Rational Design of an Epstein-Barr Virus Vaccine Targeting the Receptor-Binding Site.

Epstein-Barr virus (EBV) represents a major global health problem. Though it is associated with infectious mononucleosis and ∼200,000 cancers annually worldwide, a vaccine is not available. The major target of immunity is EBV glycoprotein 350/220 (gp350) that mediates attachment to B cells through complement receptor 2 (CR2/CD21). Here, we created self-assembling nanoparticles that displayed different domains of gp350 in a symmetric array. By focusing presentation of the CR2-binding domain on nanoparticles, potent neutralizing antibodies were elicited in mice and non-human primates. The structurally designed nanoparticle vaccine increased neutralization 10- to 100-fold compared to soluble gp350 by targeting a functionally conserved site of vulnerability, improving vaccine-induced protection in a mouse model. This rational approach to EBV vaccine design elicited potent neutralizing antibody responses by arrayed presentation of a conserved viral entry domain, a strategy that can be applied to other viruses.

Oral Probiotic Vaccine Expressing Koi Herpesvirus (KHV) ORF81 Protein Delivered by Chitosan-Alginate Capsules Is a Promising Strategy for Mass Oral Vaccination of Carps against KHV Infection.

Koi herpesvirus (KHV) is highly contagious and lethal to cyprinid fish, causing significant economic losses to the carp aquaculture industry, particularly to koi carp breeders. Vaccines delivered through intramuscular needle injection or gene gun are not suitable for mass vaccination of carp. So, the development of cost-effective oral vaccines that are easily applicable at a farm level is highly desirable. In this study, we utilized chitosan-alginate capsules as an oral delivery system for a live probiotic (Lactobacillus rhamnosus) vaccine, pYG-KHV-ORF81/LR CIQ249, expressing KHV ORF81 protein. The tolerance of the encapsulated recombinant Lactobacillus to various digestive environments and the ability of the probiotic strain to colonize the intestine of carp was tested. The immunogenicity and the protective efficacy of the encapsulated probiotic vaccine was evaluated by determining IgM levels, lymphocyte proliferation, expression of immune-related genes, and viral challenge to vaccinated fish. It was clear that the chitosan-alginate capsules protected the probiotic vaccine effectively against extreme digestive environments, and a significant level (P < 0.01) of antigen-specific IgM with KHV-neutralizing activity was detected, which provided a protection rate of ca. 85% for koi carp against KHV challenge. The strategy of using chitosan-alginate capsules to deliver probiotic vaccines is easily applicable for mass oral vaccination of fish.IMPORTANCE An oral probiotic vaccine, pYG-KHV-ORF81/LR CIQ249, encapsulated by chitosan-alginate capsules as an oral delivery system was developed for koi carp against koi herpesvirus (KHV) infection. This encapsulated probiotic vaccine can be protected from various digestive environments and maintain effectively high viability, showing a good tolerance to digestive environments. This encapsulated probiotic vaccine has a good immunogenicity in koi carp via oral vaccination, and a significant level of antigen-specific IgM was effectively induced after oral vaccination, displaying effective KHV-neutralizing activity. This encapsulated probiotic vaccine can provide effective protection for koi carp against KHV challenge, which is handling-stress free for the fish, cost effective, and suitable for the mass oral vaccination of koi carp at a farm level, suggesting a promising vaccine strategy for fish.

An HSV-2 nucleoside-modified mRNA genital herpes vaccine containing glycoproteins gC, gD, and gE protects mice against HSV-1 genital lesions and latent infection.

