gB recognized by mAb 2c. The CaP nanoparticles additionally contained a TLR-ligand CpGm and were formulated with adjuvants to facilitate the humoral immune response. This vaccine effectively protected mice from lethal HSV-1 infection by inducing cell-to-cell spread inhibiting antibodies.

A novel system was developed for generating highly attenuated vaccinia virus LC16m8 (m8, third-generation smallpox vaccine) that expresses foreign genes. The innovations in this system are its excisable selection marker, specificity of the integration site of a gene of interest, and easy identification of clones with a fluorescent signal. Using this system, recombinant m8s, which expressed herpes simplex virus 2 (HSV-2) glycoprotein B (gB)-, gD-, or both gB and gD (gB + gD), were generated, and their efficacy was evaluated. First, the induction of a specific IgG against these HSV-2 glycoproteins in mice infected with one of these recombinant m8s was confirmed by an immunofluorescent assay. Next, mice preinfected with one of the recombinant m8s were infected with HSV-2 at a lethal dose to examine the vaccine efficacy. The fatality rate among the mice preinfected with either the recombinant gB + gD- or gD-expressing m8 significantly decreased in comparison with the control. The survival rate in male and female mice preinfected with either the recombinant gB + gD- or gD-expressing m8 increased to 100% and 60%, respectively, while most of the control mice died. In summary, this new system may be applicable to creation of a novel m8-based vaccine.

We hypothesized that vigorous replication of a pathogen may be critical for eliciting the most potent and balanced immune response against it. Hence, attenuation/inactivation (as in conventional vaccines) should be avoided. Instead, the necessary safety should be provided by placing replication of the pathogen under stringent control and by activating time-limited replication of the pathogen strictly in an administration region in which pathology cannot develop. Immunization will then occur in the context of highly efficient pathogen replication and uncompromised safety. We found that localized activation in mice of efficient but limited replication of a replication-competent controlled herpesvirus vector resulted in a greatly enhanced immune response to the virus or an expressed heterologous antigen. This finding supports the above-mentioned hypothesis and suggests that the vectors may be promising novel agents worth exploring for the prevention/mitigation of infectious diseases for which efficient vaccination is lacking, in particular in immunocompromised patients., Replication-competent controlled virus vectors were derived from the virulent herpes simplex virus 1 (HSV-1) wild-type strain 17syn+ by placing one or two replication-essential genes under the stringent control of a gene switch that is coactivated by heat and an antiprogestin. Upon activation of the gene switch, the vectors replicate in infected cells with an efficacy that approaches that of the wild-type virus from which they were derived. Essentially no replication occurs in the absence of activation. When administered to mice, localized application of a transient heat treatment in the presence of systemic antiprogestin results in efficient but limited virus replication at the site of administration. The immunogenicity of these viral vectors was tested in a mouse footpad lethal challenge model. Unactivated viral vectors—which may be regarded as equivalents of inactivated vaccines—induced detectable protection against lethality caused by wild-type virus challenge. Single activation of the viral vectors at the site of administration (rear footpads) greatly enhanced protective immune responses, and a second immunization resulted in complete protection. Once activated, vectors also induced far better neutralizing antibody and HSV-1-specific cellular immune responses than unactivated vectors. To find out whether the immunogenicity of a heterologous antigen was also enhanced in the context of efficient transient vector replication, a virus vector constitutively expressing an equine influenza virus hemagglutinin was constructed. Immunization of mice with this recombinant induced detectable antibody-mediated neutralization of equine influenza virus, as well as a hemagglutinin-specific cellular immune response. Single activation of viral replication resulted in a severalfold enhancement of these immune responses. IMPORTANCE We hypothesized that vigorous replication of a pathogen may be critical for eliciting the most potent and balanced immune response against it. Hence, attenuation/inactivation (as in conventional vaccines) should be avoided. Instead, the necessary safety should be provided by placing replication of the pathogen under stringent control and by activating time-limited replication of the pathogen strictly in an administration region in which pathology cannot develop. Immunization will then occur in the context of highly efficient pathogen replication and uncompromised safety. We found that localized activation in mice of efficient but limited replication of a replication-competent controlled herpesvirus vector resulted in a greatly enhanced immune response to the virus or an expressed heterologous antigen. This finding supports the above-mentioned hypothesis and suggests that the vectors may be promising novel agents worth exploring for the prevention/mitigation of infectious diseases for which efficient vaccination is lacking, in particular in immunocompromised patients.

Induction of robust and long-term immune responses at the portal of entry remains a big challenge for HSV-2 vaccine development. The adoption of a CD4 T cell helper peptide in the vaccine is thought to be beneficial for the enhancement of immune responses, however, its effect on HSV-2 vaccines has not yet been studied. In this study, we designed a DNA vaccine (gD-TpD) simultaneously expressing HSV-2 gD ectodomain and a universal CD4 T cell helper peptide (TpD), and tested its efficacy on a murine model. Mice were immunized 3 times with gD-TpD or control DNA formulations, and then were rested until Day 150 when they were vaginally challenged with lethal doses of HSV-2. Our data showed that gD-TpD significantly increased gD-specific IgG and IgA in both sera and vaginal washes. Furthermore, the increased antibody responses showed enhanced neutralization activity in vitro. In addition, gD-TpD induced balanced Th1/2 cellular responses and CD8+ T cell-dependent CTL activity. Although immune responses dropped over time after the final immunization, robust and rapid antibody and T cell responses were induced upon virus challenge in gD-TpD group. Moreover, gD-TpD provided full protection against lethal viral challenge in immunized mice. Together, our findings indicate that the inclusion of the CD4 T cell helper peptide TpD in HSV-2 gD subunit vaccine could induce long-term protective immunity, providing information for a rational design of vaccines against HSV-2 or even other viruses.

