Live Attenuated Herpes Simplex Virus Research Articles

Cross protective efficacy of the Non-Neurotropic live attenuated herpes simplex virus type 1 vaccine VC-2 is enhanced by intradermal vaccination and deletion of glycoprotein G

Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2 respectively) cause life-long latent infections resulting in recurrent orofacial and genital blisters or sores. Ensued disease can be painful and may lead to significant mental anguish of infected individuals. Currently, there are no FDA-approved vaccines for either prophylactic or therapeutic use, and recent clinical trials of subunit vaccines failed to achieve endpoints goals. Development of a safe live-attenuated herpes simplex vaccine may provide the antigenic breadth to ultimately protect individuals from acquiring HSV disease. We have previously shown that prophylactic use of the non-neurotropic live attenuated HSV-1 vaccine, VC-2, provides potent and durable protection from genital HSV-2 disease in the guinea pig model. Here, we investigated the effects of intradermal administration as well as the deletion of the viral glycoprotein G (gG) on the efficacy of prophylactic vaccination. Vaccination with either VC-2, VC-2 gG null, or gD2 MPL/Alum offered robust protection from acute disease regardless of route of vaccination. However, both the VC-2 gG-null and the ID vaccination route were more effective compared to the parent VC2 administered by the IM route. Specifically, the VC-2 gG-null administered ID, reduced HSV-2 vaginal replication on day 2 and day 4 as well as mean recurrent lesion scores more effectively than VC2 administered IM. Most importantly, only VC-2 gG null IM and VC-2 ID significantly reduced the frequency of recurrent shedding, the most likely source for virus transmission. Similarly, while all vaccinated groups demonstrated a significant reduction in the number of animals testing PCR-positive for HSV-2 in their dorsal root ganglia following challenge only VC2 ID vaccinated animals demonstrated a significant reduction in DRG viral load. All vaccinations induced neutralizing antibodies to HSV-2 MS when compared to unvaccinated guinea pigs. Therefore, further investigation of VC-2 gG null delivered ID is warranted.

The R2 non-neuroinvasive HSV-1 vaccine affords protection from genital HSV-2 infections in a guinea pig model

Herpes simplex virus (HSV) infections are common and can cause severe illness but no vaccine is currently available. The recent failure of subunit HSV vaccines has highlighted the need for vaccines that present a diverse array of antigens, including the development of next-generation live-attenuated vaccines. However, most attenuated HSV strains propagate poorly, limiting their ability to elicit protective immune responses. A live-attenuated vaccine that replicates in non-neural tissue but is ablated for transmission into the nervous system may elicit protective immune responses without evoking neurologic complications or establishing life-long infections. Initial studies of R2, a live-attenuated vaccine that is engineered to be unable to invade the nervous system, used the guinea pig genital HSV model to evaluate the ability of R2 to replicate at the site of inoculation, cause disease and infect neural tissues. R2 was then evaluated as a vaccine using three routes of inoculation: intramuscular (IM), intradermal (ID) and intravaginal (IVag) and compared to IM administered gD2+MPL/Alum vaccine in the same model. R2 replicated in the genital tract but did not produce acute or recurrent disease and did not infect the neural tissue. The R2 vaccine-induced neutralizing antibody and decreased the severity of acute and recurrent HSV-2 disease as well as recurrent shedding. The ID route was the most effective. ID administered R2 was more effective than gD2+MPL/Alum at inducing neutralizing antibody, suppressing acute disease, and acute vaginal virus replication. R2 was especially more effective at reducing recurrent virus shedding, the most common source of HSV transmission. The live-attenuated prophylactic HSV vaccine, R2, was effective in the guinea pig model of genital HSV-2 especially when administered by the ID route. The use of live-attenuated HSV vaccines that robustly replicate in mucosal tissues but are ablated for neuroinvasion offers a promising approach for HSV vaccines.