HSV-1 causes 50% of first-time genital herpes infections in resource-rich countries and affects 190 million people worldwide. A prophylactic herpes vaccine is needed to protect against genital infections by both HSV-1 and HSV-2. Previously our laboratory developed a trivalent vaccine that targets glycoproteins C, D, and E present on the HSV-2 virion. We reported that this vaccine protects animals from genital disease and recurrent virus shedding following lethal HSV-2 challenge. Importantly the vaccine also generates cross-reactive antibodies that neutralize HSV-1, suggesting it may provide protection against HSV-1 infection. Here we compared the efficacy of this vaccine delivered as protein or nucleoside-modified mRNA immunogens against vaginal HSV-1 infection in mice. Both the protein and mRNA vaccines protected mice from HSV-1 disease; however, the mRNA vaccine provided better protection as measured by lower vaginal virus titers post-infection. In a second experiment, we compared protection provided by the mRNA vaccine against intravaginal challenge with HSV-1 or HSV-2. Vaccinated mice were totally protected against death, genital disease and infection of dorsal root ganglia caused by both viruses, but somewhat better protected against vaginal titers after HSV-2 infection. Overall, in the two experiments, the mRNA vaccine prevented death and genital disease in 54/54 (100%) mice infected with HSV-1 and 20/20 (100%) with HSV-2, and prevented HSV DNA from reaching the dorsal root ganglia, the site of virus latency, in 29/30 (97%) mice infected with HSV-1 and 10/10 (100%) with HSV-2. We consider the HSV-2 trivalent mRNA vaccine to be a promising candidate for clinical trials for prevention of both HSV-1 and HSV-2 genital herpes.

Tetrameric glycoprotein complex gH/gL/gQ1/gQ2 is a promising vaccine candidate for human herpesvirus 6B.

Primary infection of human herpesvirus 6B (HHV-6B) occurs in infants after the decline of maternal immunity and causes exanthema subitum accompanied by a high fever, and it occasionally develops into encephalitis resulting in neurological sequelae. There is no effective prophylaxis for HHV-6B, and its development is urgently needed. The glycoprotein complex gH/gL/gQ1/gQ2 (called 'tetramer of HHV-6B') on the virion surface is a viral ligand for its cellular receptor human CD134, and their interaction is thus essential for virus entry into the cells. Herein we examined the potency of the tetramer as a vaccine candidate against HHV-6B. We designed a soluble form of the tetramer by replacing the transmembrane domain of gH with a cleavable tag, and the tetramer was expressed by a mammalian cell expression system. The expressed recombinant tetramer is capable of binding to hCD134. The tetramer was purified to homogeneity and then administered to mice with aluminum hydrogel adjuvant and/or CpG oligodeoxynucleotide adjuvant. After several immunizations, humoral and cellular immunity for HHV-6B was induced in the mice. These results suggest that the tetramer together with an adjuvant could be a promising candidate HHV-6B vaccine.

Lethal Hemorrhagic Disease and Clinical Illness Associated with Elephant Endotheliotropic Herpesvirus 1 Are Caused by Primary Infection: Implications for the Detection of Diagnostic Proteins.

Elephant endotheliotropic herpesvirus (EEHV) can cause lethal hemorrhagic disease in juvenile Asian elephants, both in captivity and in the wild. Most deaths associated with the virus are caused by two chimeric variants of EEHV1 (EEHV1A and EEHV1B), while two other EEHVs endemic within Asian elephants (EEHV4 and EEHV5) have been recognized but cause death less often. Whether lethal EEHV infections are due to primary infection or reactivation of latent virus remains unknown, and knowledge of the anti-EEHV antibody levels in young elephants is limited. To close these gaps, we sought to develop a serologic assay capable of distinguishing among infections with different EEHVs using a luciferase immunoprecipitation system (LIPS) for antibody profiling and a panel of conserved EEHV recombinant proteins and proteins unique to EEHV1. The results showed that elephants dying from EEHV1 hemorrhagic disease or ill from EEHV infection were seronegative for the EEHV species that caused the disease or illness, indicating that the events were associated with primary infection rather than reactivation of latent virus. We also demonstrated that waning of EEHV1-specific antibodies can occur in the first 2 years of life, when a threshold protective level of antibody may be needed to prevent severe EEHV1-related disease. Use of the LIPS assay to identify putative "diagnostic" proteins would be a valuable asset in determining the EEHV immune status of young elephants and responses to candidate EEHV vaccines in the future.IMPORTANCE Whether clinical illness and deaths associated with elephant endotheliotropic herpesvirus (EEHV) infection result from primary infection or reactivation of latent virus is a longstanding question in the field. By applying a relatively new assay, the luciferase immunoprecipitation system (LIPS), combined with the genomic sequences of the viruses, we gained the insights and tools needed to resolve this issue. Our EEHV1-specific LIPS assay should be useful for assessing the vulnerability of elephant calves to infection with different EEHVs and evaluating antibody responses to anti-EEHV vaccines. A significant proportion of the Asian elephant population is under some form of human care. Hence, the ability to screen for EEHV immune status in elephant calves should have a major impact on the management of these animals worldwide.