Despite overwhelming experimental work, there are no licensed vaccines against the most frequent Alphaherpesviruses, namely herpes simplex virus 1 and 2 (HSV1 and 2) nor against the Epstein-Barr virus (EBV), a member of the subfamily Gammaherpesvirus.Since the DNAs of both HSVs reside in the regional sensory ganglia in a latent state (i.e. as circularized episomal molecules), a corresponding vaccine might be useful for immunotherapy rather than for prevention of primary infection. Here we describe the design of a purified subunit vaccine as well as the preparation and efficacy of a recombinant fusion protein consisting of the gD ectodomain from our domestic attenuated HSV1 strain HSZP. The EBV vaccines considered so far, were destined for prevention of infectious mononucleosis (IM) or to prevent formation of EBV related tumors. To design the EBV peptide vaccine, at least 15 carefully selected immunogenic epitopes coming from 12 virus coded proteins were bound to synthetic micro-particle carriers along with a non-specific pathogen recognizing receptor (PRR) stimulating both the T as well as B lymphocytes.The efficacy of a novel EBV peptide in the rabbit model was based on criteria such as antibody formation (EA-D detected by ELISA, early and capsid proteins tested by immunoblot), presence of LMP1 antigen and of viral DNA in peripheral white blood cells. Out of 19 peptide combinations used for vaccination, at least 6 showed a satisfactory protective effect.

Herpes simplex virus is a prevalent pathogen of humans of various age groups. The fact that no prophylactic or therapeutic vaccine is currently available suggests a significant need to further investigate the immune mechanisms induced by the virus and various vaccine candidates. We previously generated an HSV-1 mutant strain, M3, with partial deletions in ul7, ul41 and LAT that produced an attenuated phenotype in mice. In the present study, we performed a comparative analysis to characterize the immune responses induced by M3 versus wild-type HSV-1 in a mouse model. Infection with wild-type HSV-1 triggered an inflammatory-dominated response and adaptive immunity suppression and was accompanied by severe pathological damage. In contrast, infection with M3 induced a systematic immune response involving full activation of both innate and adaptive immunity and was accompanied by no obvious pathological changes. Furthermore, the immune response induced by M3 protected mice from lethal challenge with wild-type strains of HSV-1 and restrained virus proliferation and impaired latency. These data are useful for further HSV-1 vaccine development using a mutant strain construction strategy.

Varicella-zoster virus (VZV) is the first and only human herpesvirus for which a licensed live attenuated vaccine, vOka, has been developed. vOka has highly safe and effective profiles; however, worldwide herd immunity against VZV has not yet been established and it is far from eradication. Despite the successful reduction in the burden of VZV-related illness by the introduction of the vaccine, some concerns about vOka critically prevent worldwide acceptance and establishment of herd immunity, and difficulties in addressing these criticisms often relate to its ill-defined mechanism of attenuation. Advances in scientific technologies have been applied in the VZV research field and have contributed toward uncovering the mechanism of vOka attenuation as well as VZV biology at the molecular level. A subunit vaccine targeting single VZV glycoprotein, rationally designed based on the virological and immunological research, has great potential to improve the strategy for eradication of VZV infection in combination with vOka.

Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and is associated with several malignancies, including nasopharyngeal carcinoma, gastric carcinoma, Hodgkin lymphoma, Burkitt lymphoma, and lymphomas in immunocompromised persons, as well as multiple sclerosis. A vaccine is currently unavailable. While monomeric EBV gp350 was shown in a phase 2 trial to reduce the incidence of infectious mononucleosis, but not the rate of EBV infection, newer formulations of gp350 including multimeric forms, virus-like particles, and nanoparticles may be more effective. Vaccine that also include additional viral glycoproteins, lytic proteins or latency proteins might also improve the effectiveness of an EBV gp350 vaccine. Clinical trials to determine if an EBV vaccine can reduce the rate of infectious mononucleosis or post-transplant lymphoproliferative disease should be performed. The former is important since infectious mononucleosis can be associated with debilitating fatigue as well as other complications, and EBV infectious mononucleosis is associated with increased rates of Hodgkin lymphoma and multiple sclerosis. A vaccine to reduce EBV post-transplant lymphoproliferative disease would be an important proof of principle to prevent an EBV associated malignancy. Trials of an EBV vaccine to reduce the incidence of Hodgkin lymphoma, multiple sclerosis, or Burkitt lymphoma would be difficult, but feasible.

