Neutralizing Antibody Kinetics and Immune Protection Against Herpes Simplex Virus 1 Genital Disease in Vaccinated Women.

BACKGROUND: Previously, our group conducted the Herpevac Trial for Women, a randomized efficacy field trial of type 2 glycoprotein D (gD2) herpes simplex virus (HSV) vaccine adjuvanted with ASO4 in 8323 women. Study participants were selected to be seronegative for HSV-1 and HSV-2. We found that the vaccine was 82% protective against culture-positive HSV-1 genital disease but offered no significant protection against HSV-2 genital disease. Efficacy against HSV-1 was associated with higher levels of antibody to gD2 at enzyme-linked immunosorbent assay (ELISA). METHODS: To better understand the results of the efficacy study, we measured postvaccination concentrations of neutralizing antibody (nAb) to either HSV-1 and HSV-2 from HSV-infected study participants and matched uninfected controls. Statistical modeling was used to determine whether these responses were correlated with protection against HSV. RESULTS: nAbs to either HSV-1 or HSV-2 were correlated with ELISA binding antibodies to gD2. HSV-1 or HSV-2 nAb findings support the observation of protection by higher levels of antibody against HSV-1 infection, but the lack of protection against HSV-2 remains unexplained. CONCLUSIONS: The protection against HSV-1 infection observed in the Herpevac Trial for Women was associated with nAbs directed against the virus, although the power to assess this was lower in the nAb study compared with the ELISA results owing to smaller sample size. CLINICAL TRIALS REGISTRATION: NCT00057330.

Synthesis of Polyoxygenated Tropolones and their Antiviral Activity against Hepatitis B Virus and Herpes Simplex Virus-1.

Polyoxygenated tropolones possess a broad range of biological activity, and as a result are promising lead structures or fragments for drug development. However, structure-function studies and subsequent optimization have been challenging, in part due to the limited number of readily available tropolones and the obstacles to their synthesis. Oxidopyrylium [5+2] cycloaddition can effectively generate a diverse array of seven-membered ring carbocycles, and as a result can provide a highly general strategy for tropolone synthesis. Here, we describe the use of 3-hydroxy-4-pyrone-based oxidopyrylium cycloaddition chemistry in the synthesis of functionalized 3,7-dimethoxytropolones, 3,7-dihydroxytropolones, and isomeric 3-hydroxy-7-methoxytropolones through complementary benzyl alcohol-incorporating procedures. The antiviral activity of these molecules against herpes simplex virus-1 and hepatitis B virus is also described, highlighting the value of this approach and providing new structure-function insights relevant to their antiviral activity.

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.

Herpes Simplex Virus 1 Mutant with Point Mutations in UL39 Is Impaired for Acute Viral Replication in Mice, Establishment of Latency, and Explant-Induced Reactivation.