A Single-Cycle Glycoprotein D Deletion Viral Vaccine Candidate, ΔgD-2, Elicits Polyfunctional Antibodies That Protect against Ocular Herpes Simplex Virus.

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.

Transmission of infectious laryngotracheitis virus vaccine and field strains: the role of degree of contact and transmission by whole blood, plasma and poultry dust.

Understanding the mechanisms of transmission of infectious laryngotracheitis virus (ILTV) is critical to proper control as both vaccine and wild-type strains circulate within chicken flocks with potential adverse consequences. The relative efficiency of transmission by direct contact between chickens and airborne transmission has not been investigated. Furthermore, relatively high levels of ILTV DNA have been detected in poultry dust and blood but the infectivity of these is unknown. In this study, comparison of in-contact and airborne transmission of two vaccine and one field strain of ILTV revealed that all transmitted to 100% of in-contact birds by 6 days post-exposure (dpe). Airborne transmission without contact resulted in 100% transmission by 14 and 17 dpe for the wild-type and Serva vaccine virus but only 27% transmission by 21 dpe for the A20 vaccine virus. The infectivity of dust or extracts of dust and blood or plasma from infected chickens at various stages of infection was assessed by inoculation into susceptible chickens. There was no transmission by any of these materials. In conclusion, direct contact facilitated efficient ILTV transmission but the virus was unable to be transmitted by dust from infected chickens suggestive of a limited role in the epidemiology of ILTV.

Integrating Alternative Social Value Judgments Into Cost-Effectiveness Analysis of Vaccines: An Application to Varicella-Zoster Virus Vaccination.

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.

Primary vaccination in foals: a comparison of the serological response to equine influenza and equine herpesvirus vaccines administered concurrently or 2 weeks apart.

This study compared concurrent and separate primary vaccination against equid alphaherpesviruses 1 and 4, genus Varicellovirus, subfamily Alphaherpesvirinae, family Herpesviridae, and equine influenza A virus, genus Alphainfluenzavirus, family Orthomyxoviridae. Their vernacular names are equine herpesvirus 1 and 4 (EHV1/4) and equine influenza virus (EIV). Infection with these respiratory pathogens is associated with loss of performance, interruption of training schedules, and on occasion, cancellation of equestrian events. Vaccination is highly recommended, and for some activities it is a mandatory requirement of the relevant authority. As there is a dearth of information relating to the impact of concurrent vaccination on the antibody response to EHV and EIV vaccines, they are usually administered separately, often 2 weeks apart. In a previous study of booster vaccination in Thoroughbred racehorses, concurrent vaccination with whole-virus inactivated carbopol-adjuvanted EHV and EIV vaccines did not impact negatively on the antibody response. In this study, investigations were extended to concurrent versus separate primary vaccination of warmblood foals. A field study was conducted to compare the immune response to a carbopol-adjuvanted EHV vaccine and an immune stimulating complex (ISCOM)-adjuvanted EI vaccine administered concurrently and 2 weeks apart. No adverse clinical reactions were observed, the pattern of EI and EHV antibody response was similar for both groups, and there was no evidence that concurrent primary vaccination compromised the humoral response. The results are of relevance to horse owners who wish to decrease veterinary costs, limit handling of young animals, and simplify record keeping by vaccinating concurrently.

Vaccination against the Epstein-Barr virus.

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.

Membrane association of a model CD4+ T-cell vaccine antigen confers enhanced yet incomplete protection against murid herpesvirus-4 infection.

Vaccination against γ-herpesviruses has proved difficult. CD4+ T cells are essential to contain infection, but how best to prime them and whether this can reduce viral loads remain unclear. To address these questions, we used ovalbumin (OVA) as a model antigen, delivering it with murine cytomegalovirus (MCMV) to protect mice against OVA-expressing murine herpesvirus-4 (MuHV-4). Membrane-associated OVA (mOVA) was more effective than soluble OVA, both to prime CD4+ T cells and as an effector target. It was also a better target than an OVA epitope limited to infected cells, suggesting that protective CD4+ T cells recognize infected cell debris rather than infected cells themselves. While MCMV-mOVA protected acutely against MuHV-4-mOVA, long-term protection was incomplete, even when OVA-specific CD8+ T cells and B cells were also primed. Thus, even optimized single-target vaccines may poorly reduce long-term γ-herpesvirus infections.