Both varicella and herpes zoster that are caused by varicella-zoster virus (VZV), are associated with central nervous system disease. Since the introduction of polymerase chain reaction, the opportunity to detect the virus in cerebrospinal fluid (CSF) has improved dramatically. As a result VZV is diagnosed as one of the most common viruses causing CNS disease and it has become evident that this disease includes a wide spectrum of different CNS manifestations. The most evaluated CNS manifestations are encephalitis which is associated with both varicella and herpes zoster and, cerebellitis which occurs predominantly in children with varicella. Other manifestations have been less widely investigated. The incidence of cerebrovascular disease caused by VZV has been only scarcely studied and, in addition, some data indicate that vasculitis might also be involved in other VZV CNS manifestations such as herpes zoster-associated encephalitis. For this reason, VZV CNS infection must be suspected in several CNS syndromes and diagnostics should be based on CSF analysis for detection of VZV DNA by PCR and/or intrathecal antibody production. The prognosis is reported as favourable in children but few follow-up studies are available. Moreover, in adults, the prognosis is reported to be good in overall terms, but later studies indicate more serious neurological sequelae including cognition. Despite considerable mortality and morbidity, so far also in vaccinating countries, few treatment studies are available. Further treatment studies including assessments of neurological and cognitive sequelae, are warranted.

Varicella-zoster virus (VZV) is a human herpesvirus, which during primary infection typically causes varicella (chicken pox) and establishes lifelong latency in sensory and autonomic ganglia. Later in life, the virus may reactivate to cause herpes zoster (HZ; also known as shingles). To prevent these diseases, a live-attenuated heterogeneous vaccine preparation, vOka, is used routinely in many countries worldwide. Recent studies of another alphaherpesvirus, infectious laryngotracheitis virus, demonstrate that live-attenuated vaccine strains can recombine in vivo , creating virulent progeny. These findings raised concerns about using attenuated herpesvirus vaccines under conditions that favor recombination. To investigate whether VZV may undergo recombination, which is a prerequisite for VZV vaccination to create such conditions, we here analyzed 115 complete VZV genomes. Our results demonstrate that recombination occurs frequently for VZV. It thus seems that VZV is fully capable of recombination if given the opportunity, which may have important implications for continued VZV vaccination. Although no interclade vaccine-wild-type recombinant strains were found, intraclade recombinants were frequently detected in clade 2, which harbors the vaccine strains, suggesting that the vaccine strains have already been involved in recombination events, either in vivo or in vitro during passages in cell culture. Finally, previous partial and complete genomic studies have described strains that do not cluster phylogenetically to any of the five established clades. The additional VZV strains sequenced here, in combination with those previously published, have enabled us to formally define a novel sixth VZV clade. IMPORTANCE Although genetic recombination has been demonstrated to frequently occur for other human alphaherpesviruses, herpes simplex viruses 1 and 2, only a few ancient and isolated recent recombination events have hitherto been demonstrated for VZV. In the present study, we demonstrate that VZV also frequently undergoes genetic recombination, including strains belonging to the clade containing the vOKA strain.

Herpes simplex virus 2 (HSV-2) is the leading cause of genital herpes and increases the risk of HIV infection, but there is no effective vaccine. A replication-defective HSV-2 mutant virus, dl5-29, is effective in animal models and has been in a phase I trial. Previous studies have shown that dl5-29 gives higher antibody responses and better protection when inoculated intramuscularly (IM) as compared with subcutaneously (SC). However, the basis for this effect has not been defined. We confirmed that IM inoculation of dl5-29 is more immunogenic and provides better protection than SC inoculation. IM inoculation of HSV-2 strains produced higher levels of a luciferase transgene than SC inoculation, as measured by intravital bioluminescence imaging. Intramuscular immunization also showed better protection against infection with a highly pathogenic African HSV-2, demonstrating that this single vaccine can be efficacious against HSV-2 strains from different geographic regions.

It is well established that interferon gamma (IFN-γ) production by CD4 + T cells is critical for antiviral immunity against herpes simplex virus 2 (HSV-2) genital infection. However, the role of interleukin-17A (IL-17A) production by CD4 + T cells in HSV-2 antiviral immunity is yet to be elucidated. Here we demonstrate that IL-17A plays an important role in enhancing antiviral T helper type 1 (T h 1) responses in the female genital tract (FGT) and is essential for effective protection conferred by HSV-2 vaccination. While IL-17A did not play a critical role during primary genital HSV-2 infection, seen by lack of differences in susceptibility between IL-17A-deficient ( IL-17A −/− ) and wild-type (WT) C57BL/6 mice, it was critical for mediating antiviral responses after challenge/reexposure. Compared to WT mice, IL-17A −/− mice (i) infected intravaginally and reexposed or (ii) vaccinated intranasally and challenged intravaginally demonstrated poor outcomes. Following intravaginal HSV-2 reexposure or challenge, vaccinated IL-17A −/− mice had significantly higher mortality, greater disease severity, higher viral shedding, and higher levels of proinflammatory cytokines and chemokines in vaginal secretions. Furthermore, IL-17A −/− mice had impaired T h 1 cell responses after challenge/reexposure, with significantly lower proportions of vaginal IFN-γ + CD4 + T cells. The impaired T h 1 cell responses in IL-17A −/− mice coincided with smaller populations of IFN-γ + CD4 + tissue resident memory T (T RM ) cells in the genital tract postimmunization. Taken together, these findings describe a novel role for IL-17A in regulating antiviral IFN-γ + T h 1 cell immunity in the vaginal tract. This strategy could be exploited to enhance antiviral immunity following HSV-2 vaccination. IMPORTANCE T helper type 1 (T h 1) immunity, specifically interferon gamma (IFN-γ) production by CD4 + T cells, is critical for protection against genital herpesvirus (HSV-2) infection, and enhancing this response can potentially help improve disease outcomes. Our study demonstrated that interleukin-17A (IL-17A) plays an essential role in enhancing antiviral T h 1 responses in the female genital tract (FGT). We found that in the absence of IL-17A, preexposed and vaccinated mice showed poor disease outcomes and were unable to overcome HSV-2 reexposure/challenge. IL-17A-deficient mice ( IL-17A −/− ) had smaller populations of IFN-γ + CD4 + tissue resident memory T (T RM ) cells in the genital tract postimmunization than did wild-type (WT) mice, which coincided with attenuated T h 1 responses postchallenge. This has important implications for developing effective vaccines against HSV-2, as we propose that strategies inducing IL-17A in the genital tract may promote more effective T h 1 cell immunity and better overall protection.