In the process of generating herpes simplex virus 1 (HSV-1) mutations in the viral regulatory gene encoding infected cell protein 0 (ICP0), we isolated a viral mutant, termed KOS-NA, that was severely impaired for acute replication in the eyes and trigeminal ganglia (TG) of mice, defective in establishing a latent infection, and reactivated poorly from explanted TG. To identify the secondary mutation(s) responsible for the impaired phenotypes of this mutant, we sequenced the KOS-NA genome and noted that it contained two nonsynonymous mutations in UL39, which encodes the large subunit of ribonucleotide reductase, ICP6. These mutations resulted in lysine-to-proline (residue 393) and arginine-to-histidine (residue 950) substitutions in ICP6. To determine whether alteration of these amino acids was responsible for the KOS-NA phenotypes in vivo, we recombined the wild-type UL39 gene into the KOS-NA genome and rescued its acute replication phenotypes in mice. To further establish the role of UL39 in KOS-NA's decreased pathogenicity, the UL39 mutations were recombined into HSV-1 (generating UL39mut), and this mutant virus showed reduced ocular and TG replication in mice comparable to that of KOS-NA. Interestingly, ICP6 protein levels were reduced in KOS-NA-infected cells relative to the wild-type protein. Moreover, we observed that KOS-NA does not counteract caspase 8-induced apoptosis, unlike wild-type strain KOS. Based on alignment studies with other HSV-1 ICP6 homologs, our data suggest that amino acid 950 of ICP6 likely plays an important role in ICP6 accumulation and inhibition of apoptosis, consequently impairing HSV-1 pathogenesis in a mouse model of HSV-1 infection.IMPORTANCE HSV-1 is a major human pathogen that infects ∼80% of the human population and can be life threatening to infected neonates or immunocompromised individuals. Effective therapies for treatment of recurrent HSV-1 infections are limited, which emphasizes a critical need to understand in greater detail the events that modulate HSV-1 replication and pathogenesis. In the current study, we identified a neuroattenuated HSV-1 mutant (i.e., KOS-NA) that contains novel mutations in the UL39 gene, which codes for the large subunit of ribonucleotide reductase (also known as ICP6). This mutant form of ICP6 was responsible for the attenuation of KOS-NA in vivo and resulted in diminished ICP6 protein levels and antiapoptotic effect. Thus, we have determined that subtle alteration of the UL39 gene regulates expression and functions of ICP6 and severely impacts HSV-1 pathogenesis, potentially making KOS-NA a promising vaccine candidate against HSV-1.

Molecular Evolution of Herpes Simplex Virus 2 Complete Genomes: Comparison between Primary and Recurrent Infections.

Herpes simplex virus 1 (HSV-1) and HSV-2 are large, double-stranded DNA viruses that cause lifelong persistent infections characterized by periods of quiescence and recurrent disease. How HSV evolves within an infected individual experiencing multiple episodes of recurrent disease over time is not known. We determined the genome sequences of viruses isolated from two subjects in the Herpevac Trial for Women who experienced primary HSV-2 genital disease and compared them with sequences of viruses isolated from the subsequent fifth or sixth episode of recurrent disease in the same individuals. Each of the HSV-2 genome sequences was initially obtained using next-generation sequencing and completed with Sanger sequencing. Polymorphisms over the entire genomes were mapped, and amino acid variants resulting from nonsynonymous changes were analyzed based on the secondary and tertiary structures of a previously crystallized protein. A phylogenetic reconstruction was used to assess relationships among the four HSV-2 samples, other North American sequences, and reference sequences. Little genetic drift was detected in viruses shed by the same subjects following repeated reactivation events, suggesting strong selective pressure on the viral genome to maintain sequence fidelity during reactivations from its latent state within an individual host. Our results also demonstrate that some primary HSV-2 isolates from North America more closely resemble the HG52 laboratory strain from Scotland than the low-passage-number clinical isolate SD90e from South Africa or laboratory strain 333. Thus, one of the sequences reported here would be a logical choice as a reference strain for inclusion in future studies of North American HSV-2 isolates.IMPORTANCE The extent to which the HSV-2 genome evolves during multiple episodes of reactivation from its latent state within an infected individual is not known. We used next-generation sequencing techniques to determine whole-genome sequences of four viral samples from two subjects in the Herpevac Trial. The sequence of each subject's well-documented primary isolate was compared with the sequence of the isolate from their fifth or sixth episode of recurrent disease. Only 19 genetic polymorphisms unique to the primary or recurrent isolate were identified, 10 in subject A and 9 in subject B. These observations indicate remarkable genetic conservation between primary and recurrent episodes of HSV-2 infection and imply that strong selection pressures exist to maintain the fidelity of the viral genome during repeated reactivations from its latent state. The genome conservation observed also has implications for the potential success of a therapeutic vaccine.

Synthetic α-Hydroxytropolones Inhibit Replication of Wild-Type and Acyclovir-Resistant Herpes Simplex Viruses.

