A variety of rational approaches to attenuate growth and virulence of vesicular stomatitis virus (VSV) have been described previously. These include gene shuffling, truncation of the cytoplasmic tail of the G protein, and generation of noncytopathic M gene mutants. When separately introduced into recombinant VSV (rVSV), these mutations gave rise to viruses distinguished from their "wild-type" progenitor by diminished reproductive capacity in cell culture and/or reduced cytopathology and decreased pathogenicity in vivo. However, histopathology data from an exploratory nonhuman primate neurovirulence study indicated that some of these attenuated viruses could still cause significant levels of neurological injury. In this study, additional attenuated rVSV variants were generated by combination of the above-named three distinct classes of mutation. The resulting combination mutants were characterized by plaque size and growth kinetics in cell culture, and virulence was assessed by determination of the intracranial (IC) 50% lethal dose (LD 50 ) in mice. Compared to virus having only one type of attenuating mutation, all of the mutation combinations examined gave rise to virus with smaller plaque phenotypes, delayed growth kinetics, and 10-to 500-fold-lower peak titers in cell culture. A similar pattern of attenuation was also observed following IC inoculation of mice, where differences in LD 50 of many orders of magnitude between viruses containing one and two types of attenuating mutation were sometimes seen. The results show synergistic rather than cumulative increases in attenuation and demonstrate a new approach to the attenuation of VSV and possibly other viruses.Vesicular stomatitis virus (VSV) is a member of the Vesiculovirus genus of the family Rhabdoviridae. The negative-sense virus genome is 11,162 nucleotides long and contains five genes in the order 3Ј N-P-M-G-L 5Ј, encoding the five major viral proteins (1, 3). The bullet-shaped VSV particle (160 nm by 80 nm) contains a ribonucleoprotein core (nucleocapsid) composed of genomic RNA closely associated with N protein and a RNA polymerase composed of a complex of L and P proteins enveloped in a host cell-derived plasma membrane (4,18,19,44,53,56). Following uptake of the virus particle by susceptible cells, nucleocapsid and viral RNA polymerase are released into the cytoplasm and viral mRNA transcription ensues. A 3Ј-5Ј gradient of viral mRNA transcription leads to abundant N protein expression and successively decreasing levels of P, M, G, and L proteins (1,3,15,19,27,57). This gene expression gradient provides virus proteins in a suitable ratio for subsequent viral genome replication and assembly of mature virus particles. Virus replication in cell culture is rapid, and virus progeny are detectable 5 to 6 h postinfection.Since the initial recovery of infectious recombinant VSV (rVSV) from genomic cDNA (39, 61), effort has been directed towards the development of rVSV as a vaccine vector targeting a variety of different human pathogens, including human immu...

Recombinant vesicular stomatitis virus (rVSV) has shown great potential as a new viral vector for vaccination. However, the prototypic rVSV vector described previously was found to be insufficiently attenuated for clinical evaluation when assessed for neurovirulence in nonhuman primates. Here, we describe the attenuation, neurovirulence, and immunogenicity of rVSV vectors expressing human immunodeficiency virus type 1 Gag. These rVSV vectors were attenuated by combinations of the following manipulations: N gene translocations (N4), G gene truncations (CT1 or CT9), noncytopathic M gene mutations (Mncp), and positioning of the gag gene into the first position of the viral genome (gag1). The resulting N4CT1-gag1, N4CT9-gag1, and MncpCT1-gag1 vectors demonstrated dramatically reduced neurovirulence in mice following direct intracranial inoculation. Surprisingly, in spite of a very high level of attenuation, the N4CT1-gag1 and N4CT9-gag1 vectors generated robust Gag-specific immune responses following intramuscular immunization that were equivalent to or greater than immune responses generated by the more virulent prototypic vectors. MncpCT1-gag1 also induced Gag-specific immune responses following intramuscular immunization that were equivalent to immune responses generated by the prototypic rVSV vector. Placement of the gag gene in the first position of the VSV genome was associated with increased in vitro expression of Gag protein, in vivo expression of Gag mRNA, and enhanced immunogenicity of the vector. These findings demonstrate that through directed manipulation of the rVSV genome, vectors that have reduced neurovirulence and enhanced immunogenicity can be made.