Herpes simplex virus is a common causative agent of oral and genital diseases. Novel vaccines and therapeutics are needed to combat herpes infections especially after the failure of subunit vaccines in human clinical trials. We have shown that the live-attenuated HSV-1 VC2 vaccine strain is unable to establish latency in vaccinated animals and produces a robust immune response capable of completely protecting mice against lethal vaginal HSV-1 or HSV-2 infections. The guinea pig represents the best small animal model of genital HSV-2 disease. Reported here, twenty-one female Hartley guinea pigs received intramuscular injection with either the VC2 vaccine, or equal volume of conditioned tissue culture media. Animals received 2 booster vaccinations at 21 day intervals following the initial vaccination. After vaccination, animals were challenged with the highly virulent HSV-2 (G) strain. Histologically, VC2 vaccinated animals had little to no apparent inflammation/disease following challenge. Unvaccinated animals developed moderate to severe erosive and ulcerative vaginitis. Quantitative reverse-transcriptase PCR analysis in VC2 vaccinated and challenged animals identified transcriptional signatures of Th17 and regulatory Tr1 cells associated with the inflammatory response primed by VC2 vaccination. Treatment of cultured human vaginal epithelial cells (VK2 cells) with a combination of IL-17A and IL-22 resulted in the significant induction of beta-defensin 3 expression. Further, treatment of VK2 cells with IL-17A, IL-22, IL-36 or beta-defensin 3 resulted in diminished HSV-2 replication. Overall, these results suggest that intramuscular vaccination with the live-attenuated vaccine VC2 primes a mucosal immune response predisposing the adaptive expression of transcripts associated with a Th17 response to challenge and these responses contribute to antiviral immunity.

Vaccination remains the best option to combat equine herpesvirus 1 (EHV-1) infection, and several different strategies of vaccination have been investigated and developed over the past few decades. Herein, we report that the live-attenuated herpes simplex virus 1 (HSV-1) VC2 vaccine strain, which has been shown to be unable to enter into neurons and establish latency in mice, can be utilized as a vector for the heterologous expression of EHV-1 glycoprotein D (gD) and that the intramuscular immunization of mice results in strong antiviral humoral and cellular immune responses. The VC2–EHV-1–gD recombinant virus was constructed by inserting an EHV-1 gD expression cassette under the control of the cytomegalovirus immediate early promoter into the VC2 vector in place of the HSV-1 thymidine kinase (UL23) gene. The vaccines were introduced into mice through intramuscular injection. Vaccination with both the VC2–EHV-1–gD vaccine and the commercially available vaccine Vetera EHV XP 1/4 (Vetera; Boehringer Ingelheim Vetmedica) resulted in the production of neutralizing antibodies, the levels of which were significantly higher in comparison to those in VC2- and mock-vaccinated animals ( P < 0.01 or P < 0.001). Analysis of EHV-1-reactive IgG subtypes demonstrated that vaccination with the VC2–EHV-1–gD vaccine stimulated robust IgG1 and IgG2a antibodies after three vaccinations ( P < 0.001). Interestingly, Vetera-vaccinated mice produced significantly higher levels of IgM than mice in the other groups before and after challenge ( P < 0.01 or P < 0.05). Vaccination with VC2–EHV-1–gD stimulated strong cellular immune responses, characterized by the upregulation of both interferon- and tumor necrosis factor-positive CD4 + T cells and CD8 + T cells. Overall, the data suggest that the HSV-1 VC2 vaccine strain may be used as a viral vector for the vaccination of horses as well as, potentially, for the vaccination of other economically important animals. IMPORTANCE A novel virus-vectored VC2–EHV-1–gD vaccine was constructed using the live-attenuated HSV-1 VC2 vaccine strain. This vaccine stimulated strong humoral and cellular immune responses in mice, suggesting that it could protect horses against EHV-1 infection.