Herpes simplex virus 1 (HSV-1) and HSV-2 remain major human pathogens despite the development of anti-HSV therapeutics as some of the first antiviral drugs. Current therapies are incompletely effective and frequently drive the evolution of drug-resistant mutants. We recently determined that certain natural troponoid compounds such as ß-thujaplicinol readily suppress HSV-1 and HSV-2 replication. Here, we screened 26 synthetic α-hydroxytropolones with the goals of determining a preliminary structure-activity relationship for the α-hydroxytropolone pharmacophore and providing a starting point for future optimization studies. Twenty-five compounds inhibited HSV-1 and HSV-2 replication at 50 µM, and 10 compounds inhibited HSV-1 and HSV-2 at 5 µM, with similar inhibition patterns and potencies against both viruses being observed. The two most powerful inhibitors shared a common biphenyl side chain, were capable of inhibiting HSV-1 and HSV-2 with a 50% effective concentration (EC50) of 81 to 210 nM, and also strongly inhibited acyclovir-resistant mutants. Moderate to low cytotoxicity was observed for all compounds (50% cytotoxic concentration [CC50] of 50 to >100 µM). Therapeutic indexes ranged from >170 to >1,200. These data indicate that troponoids and specifically α-hydroxytropolones are a promising lead scaffold for development as anti-HSV drugs provided that toxicity can be further minimized. Troponoid drugs are envisioned to be employed alone or in combination with existing nucleos(t)ide analogs to suppress HSV replication far enough to prevent viral shedding and to limit the development of or treat nucleos(t)ide analog-resistant mutants.

Up to four distinct polypeptides are produced from the γ34.5 open reading frame of herpes simplex virus 2.

UNLABELLED: The herpes simplex virus 1 (HSV-1) ICP34.5 protein strongly influences neurovirulence and regulates several cellular antiviral responses. Despite the clinical importance of HSV-2, relatively little is known about its ICP34.5 ortholog. We found that HSV-2 produces up to four distinct forms of ICP34.5 in infected cells: a full-length protein, one shorter form sharing the N terminus, and two shorter forms sharing the C terminus. These forms appeared with similar kinetics and accumulated in cells over much of the replication cycle. We confirmed that the N-terminal form is translated from the primary unspliced transcript to a stop codon within the intron unique to HSV-2 γ34.5. We found that the N-terminal form was produced in a variety of cell types and by 9 of 10 clinical isolates. ICP27 influenced but was not required for expression of the N-terminal form. Western blotting and reverse transcription-PCR indicated the C-terminal forms did not contain the N terminus and were not products of alternative splicing or internal transcript initiation. Expression plasmids encoding methionine at amino acids 56 and 70 generated products that comigrated in SDS-PAGE with the C1 and C2 forms, respectively, and mutation of these sites abolished C1 and C2. Using a recombinant HSV-2 encoding hemagglutinin (HA)-tagged ICP34.5, we demonstrated that the C-terminal forms were also produced during infection of many human and mouse cell types but were not detectable in mouse primary neurons. The protein diversity generated from the HSV-2 γ34.5 open reading frame implies additional layers of cellular regulation through potential independent activities associated with the various forms of ICP34.5. IMPORTANCE: The herpes simplex virus 1 (HSV-1) protein ICP34.5, encoded by the γ34.5 gene, interferes with several host defense mechanisms by binding cellular proteins that would otherwise stimulate the cell's autophagic, translational-arrest, and type I interferon responses to virus infection. ICP34.5 also plays a crucial role in determining the severity of nervous system infections with HSV-1 and HSV-2. The HSV-2 γ34.5 gene contains an intron not present in HSV-1 γ34.5. A shorter N-terminal form of HSV-2 ICP34.5 can be translated from the unspliced γ34.5 mRNA. Here, we show that two additional forms consisting of the C-terminal portion of ICP34.5 are generated in infected cells. Production of these N- and C-terminal forms is highly conserved among HSV-2 strains, including many clinical isolates, and they are broadly expressed in several cell types, but not mouse primary neurons. Multiple ICP34.5 polypeptides add additional complexity to potential functional interactions influencing HSV-2 neurovirulence.

Inhibitors of nucleotidyltransferase superfamily enzymes suppress herpes simplex virus replication.

