Antiviral effect of lymphokine-activated killer cells: chemotaxis and homing to sites of virus infection

Lymphokine-activated killer (LAK) cells generated from C57BL/6 mouse spleen cells cultured with interleukin-2 are effective prophylactically against virus infection when inoculated at the site of virus injection. To predict the therapeutic efficacy of LAK cells, we determined whether LAK cells would home to sites of virus infection. In vitro, LAK cells responded chemotactically to cell-free peritoneal exudate fluids collected from virus-infected mice and to preparations of purified beta interferon. In vivo, radiolabeled LAK cells injected intravenously accumulated in the peritoneal cavities of intraperitoneally infected mice in amounts three to eight times greater than in uninfected mice. This ability to respond to chemotactic agents and migrate into sites of virus infection may make LAK cells useful as antiviral therapeutic agents

Herpes Simplex Virus Type 2 UL24 Gene Is a Virulence Determinant in Murine and Guinea Pig Disease Models

A herpes simplex virus type 2 (HSV-2) UL24 β-glucuronidase (UL24-βgluc) insertion mutant was derived from HSV-2 strain 186 via standard marker transfer techniques. Cell monolayers infected with UL24-βgluc yielded cytopathic effect with syncytium formation. UL24-βgluc replicated to wild-type viral titers in three different cell lines. UL24-βgluc was not virulent after intravaginal inoculation of BALB/c mice in that all inoculated animals survived doses up to 400 times the 50% lethal dose (LD(50)) of the parental virus. Furthermore, few UL24-βgluc-inoculated mice developed any vaginal lesions. Intravaginal inoculation of guinea pigs with UL24-βgluc at a dose equivalent to the LD(50) of parental virus (≈5 × 10(3) PFU) was not lethal (10/10 animals survived). Although genital lesions developed in some UL24-βgluc-inoculated guinea pigs, both the overall number of lesions and the severity of disease were far less than that observed for animals infected with parental strain 186

Blastogenesis of large granular lymphocytes in nonlymphoid organs

High numbers of large granular lymphocytes (LGL) accumulate in the livers and peritoneal cavities of mice during the course of viral infection. Accumulation of natural killer (NK) cells at day 3 postinfection (p.i.) was shown to be radiation-sensitive, implying that proliferation was required for this response. Accumulation occurred in splenectomized mice, indicating that the spleen, known to be an organ for mature NK cell proliferation, was not the major source for liver and peritoneal NK/LGL. Significant percentages (greater than 25%) of the LGL found in the liver and peritoneal cavity following viral infection or interferon induction with poly-inosinic:poly-cytidylic acid were defined morphologically as blasts (large cells with prominent nucleoli and intensely basophilic cytoplasms containing azurophilic granules). Most blast LGL at day 3 p.i. were sensitive to administration of anti-asialo GM1 serum in vivo, were Lyt-2-, and were enriched in populations that lysed NK cell-sensitive targets in vitro, indicating that these were NK/LGL. At day 3 p.i., leukocytes from the liver and peritoneal cavity incorporated 3H-thymidine and bound to and killed NK cell-sensitive targets in single-cell cytotoxicity assays. These data suggest that NK/LGL undergo at least one round of division in the liver and peritoneal cavity during viral infection. In contrast, blast LGL at day 7 p.i. were resistant to in vivo treatments with anti-asialo GM1 serum, were Lyt-2+, and were enriched in populations of cells that killed virus-infected histocompatible targets, indicating that they were cytotoxic T lymphocytes (CTL). These results suggest that both NK/LGL and CTL/LGL are capable of blastogenesis and presumed proliferation at sites of virus infection, providing a means for the in situ augmentation of a host\u27s cell-mediated antiviral defenses

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▿

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 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

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