The outbreak of highly pathogenic avian influenza virus in North American poultry during 2014 and 2015 demonstrated the devastating effects of the disease and highlighted the need for effective emergency vaccine prevention and control strategies targeted at currently circulating strains. This study evaluated the efficacy of experimental recombinant turkey herpesvirus vector vaccines with three different inserts targeting the hemagglutinin gene of an isolate from the recent North American influenza outbreak. White leghorn chickens were vaccinated at one day of age and challenged with A/Turkey/Minnesota/12582/2015 H5N2 at 4 weeks of age. Birds were analyzed for survival, viral shedding at two and four days after infection, and specific antibody prior to challenge and from surviving birds. The three experimental vaccines demonstrated 100%, 45% and 15% survival with the most effective vaccine significantly reducing oral and cloacal viral shedding compared to all other groups and generated specific antibody prior to challenge with highly pathogenic avian influenza virus. More studies are needed using diverse H5Nx highly pathogenic virus isolates to fully determine the breadth of coverage against possible exposure strains, as well as possible impact of maternally derived antibody on protection and vaccine efficacy.

An estimated 417 million people worldwide ages 15 to 49 are infected with herpes simplex virus type 2 (HSV-2), the most common cause of genital ulcer disease. Some individuals experience frequent recurrences of genital lesions, while others only have subclinical infection, yet all risk transmitting infection to their intimate partners. A vaccine was developed that prevents shingles, which is a recurrent infection caused by varicella-zoster virus (VZV), a closely related member of the Herpesviridae family. The success of the VZV vaccine has stimulated renewed interest in a therapeutic vaccine for genital herpes. We have been evaluating a trivalent subunit antigen vaccine for prevention of genital herpes. Here, we assess the trivalent vaccine as immunotherapy in guinea pigs that were previously infected intravaginally with HSV-2. The trivalent vaccine contains HSV-2 glycoproteins C, D, and E (gC2, gD2, gE2) subunit antigens administered with CpG and alum as adjuvants. We previously demonstrated that antibodies to gD2 neutralize the virus while antibodies to gC2 and gE2 block their immune evasion activities, including evading complement attack and inhibiting activities mediated by the IgG Fc domain, respectively. Here, we demonstrate that the trivalent vaccine significantly boosts ELISA titers and neutralizing antibody titers. The trivalent vaccine reduces the frequency of recurrent genital lesions and vaginal shedding of HSV-2 DNA by approximately 50% and almost totally eliminates vaginal shedding of replication-competent virus, suggesting that the trivalent vaccine is a worthy candidate for immunotherapy of genital herpes.

While HSV-2 typically causes genital lesions, HSV-1 is increasingly the cause of genital herpes. In addition, neonatal HSV infections are associated with a high rate of mortality and HSV-2 may increase the risk for HIV or Zika infections, reinforcing the need to develop an effective vaccine. In the GSK Herpevac trial, doubly sero-negative women were vaccinated with a truncated form of gD2 [gD2(284t)], then examined for anti-gD serum titers and clinical manifestations of disease. Surprisingly, few vaccinees were protected against genital HSV-2 but 86% were protected from genital HSV-1. These observations suggest that subtle differences in gD structure might influence a protective response. To better understand the antigenic structure of gD and how it impacts a protective response, we previously utilized several key anti-gD monoclonal antibodies (mAbs) to dissect epitopes in vaccinee sera. Several correlations were observed but the methodology limited the number of sera and mAbs that could be tested. Here, we used array-based surface plasmon imaging (SPRi) to simultaneously measure a larger number of protein-protein interactions. We carried out cross-competition or “epitope binning” studies with 39 anti-gD mAbs and four soluble forms of gD, including a form [gD2(285t)] that resembles the Herpevac antigen. The results from these experiments allowed us to organize the mAbs into four epitope communities. Notably, relationships within and between communities differed depending on the form of gD, and off-rate analysis suggested differences in mAb-gD avidity depending on the gD serotype and length. Together, these results show that gD1 and gD2 differ in their structural topography. Consistent with the Herpevac results, several mAbs that bind both gD1 and gD2 neutralize only HSV-1. Thus, this technology provides new insights into the antigenic structure of gD and provides a rationale as to how vaccination with a gD2 subunit may lead to protection from HSV-1 infection., Author summary Understanding the complex steps of herpes simplex virus (HSV) entry into susceptible cells is key for developing an effective vaccine to prevent clinical disease and control recurrences of this important human pathogen. For either serotype (HSV-1 or HSV-2), it is the interaction of glycoprotein D (gD) with receptor that initiates the cascade of events to begin infection. Importantly, gD stimulates high titers of virus-neutralizing antibodies that predict its importance in a protective response. Many HSV vaccines under development center around gD. In one recent clinical trial, subjects vaccinated with gD2 were protected against HSV-1 genital infection but not HSV-2, leading us to address how type specificity affects the antigenic topography of gD. Using a large panel of monoclonal antibodies (mAbs), we employed a new high-throughput, array-based approach to organize the mAbs into communities. Relationships within and between communities differed depending on the isotype and length of gD, suggesting significant differences in the epitope topography. We found that a subset of mAbs bound both gD1 and gD2 yet only neutralized HSV-1, possibly accounting for the anomalous results of the clinical trial. This new technology has proven to yield significant insights about how different regions of gD contribute to immune responses.