Therapeutic Mucosal Vaccination of Herpes Simplex Virus 2-Infected Guinea Pigs with Ribonucleotide Reductase 2 (RR2) Protein Boosts Antiviral Neutralizing Antibodies and Local Tissue-Resident CD4+ and CD8+ TRM Cells Associated with Protection against Recurrent Genital Herpes.

Reactivation of herpes simplex virus 2 (HSV-2) from latency causes viral shedding that develops into recurrent genital lesions. The immune mechanisms of protection against recurrent genital herpes remain to be fully elucidated. In this preclinical study, we investigated the protective therapeutic efficacy, in the guinea pig model of recurrent genital herpes, of subunit vaccine candidates that were based on eight recombinantly expressed HSV-2 envelope and tegument proteins. These viral protein antigens (Ags) were rationally selected for their ability to recall strong CD4+ and CD8+ T-cell responses from naturally "protected" asymptomatic individuals, who, despite being infected, never develop any recurrent herpetic disease. Out of the eight HSV-2 proteins, the envelope glycoprotein D (gD), the tegument protein VP22 (encoded by the UL49 gene), and ribonucleotide reductase subunit 2 protein (RR2; encoded by the UL40 gene) produced significant protection against recurrent genital herpes. The RR2 protein, delivered either intramuscularly or intravaginally with CpG and alum adjuvants, (i) boosted the highest neutralizing antibodies, which appear to cross-react with both gB and gD, and (ii) enhanced the numbers of functional gamma interferon (IFN-γ)-producing CRTAM+ CFSE+ CD4+ and CRTAM+ CFSE+ CD8+ TRM cells, which express low levels of PD-1 and TIM-3 exhaustion markers and were localized to healed sites of the vaginal mucocutaneous (VM) tissues. The strong B- and T-cell immunogenicity of the RR2 protein was associated with a significant decrease in virus shedding and a reduction in both the severity and frequency of recurrent genital herpes lesions. In vivo depletion of either CD4+ or CD8+ T cells significantly abrogated the protection. Taken together, these preclinical results provide new insights into the immune mechanisms of protection against recurrent genital herpes and promote the tegument RR2 protein as a viable candidate Ag to be incorporated in future genital herpes therapeutic mucosal vaccines.IMPORTANCE Recurrent genital herpes is one of the most common sexually transmitted diseases, with a global prevalence of HSV-2 infection predicted to be over 536 million worldwide. Despite the availability of many intervention strategies, such as sexual behavior education, barrier methods, and the costly antiviral drug treatments, eliminating or at least reducing recurrent genital herpes remains a challenge. Currently, no FDA-approved therapeutic vaccines are available. In this preclinical study, we investigated the immunogenicity and protective efficacy, in the guinea pig model of recurrent genital herpes, of subunit vaccine candidates that were based on eight recombinantly expressed herpes envelope and tegument proteins. We discovered that similar to the dl5-29 vaccine, based on a replication-defective HSV-2 mutant virus, which has been recently tested in clinical trials, the RR2 protein-based subunit vaccine elicited a significant reduction in virus shedding and a decrease in both the severity and frequency of recurrent genital herpes sores. This protection correlated with an increase in numbers of functional tissue-resident IFN-γ+ CRTAM+ CFSE+ CD4+ and IFN-γ+ CRTAM+ CFSE+ CD8+ TRM cells that infiltrate healed sites of the vaginal tissues. Our study sheds new light on the role of TRM cells in protection against recurrent genital herpes and promotes the RR2-based subunit therapeutic vaccine to be tested in the clinic.

An Equine Herpesvirus Type 1 (EHV-1) Ab4 Open Reading Frame 2 Deletion Mutant Provides Immunity and Protection from EHV-1 Infection and Disease.