Herpesviruses are large double-stranded DNA viruses that cause serious human diseases. Herpesvirus DNA replication depends on multiple processes typically catalyzed by nucleotidyltransferase superfamily (NTS) enzymes. Therefore, we investigated whether inhibitors of NTS enzymes would suppress replication of herpes simplex virus 1 (HSV-1) and HSV-2. Eight of 42 NTS inhibitors suppressed HSV-1 and/or HSV-2 replication by >10-fold at 5 µM, with suppression at 50 µM reaching ∼1 million-fold. Five compounds in two chemical families inhibited HSV replication in Vero and human foreskin fibroblast cells as well as the approved drug acyclovir did. The compounds had 50% effective concentration values as low as 0.22 µM with negligible cytotoxicity in the assays employed. The inhibitors suppressed accumulation of viral genomes and infectious particles and blocked events in the viral replication cycle before and during viral DNA replication. Acyclovir-resistant mutants of HSV-1 and HSV-2 remained highly sensitive to the NTS inhibitors. Five of six NTS inhibitors of the HSVs also blocked replication of another herpesvirus pathogen, human cytomegalovirus. Therefore, NTS enzyme inhibitors are promising candidates for new herpesvirus treatments that may have broad efficacy against members of the herpesvirus family.

A proautophagic antiviral role for the cellular prion protein identified by infection with a herpes simplex virus 1 ICP34.5 mutant.

The cellular prion protein (PrP) often plays a cytoprotective role by regulating autophagy in response to cell stress. The stress of infection with intracellular pathogens can stimulate autophagy, and autophagic degradation of pathogens can reduce their replication and thus help protect the infected cells. PrP also restricts replication of several viruses, but whether this activity is related to an effect on autophagy is not known. Herpes simplex virus 1 (HSV-1) effectively counteracts autophagy through binding of its ICP34.5 protein to the cellular proautophagy protein beclin-1. Autophagy can reduce replication of an HSV-1 mutant, Δ68H, which is incapable of binding beclin-1. We found that deletion of PrP in mice complements the attenuation of Δ68H, restoring its capacity to replicate in the central nervous system (CNS) to wild-type virus levels after intracranial or corneal infection. Cultured primary astrocytes but not neurons derived from PrP(-/-) mice also complemented the attenuation of Δ68H, enabling Δ68H to replicate at levels equivalent to wild-type virus. Ultrastructural analysis showed that normal astrocytes exhibited an increase in the number of autophagosomes after infection with Δ68H compared with wild-type virus, but PrP(-/-) astrocytes failed to induce autophagy in response to Δ68H infection. Redistribution of EGFP-LC3 into punctae occurred more frequently in normal astrocytes infected with Δ68H than with wild-type virus, but not in PrP(-/-) astrocytes, corroborating the ultrastructural analysis results. Our results demonstrate that PrP is critical for inducing autophagy in astrocytes in response to HSV-1 infection and suggest that PrP positively regulates autophagy in the mouse CNS.

Genome sequence of herpes simplex virus 1 strain KOS.

Herpes simplex virus type 1 (HSV-1) strain KOS has been extensively used in many studies to examine HSV-1 replication, gene expression, and pathogenesis. Notably, strain KOS is known to be less pathogenic than the first sequenced genome of HSV-1, strain 17. To understand the genotypic differences between KOS and other phenotypically distinct strains of HSV-1, we sequenced the viral genome of strain KOS. When comparing strain KOS to strain 17, there are at least 1,024 small nucleotide polymorphisms (SNPs) and 172 insertions/deletions (indels). The polymorphisms observed in the KOS genome will likely provide insights into the genes, their protein products, and the cis elements that regulate the biology of this HSV-1 strain.

Genome sequence of herpes simplex virus 1 strain McKrae.