No vaccines are approved for prevention or treatment of genital herpes. The focus of genital herpes vaccine trials has been on prevention using herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) alone or combined with glycoprotein B. These prevention trials did not achieve their primary end points. However, subset analyses reported some positive outcomes in each study. The most recent trial was the Herpevac Trial for Women that used gD2 with monophosphoryl lipid A and alum as adjuvants in herpes simplex virus type 1 (HSV-1) and HSV-2 seronegative women. Unexpectedly, the vaccine prevented genital disease by HSV-1 but not HSV-2. Currently, HSV-1 causes more first episodes of genital herpes than HSV-2, highlighting the importance of protecting against HSV-1. The scientific community is conflicted between abandoning vaccine efforts that include gD2 and building upon the partial successes of previous trials. We favor building upon success and present approaches to improve outcomes of gD2-based subunit antigen vaccines.

The development of immunization strategies to protect against ocular infection with herpes simplex virus 1 (HSV-1) must address the issue of the effects of the strategy on the establishment of latency in the trigeminal ganglia (TG). It is the reactivation of this latent virus that can cause recurrent disease and corneal scarring. CD8 + T cells and dendritic cells (DCs) have been implicated in the establishment and maintenance of latency through several lines of inquiry. The objective of the current study was to use CD8α −/− and CD8β −/− mice to further evaluate the contributions of CD8 + T cells and the CD8α + and CD8α − subpopulations of DCs to the protection afforded against ocular infection by immunization against HSV-1 and their potential to increase latency. Neutralizing antibody titers were similar in immunized CD8α −/− , CD8β −/− , and wild-type (WT) mice, as was virus replication in the eye. However, on day 3 postinfection (p.i.), the copy number of HSV-1 glycoprotein B (gB) was higher in the corneas and TG of CD8α −/− mice than those of WT mice, whereas on day 5 p.i. it was lower. As would be anticipated, the lack of CD8α + or CD8β + cells affected the levels of type I and type II interferon transcripts, but the effects were markedly time dependent and tissue specific. The levels of latent virus in the TG, as estimated by measurement of LAT transcripts and in vitro explant reactivation assays, were lower in the immunized, ocularly challenged CD8α −/− and WT mice than in their CD8β −/− counterparts. Immunization reduced the expression of PD-1, a marker of T-cell exhaustion, in the TG of ocularly challenged mice, and mock-immunized CD8α −/− mice had lower levels of PD-1 expression and latency than mock-immunized WT or CD8β −/− mice. The expansion of the CD8α − subpopulation of DCs through injection of WT mice with granulocyte-macrophage colony-stimulating factor (GM-CSF) DNA reduced the amount of latency and PD-1 expression in the TG of infected mice. In contrast, injection of FMS-like tyrosine kinase 3 ligand (Flt3L) DNA, which expanded both subpopulations, was less effective. Our results suggest that the absence of both CD8α + T cells and CD8α + DCs does not reduce vaccine efficacy, either directly or indirectly, in challenged mice and that administration of GM-CSF appears to play a beneficial role in reducing latency and T-cell exhaustion. IMPORTANCE In the past 2 decades, two large clinical HSV vaccine trials were performed, but both vaccine studies failed to reach their goals. Thus, as an alternative to conventional vaccine studies, we have used a different strategy to manipulate the host immune responses in an effort to induce greater protection against HSV infection. In lieu of the pleiotropic effect of CD8α + DCs in HSV-1 latency, in this report, we show that the absence of CD8α + T cells and CD8α + DCs has no adverse effect on vaccine efficacy. In line with our hypothesis, we found that pushing DC subpopulations from CD8α + DCs toward CD8α − DCs by injection of GM-CSF reduced the amount of latent virus and T-cell exhaustion in TG. While these studies point to the lack of a role for CD8α + T cells in vaccine efficacy, they in turn point to a role for GM-CSF in reducing HSV-1 latency.

Nasopharyngeal carcinoma (NPC) of the undifferentiated subtype remains endemic in southern China, with a peak incidence in this region approaching 30 cases per 100,000 population per year. Despite advances in chemotherapy and radiation delivery techniques in localized disease, distant metastasis is still common and NPC remains the seventh leading cause of cancer death in the region. There is great need for early diagnosis, developing novel therapies, and identifying patients with localized disease at higher risk of future recurrence or metastasis to appropriately tailor their treatment and improve outcomes. Knowledge of the integral involvement of Epstein-Barr virus (EBV) in the pathogenesis of undifferentiated NPC has been of seminal importance in developing strategies to optimize disease management. The close association with EBV is being evaluated in multiple settings including screening of at-risk populations, disease prognostication, development of targeted therapies, optimizing adjuvant treatment, and early recurrence detection. These translational studies are likely to have an enormous effect on management of undifferentiated NPC and significantly improve the landscape of the disease in years to come.