Type-1 interferon receptor knockout mice generate high titers of ADCC antibodies in response to vaccination with ΔgD-2 but display defects in executing ADCC

Abstract ΔgD-2 is a live-attenuated Herpes Simplex Virus type-2 (HSV-2) strain genetically deleted for glycoprotein D (gD) that elicits complete protection against HSV-1 and HSV-2 through antibody-dependent cell-mediated cytotoxicity and phagocytosis (ADCC and ADCP). The mechanisms by which immunogens elicit ADCC/ADCP activating antibodies are not well understood but type-1 interferons (IFNα/β) have been shown to play a role in activating humoral immunity and FcγR-mediated immune modulation. To investigate the role of IFNα/β in generating ADCC/ADCP antibodies, we vaccinated IFNα/β receptor knockout mice (IFNAR−/−−) with ΔgD-2 and compared the resulting immunity to that of WT mice. IFNAR−/− mice vaccinated with ΔgD-2 generated similar overall and isotype specific anti-HSV IgG titers to WT mice. Serum from vaccinated IFNAR−/− mice also induced significantly more ADCC/ADCP killing than serum from vaccinated WT mice (p<0.05). To determine whether this serum initiated ADCC/ADCP in vivo, serum from vaccinated IFNAR−/− mice was passively transferred into naive WT and FcγRIV−/− mice. WT mice that received the vaccinated serum were partially protected from 10x LD90 challenge with HSV-2 but FcγRIV−/− mice were not (p<0.05). Additionally, serum from vaccinated WT mice did not confer any protection to naive IFNAR−/− mice, indicating difficulty in mobilizing ADCC/ADCP (p<0.01). However, IFNAR−/− mice that were vaccinated with ΔgD-2 were completely protected from morbidity and mortality following a 10x LD90 challenge with HSV-2 (p<0.0001). Together, our data show that the production of ADCC/ADCP antibodies in response to ΔgD-2 is independent of IFNα/β, but IFNAR signaling is important for executing ADCC/ADCP protection.

Intramuscular Immunization of Mice with the Live-Attenuated Herpes Simplex Virus 1 Vaccine Strain VC2 Expressing Equine Herpesvirus 1 (EHV-1) Glycoprotein D Generates Anti-EHV-1 Immune Responses in Mice.

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 EHVXP 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.

Impact of Type I Interferon on the Safety and Immunogenicity of an Experimental Live-Attenuated Herpes Simplex Virus 1 Vaccine in Mice.

Viral fitness dictates virulence and capacity to evade host immune defenses. Understanding the biological underpinnings of such features is essential for rational vaccine development. We have previously shown that the live-attenuated herpes simplex virus 1 (HSV-1) mutant lacking the nuclear localization signal (NLS) on the ICP0 gene (0ΔNLS) is sensitive to inhibition by interferon beta (IFN-β) in vitro and functions as a highly efficacious experimental vaccine. Here, we characterize the host immune response and in vivo pathogenesis of HSV-1 0ΔNLS relative to its fully virulent parental strain in C57BL/6 mice. Additionally, we explore the role of type 1 interferon (IFN-α/β) signaling on virulence and immunogenicity of HSV-1 0ΔNLS and uncover a probable sex bias in the induction of IFN-α/β in the cornea during HSV-1 infection. Our data show that HSV-1 0ΔNLS lacks neurovirulence even in highly immunocompromised mice lacking the IFN-α/β receptor. These studies support the translational viability of the HSV-1 0ΔNLS vaccine strain by demonstrating that, while it is comparable to a virulent parental strain in terms of immunogenicity, HSV-1 0ΔNLS does not induce significant tissue pathology.IMPORTANCE HSV-1 is a common human pathogen associated with a variety of clinical presentations ranging in severity from periodic "cold sores" to lethal encephalitis. Despite the consistent failures of HSV subunit vaccines in clinical trials spanning the past 28 years, opposition to live-attenuated HSV vaccines predicated on unfounded safety concerns currently limits their widespread acceptance. Here, we demonstrate that a live-attenuated HSV-1 vaccine has great translational potential.

Live Attenuated Herpes Simplex Virus 2 Glycoprotein E Deletion Mutant as a Vaccine Candidate Defective in Neuronal Spread