The herpes simplex virus 1 (HSV-1) strain McKrae is highly virulent compared to other wild-type strains of HSV-1. To help us better understand the genetic determinants that lead to differences in the pathogenicity of McKrae and other HSV-1 strains, we sequenced its genome. Comparing the sequence of McKrae's genome to that of strain 17 revealed that the genomes differ by at least 752 single nucleotide polymorphisms (SNPs) and 86 insertion/deletion events (indels). Although the majority of these polymorphisms reside in noncoding regions, 241 SNPs and 10 indels alter the protein-coding sequences of 58 open reading frames. Some of these variations are expected to contribute to the pathogenic phenotype of McKrae.

Deletion of the herpes simplex virus 1 UL49 gene results in mRNA and protein translation defects that are complemented by secondary mutations in UL41.

Herpes simplex virus 1 (HSV-1) virions, like those of all herpesviruses, contain a protein layer termed the tegument localized between the capsid and the envelope. VP22, encoded by the U(L)49 gene, is one of the most abundant tegument proteins in HSV-1 virions. Studies with a U(L)49-null mutant showed that the absence of VP22 resulted in decreased protein synthesis at late times in infection. VP22 is known to form a tripartite complex with VP16 and vhs through direct interactions with VP16. Given that U(L)49-null mutants have been shown to acquire spontaneous secondary mutations in the U(L)41 gene, which encodes vhs, we hypothesized that VP22 and vhs may play antagonistic roles during HSV-1 infections. In the present study, we show that the protein synthesis defect observed in U(L)49-null virus infections was rescued by a secondary, compensatory frameshift mutation in U(L)41. A double mutant bearing a deletion of U(L)49 and a point mutation in vhs previously shown to specifically abrogate vhs's RNase activity also resulted in a rescue of protein synthesis. To determine whether the U(L)49(-) protein synthesis defect, and the rescue by secondary mutations in vhs, occurred at the mRNA and/or translational levels, quantitative reverse transcriptase PCR (qRT-PCR) and polysome analyses were performed. We found that the absence of VP22 caused a small decrease in mRNA levels as well as a defect in polysome assembly that was independent of mRNA abundance. Both defects were complemented by the secondary mutations in vhs, indicating functional interplay between VP22 and vhs in both accumulation and translation of viral mRNAs.

ICP8-vhs- HSV-2 Vaccine Expressing B7 Costimulation Molecules Optimizes Safety and Efficacy against HSV-2 Infection in Mice.

Herpes simplex virus 2 (HSV-2) causes most sexually transmitted genital ulcerative disease. No effective prophylactic vaccine is currently available. Replication-defective (ICP8-) HSV stimulates immune responses in animals without producing progeny virus, making it potentially useful as a safe form of a live vaccine against HSV. We previously demonstrated that mice generate a stronger response to ICP8- virus encoding B7-2 costimulation molecules than to the parental replication-defective virus. We have also demonstrated enhanced immunogenicity of an ICP8-, virion host shutoff (vhs)- virus which can no longer destabilize viral and host mRNAs. Here, we constructed a triple mutant, ICP8-vhs-B7-2+ strain, and compared it to both double mutant viruses. Immunization of mice with a single dose of ICP8-B7-2+ or ICP8-vhs-B7-2+ virus decreased challenge virus replication in the vaginal mucosa, genital disease, and mortality more effectively than immunization with the ICP8-vhs- virus. Immunization with ICP8-B7-2+ or ICP8-vhs-B7-2+ virus also effectively suppressed subsequent HSV-2 infection of the nervous system compared to immunization with the ICP8-vhs- virus. ICP8-B7-2+ and ICP8-vhs-B7-2+ strains induced more IFN gamma-producing CD8 T cells and memory CD8 T cells than did ICP8-vhs- virus, potentially explaining the enhanced protective effects. Thus, B7 costimulation molecules expressed from a replication-defective vaccine can enhance vaccine efficacy, even in an immunocompetent host.

Antiviral activity of amide-appended α-hydroxytropolones against herpes simplex virus-1 and -2.