The investigational AS04-adjuvanted herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) subunit prophylactic vaccine ('HSV vaccine'; GlaxoSmithKline Vaccines) has been shown to be well tolerated in adults, but limited data exist for pre-teen and adolescent girls, a likely target population. The primary objective of this study was to compare the occurrence of serious adverse events (SAEs) over 12 months between HSV vaccine recipients and saline recipients (placebo control group) in pre-teen and adolescent girls. The immunogenicity of the HSV vaccine was also assessed.Healthy girls aged 10-17 years, stratified by age (10-15 years; 16-17 years), were randomised 2:1:1 to receive the HSV vaccine, a hepatitis A vaccine (Havrix™; HAV control) or placebo (saline) according to a 0-, 1-, 6-month schedule. Participants and study personnel not involved in the preparation or administration of vaccines were blinded to treatment. Safety and immunogenicity analyses were performed overall and by age (10-15 years; 16-17 years) and HSV serostatus.No statistically significant difference in the percentage of subjects with SAEs was observed between the HSV and saline group, or between the HSV and pooled control (HAV and saline) groups. The HSV vaccine was well tolerated, although a higher incidence of solicited local symptoms was observed in the HSV group than in the control group. Neither age nor HSV serostatus at the time of study entry had an impact on the safety profile of this vaccine. The HSV vaccine was immunogenic regardless of pre-vaccination HSV serostatus. Higher anti-gD geometric mean concentrations were observed in HSV-1 seropositive participants than in HSV-1 seronegative participants.The HSV vaccine had an acceptable safety profile, and was well tolerated and immunogenic when administered to girls aged 10-17 years regardless of age or HSV pre-vaccination serostatus.

Despite recent advances in vaccine design and strategies, latent infection with herpes simplex virus (HSV) remains a formidable challenge. Approaches involving live-attenuated viruses and inactivated viral preparations were popular throughout the twentieth century. In the past ten years, many vaccine types, both prophylactic or therapeutic, have contained a replication-defective HSV, viral DNA or glycoproteins. New research focused on the mechanism of immune evasion by the virus has involved developing vaccines with various gene deletions and manipulations combined with the use of new and more specific adjuvants. In addition, new "prime-boost" methods of strengthening the vaccine efficacy have proven effective, but there have also been flaws with some recent strategies that appear to have compromised vaccine efficacy in humans. Given the complicated lifecycle of HSV and its unique way of spreading from cell-to-cell, it can be concluded that the development of an ideal vaccine needs new focus on cell-mediated immunity, better understanding of the latent viral genome and serious consideration of gender-based differences in immunity development among humans. This review summarizes recent developments made in the field and sheds light on some potentially new ways to conquer the problem including development of dual-action prophylactic microbicides that prohibit viral entry and, in addition, induce a strong antigen response.

Open reading frame 4 (ORF4) of varicella-zoster virus (VZV) encodes an immediate-early protein that is believed to be important for viral infectivity and establishing latency. Evidence suggests that VZV-specific T cells are crucial in the control of viral replication, but there are no data addressing the existence of potential ORF4 protein-specific CD4 + T cells. We tested the hypothesis that VZV ORF4 protein-specific CD4 + T cells could be identified and characterized within the peripheral blood of healthy immune donors following primary infection. Gamma interferon (IFN-γ) immunosorbent assays were used to screen peripheral blood mononuclear cells obtained from healthy seropositive donors for responses to overlapping ORF4 peptides, viral lysate, and live vaccine. High frequencies of ORF4 protein-specific T cells were detected ex vivo in individuals up to 52 years after primary infection. Several immunogenic regions of the ORF4 protein were identified, including a commonly recognized epitope which was restricted through HLA-DRB1*07. Total ORF4 protein-specific responses comprised 19.7% and 20.7% of the total lysate and vaccine responses, respectively, and were dominated by CD4 + T cells. Indeed, CD4 + T cells were found to dominate the overall virus-specific IFN-γ cellular immune response both ex vivo and after expansion in vitro. In summary, we have identified an ORF4 protein as a novel target antigen for persistent VZV-specific CD4 + T cells, with implications for disease pathogenesis and future vaccine development.

Herpes simplex virus (HSV)-2 is the predominant cause of genital herpes and has been implicated in HIV infection and transmission. Thus far, vaccines developed against HSV-2 have been clinically ineffective in preventing infection. This review aims to summarize the innate and adaptive immune responses against HSV-2 and examines the current status of vaccine development.Both innate and adaptive immune responses are essential for an effective primary immune response and the generation of immunity. The innate response involves Toll-like receptors, natural killer cells, plasmacytoid dendritic cells, and type I, II, and III interferons. The adaptive response requires a balance between CD4+ and CD8+ T-cells for optimal viral clearance. T-regulatory cells may be involved, although their exact function has yet to be determined. Current vaccine development involves the use of HSV-2 peptides or attenuated/replication-defective HSV-2 to generate adaptive anti-HSV-2 immune responses, however the generation of innate responses may also be an important consideration.Although vaccine development has primarily focused on the adaptive response, arguments for innate involvement are emerging. A greater understanding of the innate and adaptive processes underlying the response to HSV-2 infection will provide the foundation for the development of an effective vaccine.