Equine herpesvirus type 1 (EHV-1) outbreaks continue to occur despite widely used vaccination. Therefore, development of EHV-1 vaccines providing improved immunity and protection is ongoing. Here, an open reading frame 2 deletion mutant of the neuropathogenic EHV-1 strain Ab4 (Ab4ΔORF2) was tested as a vaccine candidate. Three groups of horses (n = 8 each) were infected intranasally with Ab4ΔORF2 or the parent Ab4 virus or were kept as noninfected controls. Horses infected with Ab4ΔORF2 had reduced fever and nasal virus shedding compared to those infected with Ab4 but mounted similar adaptive immunity dominated by antibody responses. Nine months after the initial infection, all horses were challenged intranasally with Ab4. Previously noninfected horses (control/Ab4) displayed clinical signs, shed large amounts of virus, and developed cell-associated viremia. In contrast, 5/8 or 3/8 horses previously infected with Ab4ΔORF2 or Ab4, respectively, were fully protected from challenge infection as indicated by the absence of fever, clinical disease, nasal virus shedding, and viremia. All of these outcomes were significantly reduced in the remaining, partially protected 3/8 (Ab4ΔORF2/Ab4) and 5/8 (Ab4/Ab4) horses. Protected horses had EHV-1-specific IgG4/7 antibodies prior to challenge infection, and intranasal antibodies increased rapidly postchallenge. Intranasal inflammatory markers were not detectable in protected horses but quickly increased in control/Ab4 horses during the first week after infection. Overall, our data suggest that preexisting nasal IgG4/7 antibodies neutralize EHV-1, prevent viral entry, and thereby protect from disease, viral shedding, and cell-associated viremia. In conclusion, improved protection from challenge infection emphasizes further evaluation of Ab4ΔORF2 as a vaccine candidate.IMPORTANCE Nasal equine herpesvirus type 1 (EHV-1) shedding is essential for virus transmission during outbreaks. Cell-associated viremia is a prerequisite for the most severe disease outcomes, abortion and equine herpesvirus myeloencephalopathy (EHM). Thus, protection from viremia is considered essential for preventing EHM. Ab4ΔORF2 vaccination prevented EHV-1 challenge virus replication in the upper respiratory tract in fully protected horses. Consequently, these neither shed virus nor developed cell-associated viremia. Protection from virus shedding and viremia during challenge infection in combination with reduced virulence at the time of vaccination emphasizes ORF2 deletion as a promising modification for generating an improved EHV-1 vaccine. During this challenge infection, full protection was linked to preexisting local and systemic EHV-1-specific antibodies combined with rapidly increasing intranasal IgG4/7 antibodies and lack of nasal type I interferon and chemokine induction. These host immune parameters may constitute markers of protection against EHV-1 and be utilized as indicators for improved vaccine development and informed vaccination strategies.

Immunogenic particles with a broad antigenic spectrum stimulate cytolytic T cells and offer increased protection against EBV infection ex vivo and in mice.

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.

Carbon nanotube-based DNA vaccine against koi herpesvirus given by intramuscular injection.

Koi herpesvirus (KHV) also named Cyprinid Herpesvirus 3 (CyHV-3) is one of the most threatening pathogens affecting common carp production as well as the valued ornamental koi carp. The current commercial vaccines available are costly and potentially cause severe stress caused by live virus. KHV ORF149 gene has been proved encoding one of the main immunogenic proteins for KHV. In this study, we coupled a plasmid expression vector for ORF149 to single walled carbon nanotubes (SWCNTs) for an anti-KHV vaccine. The vaccine conferred an 81.9% protection against intraperitoneal challenge with KHV. Importantly, SWCNTs as a promising vehicle can enhanced the protective effects 33.9% over that of the naked DNA vaccine at the same dose. The protection was longer and serum antibody production, enzyme activities and immune-related gene expression were all induced in fish vaccinated with the nanotube-DNA vaccine compared with the DNA alone. Thereby, this study demonstrates that the ORF149 DNA vaccine loaded onto SWCNTs as a novel vaccine might provide an effective method of coping with KHV disease using intra-muscular vaccination.

Protective immunity against CyHV-3 infection via different prime-boost vaccination regimens using CyHV-3 ORF131-based DNA/protein subunit vaccines in carp Cyprinus carpio var. Jian.

