R. Michael Hendry

Neurovirulence Properties of Recombinant Vesicular Stomatitis Virus Vectors in Non-Human Primates

n Abstractn n Although vesicular stomatitis virus (VSV) neurovirulence and pathogenicity in rodents have been well studied, little is known about VSV pathogenicity in non-human primates. To address this question, we measured VSV viremia, shedding, and neurovirulence in macaques. Following intranasal inoculation, macaques shed minimal recombinant VSV (rVSV) in nasal washes for 1xa0day post-inoculation; viremia was not detected. Following intranasal inoculation of macaques, wild type (wt) VSV, rVSV, and two rVSV-HIV vectors showed no evidence of spread to CNS tissues. However, macaques inoculated intrathalamically with wt VSV developed severe neurological disease. One of four macaques receiving rVSV developed clinical and histological signs similar to the wt group, while the remaining three macaques in this group and all of the macaques in the rVSV-HIV vector groups showed no clinical signs of disease and reduced severity of histopathology compared to the wt group. The implications of these findings for rVSV vaccine development are discussed.n n

Attenuation of Recombinant Vesicular Stomatitis Virus-Human Immunodeficiency Virus Type 1 Vaccine Vectors by Gene Translocations and G Gene Truncation Reduces Neurovirulence and Enhances Immunogenicity in Mice

ABSTRACT 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.

Synergistic Attenuation of Vesicular Stomatitis Virus by Combination of Specific G Gene Truncations and N Gene Translocations

ABSTRACT 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 (LD50) 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 LD50 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.

Immobilization of antibodies on nylon for use in enzyme-linked immunoassay

Antibodies were immobilized by covalent linkage on nylon balls and powder for use in solid-phase enzyme-linked immunoassays. Covalent linkage of antibody to nylon was accomplished by treatment of partially hydrolyzed nylon with glutaraldehyde or carbodiimides. Up to 0.74 microgram of immunoglobulin G per mm2 nylon could be immobilized, whereas only 0.02 microgram per mm2 could be adsorbed to polystyrene, and the binding to nylon was stable. This eliminated the problem of antibody desorption noted in conventional enzyme-linked immunosorbent assay which are based on simple adsorption to plastics, and gave more reproducible results. The method was also more sensitive, detecting levels of approximately 1 ng per ml of immunoglobulin E in clinical samples. Further, antibodies coupled to nylon balls remained bound under conditions that dissociate antibody-antigen complexes, which permitted reuse of the immobilized antibodies for immunoassays.

Recombinant vesicular stomatitis virus as an HIV-1 vaccine vector

Recombinant vesicular stomatitis virus (rVSV) is currently under evaluation as a human immunodeficiency virus (HIV)-1 vaccine vector. The most compelling reasons to develop rVSV as a vaccine vector include a very low seroprevalence in humans, the ability to infect and robustly express foreign antigens in a broad range of cells, and vigorous growth in continuous cell lines used for vaccine manufacture. Numerous preclinical studies with rVSV vectors expressing antigens from a variety of human pathogens have demonstrated the versatility, flexibility, and potential efficacy of the rVSV vaccine platform. When administered to nonhuman primates (NHPs), rVSV vectors expressing HIV-1 Gag and Env elicited robust HIV-1-specific cellular and humoral immune responses, and animals immunized with rVSV vectors expressing simian immunodeficiency virus (SIV) Gag and HIV Env were protected from AIDS after challenge with a pathogenic SIV/HIV recombinant. However, results from an exploratory neurovirulence study in NHPs indicated that these prototypic rVSV vectors might not be adequately attenuated for widespread use in human populations. To address this safety concern, a variety of different attenuation strategies, designed to produce a range of further attenuated rVSV vectors, are currently under investigation. Additional modifications of further attenuated rVSV vectors to upregulate expression of HIV-1 antigens and coexpress molecular adjuvants are also being developed in an effort to balance immunogenicity and attenuation.

Recombinant Vesicular Stomatitis Virus Vectors Expressing Herpes Simplex Virus Type 2 gD Elicit Robust CD4+ Th1 Immune Responses and Are Protective in Mouse and Guinea Pig Models of Vaginal Challenge

ABSTRACT 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.

Monoclonal capture antibody ELISA for respiratory syncytial virus: detection of individual viral antigens and determination of monoclonal antibody specificities.

An enzyme-linked immunosorbent assay (ELISA) for respiratory syncytial virus (RSV) that employs solid-phase monoclonal antibodies was developed. RSV antigens bound by these monoclonal capture antibodies were detected by addition of a polyclonal bovine antiserum, followed anti-bovine enzyme conjugate and enzyme substrate. The sensitivity and specific of the assay were determined by titrations of the solid-phase antibodies and by antigen titrations with both unpurified RSV-infected cell culture material and purified RSV nucleocapsids. The addition of a competitive binding step prior to the addition of antigen to the solid-phase antibody provides further evidence of the assays specificity. Furthermore, the competitive binding assay enables the antigen specificity of monoclonal antibodies to be determined or compared without the use of purified antigens. Monoclonal capture ELISA is a convenient, rapid, and sensitive assay that can be used to measure specific RSV antigens in unpurified preparations as well as to determine anti-RSV antibody specificity and should prove useful in examining other complex antigen-antibody systems.

Detection and identification of influenza virus antigens by nylon-coupled enzyme-linked immunoassay

A direct solid-phase enzyme-linked immunoassay for rapid detection and typing of influenza virus was developed utilizing antibodies immobilized by covalent linkage to nylon beads. Covalent linkage of antibody to nylon was accomplished by treatment of partially hydrolyzed nylon with glutaraldehyde. For comparison to conventional enzyme-linked immunosorbent assays (ELISA), IgG fractions were adsorbed to polystyrene beads. Influenza type-specific immunoglobulins coupled to nylon beads were used in an enzyme-linked immunoassay to identify influenza A/USSR/77(H1N1), and A/Texas/75 (H3N2). In titrations of viral antigen, antibody coupled to nylon beads detected 1.9 X 10(4) plaque-forming units (PFU) per assay, whereas 2.2 X 10(5) PFU were required in assays utilizing antibody adsorbed to polystyrene beads. Use of fluorogenic or radioactive substrates for alkaline phosphatase-labeled antibodies increased the sensitivity for virus detection 10-fold with this enzyme, but were only slightly more sensitive than chromogenic substrates with peroxidase-labeled antibody.