Virulent varicella-zoster virus (VZV) can spread in immunocompetent humans, resulting in symptoms mostly of the skin. In contrast, vaccine Oka (V-Oka), the attenuated VZV vaccine strain, only rarely causes clinical reactions. The mechanisms underlying these pathogenetic differences are unclear. In this study, we comparatively analyzed the ability of virulent VZV and V-Oka to modulate instruction of dendritic cells (DCs) by innate signals. DCs isolated from normal human skin were susceptible to infection with VZV and V-Oka. Moreover, inflammatory DCs, which play a crucial role in the stimulation of Th1 immune responses, accumulated in herpes zoster lesions. Infection of inflammatory DCs generated in vitro with virulent VZV or V-Oka resulted in upregulation of CD1c. Upon coculture with CD1c-restricted innate cells, DCs developed a mature phenotype whether infected with virulent VZV or V-Oka. Intriguingly, a striking difference was detected on the functional level. The release of IFN-γ and IL-12, the signature cytokines of Th1 responses, was enhanced by V-Oka but blocked by virulent VZV. V-Oka and virulent VZV efficiently synergized with CD40L, eliminating the possibility that CD40 signaling was a target of VZV-associated immune evasion. Instead, virulent VZV selectively interfered with signaling through TLR2, which is known to sense VZV. Thus, virulent VZV subverts Th1-promoting instruction of human DCs by blocking TLR2-mediated innate signals that prime IL-12 production by DCs. Taken together, our results demonstrate a novel immune-evasion mechanism of virulent VZV that has been lost during the attenuation process leading to the VZV vaccine strain.

Herpes simplex virus type 1(HSV-1) presents a conundrum to public health worldwide because of its specific pathogenicity and clinical features. Some experimental vaccines, such as the recombinant viral glycoproteins, exhibit the viral immunogenicity of a host-specific immune response, but none of these has achieved a valid epidemiological protective efficacy in the human population. In the present study, we constructed an attenuated HSV-1 strain M3 through the partial deletion of UL7, UL41, and the latency-associated transcript (LAT) using the CRISPR/Cas9 system. The mutant strain exhibited lowered infectivity and virulence in macaques. Neutralization testing and ELISpot detection of the specific T-cell responses confirmed the specific immunity induced by M3 immunization and this immunity defended against the challenges of the wild-type strain and restricted the entry of the wild-type strain into the trigeminal ganglion. These results in rhesus macaques demonstrated the potential of the attenuated vaccine for the prevention of HSV-1 in humans.

Herpes simplex virus (HSV) serotypes 1 and 2 establish lifelong infections that can produce reactivated pools of virus at mucosal sites where primary infections were initiated. No approved vaccines are available. To break the transmission cycle, interventions must either prevent infection or reduce infectivity at mucosal sites. This article discusses the recent experimental successes of immunoprophylactic and therapeutic compounds that enhance resistance and/or reduce viral loads at genital and ocular mucosa. Current data indicate Toll-like receptor agonists and selected immunomodulating compounds effectively increase the HSV infection threshold and hold promise for genital prophylaxis. Similarly, immunization at genital and extragenital mucosal sites is discussed. Finally, preclinical success with novel immunotherapies for ocular HSV that address herpetic keratitis and corneal blindness is reviewed.

CJ9-gD belongs to a new class of replication-defective recombinant herpes simplex viruses (HSVs) type 1 that can function in trans to prevent the replication of wild-type HSV in co-infected cells. Furthermore, CJ9-gD cannot establish latent infection in vivo and it expresses high levels of the major HSV-1 antigen glycoprotein D immediately following infection. In this study we show that guinea pigs immunized with CJ9-gD developed at least 9,600-fold higher titers of HSV-1-specific neutralization antibodies than mock-immunized controls. After challenge with wild-type HSV-1, all 10 mock-immunized guinea pigs developed multiple skin lesions with an average of 53.3 lesions per animal, whereas only 2 minor lesions were found in 1 of 10 CJ9-gD-immunized animals, representing a 267-fold reduction on the incidence of primary herpetic skin lesions in immunized animals. Quantitative PCR analysis revealed that the amount and frequency of wild-type HSV-1 viral DNA present in dorsal root ganglia of immunized animals was significantly lower than that in mock-immunized controls. Collectively, we demonstrate that vaccination with CJ9-gD elicits strong and protective immune responses against primary HSV-1 skin disease and reduces the extent of latent infection by challenge virus.

In recent clinical trials, a herpes simplex virus (HSV) recombinant glycoprotein D (gD) vaccine was more efficacious in woman than in men. Here we report six HLA-DR-restricted T-cell gD epitope peptides that bind to multiple HLA-DR (DR1, DR4, DR7, DR13, DR15, and DRB5) molecules that represent a large proportion of the human population. Four of these peptides recalled naturally primed CD4+T cells in up to 45% of the 46 HSV-seropositive, asymptomatic individuals studied. For the gD49-82, gD77-104, and gD121-152peptides, the CD4+T-cell responses detected in HSV-seropositive, asymptomatic women were higher and more frequent than the responses detected in men. Immunization of susceptible DRB1*0101 transgenic mice with a mixture of three newly identified, gender-dependent, immunodominant epitope peptides (gD49-82, gD77-104, and gD121-152) induced a gender- and CD4+T-cell-dependent immunity against ocular HSV type 1 challenge. These results revealed a gender-dependent T-cell response to a discrete set of gD epitopes and suggest that while a T-cell epitope-based HSV vaccine that targets a large percentage of the human population may be feasible with a limited number of immunodominant promiscuous HLA-DR-restricted epitopes, gender should be taken into account during evaluations of such vaccines.