α-Hydroxytropolones (αHTs) have potent antiviral activity against herpes simplex virus-1 and -2 (HSV-1 and HSV-2) in cell culture, including against acyclovir-resistant mutants, and as a result have the potential to be developed as antiviral drugs targeting these viruses. We recently described a convenient final-step amidation strategy to their synthesis, and this was used to generate 57 amide-substituted αHTs that were tested against hepatitis B virus. The following manuscript describes the evaluation of this library against HSV-1, as well as a subset against HSV-2. The structure-function analysis obtained from these studies demonstrates the importance of lipophilicity and rigidity to αHT-based anti-HSV potency, consistent with our prior work on smaller libraries. We used this information to synthesize and test a targeted library of 4 additional amide-appended αHTs. The most potent of this new series had a 50% effective concentration (EC50) for viral inhibition of 72 nM, on par with the most potent αHT antivirals we have found to date. Given the ease of synthesis of amide-appended αHTs, this new class of antiviral compounds and the chemistry to make them should be highly valuable in future anti-HSV drug development.

Efficacy of an HSV-1 Neuro-Attenuated Vaccine in Mice Is Reduced by Preventing Viral DNA Replication.

We previously isolated an HSV-1 mutant, KOS-NA, that contains two non-synonymous mutations in UL39. One of the mutations, resulting in an R950H amino acid substitution in ICP6, renders KOS-NA severely neuro-attenuated and significantly reduces HSV-1 latency. Vaccination of mice with KOS-NA prior to corneal challenge provides significant protection against HSV-1-mediated eye diseases even at a very low immunizing dose, indicating its utility as a vaccine scaffold. Because KOS-NA contains a neuro-attenuating mutation in a single gene, we sought to improve its safety by deleting a portion of the UL29 gene whose protein product, ICP8, is essential for viral DNA replication. Whereas KOS-NA reduced replication of HSV-1 challenge virus in the corneal epithelium and protected mice against blepharitis and keratitis induced by the challenge virus, KOS-NA/8- and an ICP8- virus were significantly less efficacious except at higher doses. Our results suggest that the capacity to replicate, even at significantly reduced levels compared with wild-type HSV-1, may be an important feature of an effective vaccine. Means to improve safety of attenuated viruses as vaccines without compromising efficacy should be sought.

Synthetic derivatives of the antifungal drug ciclopirox are active against herpes simplex virus 2.

We previously showed that the anti-fungal drug ciclopirox olamine effectively inhibits replication of herpes simplex virus (HSV)-1 and HSV-2. Given the rise of HSV strains that are resistant to nucleos(t)ide analog treatment, as well as the incomplete efficacy of nucleos(t)ide analogs, new inhibitory compounds must be explored for potential use in the treatment of HSV infection. In the present study, we analyzed 44 compounds derived from the core structure of ciclopirox olamine for inhibitory activity against HSV. Thirteen of these derivative compounds inhibited HSV-2 replication by > 1000- to ∼100,000-fold at 1 µM and displayed EC50 values lower than that of acyclovir, as well as low cytotoxicity, indicating their strong therapeutic potential. Through structural comparison, we also provide evidence for the importance of various structural motifs to the efficacy of ciclopirox and its derivatives, namely hydrophobic groups at R4 and R6 of the ciclopirox core structure. Like ciclopirox, representative analogs exhibit some oral bioavailability but are rapidly cleared in vivo. Together, these results will guide further development of N-hydroxypyridones as HSV therapeutics.

Increased eIF2alpha phosphorylation attenuates replication of herpes simplex virus 2 vhs mutants in mouse embryonic fibroblasts and correlates with reduced accumulation of the PKR antagonist ICP34.5.