A herpes simplex virus 2 (HSV-2) glycoprotein E deletion mutant (gE2-del virus) was evaluated as a replication-competent, attenuated live virus vaccine candidate. The gE2-del virus is defective in epithelial cell-to-axon spread and in anterograde transport from the neuron cell body to the axon terminus. In BALB/c and SCID mice, the gE2-del virus caused no death or disease after vaginal, intravascular, or intramuscular inoculation and was 5 orders of magnitude less virulent than wild-type virus when inoculated directly into the brain. No infectious gE2-del virus was recovered from dorsal root ganglia (DRG) after multiple routes of inoculation; however, gE2-del DNA was detected by PCR in lumbosacral DRG at a low copy number in some mice. Importantly, no recurrent vaginal shedding of gE2-del DNA was detected in immunized guinea pigs. Intramuscular immunization outperformed subcutaneous immunization in all parameters evaluated, although individual differences were not significant, and two intramuscular immunizations were more protective than one. Immunized animals had reduced vaginal disease, vaginal titers, DRG infection, recurrent genital lesions, and recurrent vaginal shedding of HSV-2 DNA; however, protection was incomplete. A combined modality immunization using live virus and HSV-2 glycoprotein C and D subunit antigens in guinea pigs did not totally eliminate recurrent lesions or recurrent vaginal shedding of HSV-2 DNA. The gE2-del virus used as an immunotherapeutic vaccine in previously HSV-2-infected guinea pigs greatly reduced the frequency of recurrent genital lesions. Therefore, the gE2-del virus is safe, other than when injected at high titer into the brain, and is efficacious as a prophylactic and immunotherapeutic vaccine.

Effect of an immune enhancer, GPI-0100, on vaccination with live attenuated herpes simplex virus (HSV) type 2 or glycoprotein D on genital HSV-2 infections of guinea pigs

These studies were performed to determine the effect of AD-472, an attenuated human herpes simplex virus (HSV) type 2 or HSV-2 glycoprotein D (gD) when combined with an adjuvant, GPI-0100, a semi-synthetic Quillaja Saponin analog in a genital HSV-2 infection in guinea pigs. While animals immunized with either vaccine had reduced clinical disease, GPI-0100 only improved the efficacy of gD and did not affect the efficacy of the live vaccine. Neither vaccine had any therapeutic effect if administered 24 h after viral infection.

Rectal immunization generates protective immunity in the female genital tract against herpes simplex virus type 2 infection: Relative importance of myeloid differentiation factor 88

The present study was undertaken to examine the potential of rectal route of immunization for induction of protective immunity in the female genital tract against genital herpes infection in mice. A single rectal immunization of female C57Bl/6 mice with live attenuated herpes simplex virus type 2 lacking thymidine kinase (HSV-2 TK −) was shown to confer HSV-specific cellular and humoral immune responses as well as protection against an otherwise lethal vaginal challenge with a virulent HSV-2 strain. The immunity afforded by rectal immunization with HSV-2 TK − was shown to be independent of sex hormonal influence and the usage of the adaptor protein myeloid differentiation factor 88 (MyD88). Next, the impact of rectal immunization with HSV-2 glycoprotein D (gD) in combination with CpG oligodeoxynucleotide (ODN) or cholera toxin (CT) on induction of immunity against HSV-2 was investigated. Rectal immunization of mice with gD + CpG failed to generate gD specific immune responses and protection against genital herpes infection. Conversely, rectal immunization with gD + CT elicited potent gD-specific cellular immune responses and protection against genital herpes infection through a MyD88-dependent manner. These results highlight the potential of rectal route for the development of novel immunization strategies to elicit immunity in the female genital tract against genital herpes and presumably other sexually transmitted diseases.

Herpes simplex virus‐induced, death receptor‐dependent apoptosis and regression of transplanted human cancers

Inoculation of a live attenuated herpes simplex virus (HSV) vector, betaH1, into human U87MG glioblastoma cells transplanted into athymic nude mice induced complete regression of tumors. The infected cells underwent histochemically confirmed apoptosis without lymphocyte infiltration after expressing CD30, CD30 ligand (CD30L), tumor necrosis factor (TNF)-alpha, TNF receptor 1 (TNF-R1), FAS, and FAS ligand (FAS-L) with activation of caspases 3 and 8. Induction of the transcripts of these receptors and ligands in inoculated tumors was confirmed by quantitative RT-PCR. To examine the specificity of apoptosis in the transplanted tumor, we inoculated betaH1 into transplanted human lung, breast, gastric, and colon cancer tumors, and similar tumor regression with apoptosis was observed in all tumors. We analyzed the roles of expression of CD30, CD30L, TNF-alpha, TNF-R1, FAS, and FAS-L in the tumors, and found that HSV-induced apoptosis was suppressed by the respective antibodies. These findings indicate that the CD30/CD30L, TNF-alpha/TNF-R1, and FAS/FAS-L interactions resulted in apoptosis and tumor regression in immunocompromised mice. In addition to the death receptor-dependent apoptosis induced by HSV, the expressed ligands and receptors might enhance the susceptibility of tumor cells to cell-mediated cyto-toxicity and augment the activation of tumor-killing lymphocytes in immunocompetent models.