Recombinant vesicular stomatitis virus (rVSV) vectors offer an attractive approach for the induction of robust cellular and humoral immune responses directed against human pathogen target antigens. We evaluated rVSV vectors expressing full-length glycoprotein D (gD) from herpes simplex virus type 2 (HSV-2) in mice and guinea pigs for immunogenicity and protective efficacy against genital challenge with wild-type HSV-2. Robust Th1-polarized anti-gD immune responses were demonstrated in the murine model as measured by induction of gD-specific cytotoxic T lymphocytes and increased gamma interferon expression. The isotype makeup of the serum anti-gD immunoglobulin G (IgG) response was consistent with the presence of a Th1-CD4 ؉ anti-gD response, characterized by a high IgG2a/IgG1 IgG subclass ratio. Functional anti-HSV-2 neutralizing serum antibody responses were readily demonstrated in both guinea pigs and mice that had been immunized with rVSV-gD vaccines. Furthermore, guinea pigs and mice were prophylactically protected from genital challenge with high doses of wild-type HSV-2. In addition, guinea pigs were highly protected against the establishment of latent infection as evidenced by low or absent HSV-2 genome copies in dorsal root ganglia after virus challenge. In summary, rVSV-gD vectors were successfully used to elicit potent anti-gD Th1-like cellular and humoral immune responses that were protective against HSV-2 disease in guinea pigs and mice.Herpes simplex virus type 2 (HSV-2) infections remain a serious public health problem worldwide. HSV-2 genital lesions are not only painful and disfiguring but also facilitate the transmission of human immunodeficiency virus (HIV) (7). The seroprevalence in the United States has increased by 30% between 1976 and 1994, and roughly one of every five people over the age of 12 in the United States is infected with HSV-2 (15). Individuals latently infected with HSV-2 remain infected for life and can exhibit asymptomatic viral shedding. It is therefore believed that, without intervention, such as the development of prophylactic and/or therapeutic HSV-2 vaccines, HSV-2 prevalence will continue to rise in the future.Small experimental animal vaginal challenge models in mice and guinea pigs have been used for preclinical evaluation of a number of HSV-2 vaccine strategies, including subunit vaccines (gB and/or gD with or without interleukin-12 [IL-12]), plasmid HSV DNA vaccines (gD and/or gB with or without cytokine DNA (IL-2, IL-4, IL-10, IL-12, IL-15, or IL-18), attenuated HSV-2 vaccines (TKϪ, BlacZ, dl5-29, RAV 9395, ICP10⌬PK, or AD472), and virus-vectored HSV-2 vaccines (adenovirus, varicella-zoster virus, or vaccinia virus) (1,9,12,17,21,22, 34, 39, 40,45,60,62,66). Various degrees of success have been achieved in these preclinical studies, but limited success has carried over to the clinical setting, where the experience with HSV-2 subunit vaccines has had mixed results (10). Nonetheless, an adjuvanted gD subunit approach has achieved some success in early clinical trials...

Recombinant vesicular stomatitis viruses (rVSVs) are being developed as potential HIV-1 vaccine candidates. To characterize the in vivo replication and dissemination of rVSV vectors in mice, high doses of a highly attenuated vector expressing HIV-1 Gag, rVSV IN -N4CT9-Gag1, and a prototypic reference virus, rVSV IN -HIVGag5, were delivered intramuscularly (IM), intranasally (IN), or intravenously (IV). We used quantitative, real-time RT-PCR (Q-PCR) and standard plaque assays to measure the temporal dissemination of these viruses to various tissues. Following IM inoculation, both viruses were detected primarily at the injection site as well as in draining lymph nodes; neither virus induced significant weight loss, pathologic signs, or evidence of neuroinvasion. In contrast, following IN inoculation, the prototypic virus was detected in all tissues tested and caused significant weight loss leading to death. IN administration of rVSV IN -N4CT9-Gag1 resulted in detection in numerous tissues (brain, lung, nasal turbinates, and lymph nodes) albeit in significantly reduced levels, which caused little or no weight loss nor any mortality. Following IV inoculation, both prototypic and attenuated viruses were detected by Q-PCR in all tissues tested. In contrast to the prototype, rVSV IN -N4CT9-Gag1 viral loads were significantly lower in all organs tested, and no infectious virus was detected in the brain following IV inoculation, despite the presence of viral RNA. These studies demonstrated significant differences in the biodistribution patterns of and the associated pathogenicity engendered by the prototypic and attenuated vectors in a highly susceptible host.

Highly attenuated recombinant vesicular stomatitis virus (rVSV) vectors expressing HIV-1 Gag have been shown to be immunogenic in preclinical animal models. Here we explored the potential to enhance immunogenicity through co-delivery of either replication-competent (rVSV-N3CT9) or non-propagating (rVSV-Gstem) vectors expressing GM-CSF. A construct expressing both antigen and cytokine, Gstem-Gag1-GMCSF6, was also generated. BALB/c mice were injected i.m. with Indiana rVSV vectors and boosted 8 weeks later with New Jersey glycoprotein-exchange vectors. Co-delivery of 105pfu of N3CT9-GMCSF5 and 105 pfu of N4CT9-Gag1 resulted in Gag specific IFN-g responses that were approximately two-fold higher than responses elicited by 105pfu of N4CT9-Gag1 alone. These results were confirmed by CBA analysis of supernatants from peptide-restimulated cells. Interestingly, MHC-pentamer staining revealed no significant difference in the frequency of Gag specific CD8+ cells between the two immunization groups. Similar observations were made in tests of the Gstem vector, where 106pfu of Gstem-Gag1-GMCSF6 elicited a more robust IFN-g response after boost than 106pfu of Gstem-Gag1, while the percentage of Gag specific CD8+ cells remained equivalent. In conclusion, GM-CSF exerted immunomodulatory effects in vivo, distinguished here by the enhanced functional differentiation of vaccine elicited effector cells. This work was supported by NIH contract NO1-AI-25458