Cyprinid Herpesvirus 3 (CyHV-3), also known as Koi Herpesvirus (KHV), causes Koi Herpesvirus Disease (KHVD) which leads to serious economic losses worldwide. To exploit DNA/subunit vaccine candidates, CyHV-3 ORF131 gene and cDNA was cloned and analyzed in the present study. Major B cell epitopes of deduced CyHV-3 pORF131 was also predicted. Then the complete CDS of CyHV-3 ORF131 was inserted into pEGFP-N1 vector and a modified pYD1/EBY100 system to construct the DNA and subunit vaccine, respectively. Subsequently, carp were immunized with homologous and heterologous prime-boost regimens relying on the constructed DNA and oral subunit vaccines. Then the protective immunity generated from different vaccines and regimens as well as the capacity of yeast (Saccharomyces cerevisiae) as an oral vaccine vehicle was evaluated. Our study confirmed that CyHV-3 ORF131 gene consisted of 2 introns and 3 exons encoding a 428 amino acids peptide. Further analysis indicated that four fragments of CyHV-3 pORF131 contained the major B cell epitopes (Cys20~Val140, Ser169~Tyr245, Thr258~Pro390, Phe414~Gln428), which could be linked and expressed in E. coli (BL21) as a truncated pORF131. The expression of full-length CyHV-3 pORF131 by pEGFP-N1 and yeast surface display was verified by In vitro assays before vaccination. Immunization of carp with CyHV-3 ORF131 DNA and subunit vaccines could evoke the activation of immune-related genes such as CXCa, CXCR1, IL-1ß, TNF-α, INF-a1, Mx-1, IgM, IgT1 and production of specific serum IgM measured by ELISA. RPS (relative percent of survival) ranging from 53.33% to 66.67% was acquired post challenge test. Moreover, flow cytometry analysis illustrated the delivery of surface-displayed CyHV-3 pORF131 to midgut after oral gavage. Thus, our findings suggest that CyHV-3 ORF131 can serve as DNA/subunit vaccines candidate and the yeast as an ideal oral vaccine vehicle.

Protective immunity following vaccination with a recombinant multiple-epitope protein of bovine herpesvirus type I in a rabbit model.

Bovine herpesvirus type 1 (BoHV-1) causes considerable economic losses to the cow industry. Vaccination remains an effective strategy to control the diseases associated with BoHV-1. However, live vaccines present safety concerns, especially in pregnant cows; thus, nonreplicating vaccines have been developed to control the disease. The envelope glycoproteins of BoHV-1 induce a protective immune response. In this work, selected epitopes on glycoproteins gD, gC, and gB were constructed in triplicate with linker peptides. Vaccination of rabbits demonstrated that P2-gD/gC/gB with AAYAAY induced higher specific antibodies than that with GGGGS linker. P2-gD/gC/gB with AAYAAY linker was fused with bovine interleukin-6 (BoIL-6) or rabbit IL-6 (RaIL-6) and bacterially expressed. Rabbits were intramuscularly immunized with 100 µg of P2-gD/gC/gB-BoIL-6, P2-gD/gC/gB-RaIL-6, P2-gD/gC/gB, P2-gD/gC/gB plus BoIL-6, P2-(gD-a)3-BoIL-6, or P2-(gD-a)3 emulsified with ISA 206 adjuvant thrice at 3-week intervals. P2-gD/gC/gB-BoIL-6 generated a higher titer of BoHV-1-specific antibodies, neutralizing antibodies, interferon (IFN)-γ, and IL-4 compared with P2-gD/gC/gB plus BoIL-6, P2-gD/gC/gB-RaIL-6, or other formulation. P2-gD/gC/gB-BoIL-6 triggered similar levels of antibodies and significantly higher titer of IFN-γ and IL-4 compared with inactivated bovine viral diarrhea (BVD)-infectious bovine rhinotracheitis (IBR) vaccine. Rabbits vaccinated with P2-gD/gC/gB-BoIL-6 dramatically reduced viral shedding and tissue lesions in lungs and trachea after viral challenge and reactivation compared with those with P2-gD/gC/gB plus BoIL-6 or P2-gD/gC/gB-RaIL-6. P2-gD/gC/gB-BoIL-6 provided protective effects against viral shedding and tissue pathogenesis similar to those of the inactivated vaccine. The data confirmed the safety and immunogenicity of multiple-epitope recombinant protein and a potential vaccine candidate to control the disease, especially for pregnant cattle.

Attenuated Herpes Simplex Virus 1 (HSV-1) Expressing a Mutant Form of ICP6 Stimulates a Strong Immune Response That Protects Mice against HSV-1-Induced Corneal Disease.

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.

Immunization by Replication-Competent Controlled Herpesvirus Vectors.

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.