Regulated transgene delivery by ganciclovir in the brain without physiological alterations by a live attenuated herpes simplex virus vector

The distribution of a live attenuated herpes simplex virus (βH1)-mediated gene delivery into the central nervous system (CNS) was regulated by growth inhibition with ganciclovir (GCV) and the effect of this transgene expression system on the physiologic response was characterized by the acoustic startle response and its prepulse inhibition. We inoculated βH1 expressing β-galactosidase (β-gal) driven by the latency associated transcripts promoter into the right caudate putamen of rats. Histochemical analysis demonstrated that the inoculation of βH1 in the right caudate putamen resulted in a high level of β-gal expression in the neurons of the area projecting to the inoculation site. On 14 days after inoculation without GCV-treatment, β-gal activity localized in the anterior olfactory nucleus, frontal, insular, orbital, parietal, perirhinal, piriform cortices and the temporal region including the amygdala. In contrast, the distribution of β-gal activity was regulated by the interval between virus inoculation and GCV-treatment and maintained after its cessation without significant alteration. The whole process of transgene expression did not influence the emotional behavior, indicating that this vector system is a suitable model for analyzing the transgene function or applying the gene therapy for the CNS diseases.

Effect of immunity on gene delivery into anterior horn motor neurons by live attenuated herpes simplex virus vector.

Efficient and prolonged foreign gene expression has been demonstrated in the bilateral anterior horn motor neurons of the spinal cord by intramuscular inoculation with attenuated herpes simplex virus (HSV) expressing latency associated transcript promoter-driven beta-galactosidase (betaH1). To examine the effect of immunity on the gene delivery, betaH1 was applied in rats immunized subcutaneously or intramuscularly with the parent HF strain. Rats were immunized subcutaneously with HF strain and 28 days later when the high antibody titer was maintained, betaH1 was inoculated into the right gastrocnemius muscle. Second, 35 days after inoculation with HF strain into the right gastrocnemius muscle, betaH1 was inoculated at the same site. In both ways of immunization, immunity did not abolish or prevent the transgene expression in the anterior horn motor neurons, but attenuated the range and the number of the beta-galactosidase-positive neurons from about 85% to 50-65% on 28 days after inoculation with betaH1. However, beta-galactosidase activity was observed in a wide range of the bilateral anterior horn motor neurons without significant pathological changes. These findings support the feasibility of the attenuated HSV vector in gene delivery into the central nervous system, even in the presence of immunity.

Pathogenesis of experimental skin infections induced by drug-resistant herpes simplex virus mutants.

The comparative analysis of the pathogenicity of a parental herpes simplex virus type 1 strain and its phosphonoacetic acid (PAA)-resistant and acyclovir (ACV)-resistant mutants showed marked differences among them. After orofacial skin inoculation of hairless mice the parental and PAA-resistant viruses were detected during the first 4 days after infection at high and increasing titers in the trigeminal ganglia; the ACV-resistant mutant was present at low and decreasing titers in the ganglia. Severe and slow-healing skin lesions were produced by the parental and PAA-resistant viruses; mild and rapidly healing lesions were produced by the ACV-resistant mutant. Virus titers in ganglia and the intensity of skin lesions were related to the virus dose used in the primary infection. Latent infections became established in trigeminal ganglia of mice inoculated with 10(6.0) plaque-forming units of the parental or PAA-resistant virus; no latent infections were detected in ganglia of mice inoculated with 10(7.0) plaque-forming units of the ACV-resistant mutant. Serum antibody titers attained similar values 4 weeks after primary infection with both mutants and the parental virus. Mice infected with the ACV-resistant mutant were reinfected with the parental and PAA-resistant viruses; the degree of protection against development of skin lesions, mortality, and latency was related to the dose of ACV-resistant virus used in the primary infection. Mortality was prevented by a dose of 10(6.0) plaque-forming units, skin lesions were prevented by a dose of 10(6.5) plaque-forming units, and latency was prevented by a dose of 10(7.0) plaque-forming units of the ACV-resistant mutant. Protection against reinfection with the PAA-resistant mutant was achieved with lower doses than protection against the parental virus. Serum antibody titers showed a 4- to 15-fold increase after reinfection. The results suggest that the ACV-resistant, latency-negative mutant has many attributes of a live attenuated herpes simplex virus vaccine.