Herpes simplex virus 2 (HSV-2) strains containing mutations in the virion host shutoff (vhs) protein are attenuated for replication compared with wild-type virus in mouse embryonic fibroblasts (MEFs). However, HSV-2 vhs mutants replicate to near wild-type levels in the absence of the RNA-activated protein kinase (PKR). PKR is one of several kinases that phosphorylates the eukaryotic initiation factor 2alpha (eIF2alpha) to inhibit translation initiation, and we previously found that more of the phosphorylated form of eIF2alpha accumulates in MEFs infected with HSV-2 vhs mutants than with wild-type virus. Here, we show that this increase in phosphorylated eIF2alpha is primarily PKR dependent. Using MEFs expressing nonphosphorylatable eIF2alpha, we demonstrate that phosphorylated eIF2alpha is the primary cause of attenuated replication of HSV-2 vhs mutants and that attenuation correlates with decreased accumulation of viral proteins. Normally, HSV antagonizes eIF2alpha phosphorylation through the action of ICP34.5, which redirects protein phosphatase 1alpha (PP1alpha) to dephosphorylate eIF2alpha during infection. We show that ICP34.5 does not accumulate efficiently in MEFs infected with HSV-2 vhs mutant viruses, suggesting that the accumulation of phosphorylated eIF2alpha and the attenuated phenotype of HSV-2 vhs mutants in MEFs result from a deficiency in ICP34.5.

Virus-encoded b7-2 costimulation molecules enhance the protective capacity of a replication-defective herpes simplex virus type 2 vaccine in immunocompetent mice.

Herpes simplex virus 2 (HSV-2) and, to a lesser extent, HSV-1 cause the majority of sexually transmitted genital ulcerative disease. No effective prophylactic vaccine is currently available. Replication-defective HSV stimulates immune responses in animals but produces no progeny virus, making it potentially useful as a safe form of live vaccine against HSV. Because it does not replicate and spread in the host, however, replication-defective virus may have relatively limited capacity to solicit professional antigen presentation. We previously demonstrated that in mice devoid of B7-1 and B7-2 costimulation molecules, replication-defective HSV-2 encoding B7-1 or B7-2 induces stronger immune responses and protection against HSV-2 challenge than immunization with replication-defective virus alone. Here, we vaccinated wild-type mice fully competent to express endogenous B7 costimulation molecules with replication-defective HSV-2 or replication-defective virus encoding B7-2 and compared their capacities to protect against vaginal HSV-2 infection and disease. Replication-defective virus encoding B7-2 induced more IFN-gamma-producing CD4 T cells than did replication-defective virus alone. Immunization with B7-2-expressing virus decreased challenge virus replication in the vaginal mucosa, genital and neurological disease, and mortality more effectively than did immunization with the parental replication-defective virus. Prior immunization with B7-expressing, replication-defective virus also effectively suppressed infection of the nervous system compared to immunization with the parental virus. Thus, B7 costimulation molecules expressed at the site of HSV infection can enhance vaccine efficacy even in a fully immunocompetent host.

Antiviral activity of Α-hydroxytropolones on caprine alphaherpesvirus 1 in vitro.

The emergence of human alphaherpesvirus strains (i.e. HHV-1 and -2) resistant to commonly used antiviral drugs has prompted the research for alternative, biologically active anti-herpetic agents. Natural-product and synthetic α-hydroxytropolones (αHTs) have been identified as lead therapeutic agents for a number of infections, including HHV-1 and -2, and several veterinary herpesviruses, i.e. bovine alphaherpesvirus 1 (BoHV-1), equine alphaherpesvirus 1 (EHV-1) and feline alphaherpesvirus 1 (FHV-1). In the present study we evaluated the activity in vitro of two natural and two synthetic α-hydroxytropolones (αHTs) against Caprine alphaherpesvirus 1 (CpHV-1) which is regarded as a useful homologous animal model for the study of HSV-2 infection, chiefly for the assessment of antiviral drugs in in vivo studies. AlphaHTs were able to decrease significantly CpHV-1 viral titres up to 4.25 log10 TCID50/50 µl and suppressed extensively CpHV-1 nucleic acids up to 8.71 log10 viral DNA copy number/10 µl. This study demonstrated the efficacy of αHTs against CpHV-1 in vitro, adding to their activity observed against the human and animal alphaherpesviruses in vitro. The activity of αHTs against CpHV-1 appeared similar but not identical to the patterns of activity observed against other alphaherpesviruses, suggesting virus-related variability in terms of response to specific αHT molecules. These findings open several perspectives in terms of future studies using the CpHV-1 homologous animal model, for the development of therapeutic tools against herpesviruses.