2, and the unique short segment of HSV-2. A 12-kB deletion of internal repeats from the HSV-2 short domain, mapping for a series of glycoproteins (including G, J, GD, GI, and GE), was inserted. This vaccine considerably reduces virulence in rodent models, is stable upon serial passage in mouse brains, and is safe and efficacious in aotis monkeys at concentrations up to 107 PFU administered by any route, including intracerebrally. It was safe in humans at up to 104.5 PFU (higher doses were not studied). This vaccine required two doses, implying that it was overly attenuated for administration to humans.
HSV can also be attenuated through deletions and stop codes. The gamma 134.5 gene resides in the inverted repeats in two copies on the unique long segment of the HSV-2 genome; when 134.5 is deleted, it leaves a virus with an LD-50 upon inoculation directly into the central nervous system of a mouse of 106 PFU, compared with 102 PFU for the restored wild-type virus. A stop-code on the carboxy terminus of the genome can similarly attenuate the virus.
Using the latter technique with HSV-2, a recombinant vaccine was generated that also deleted the structural components of UL-55 and UL-56. This vaccine has significantly reduced neurovirulence in mice—LD-50 is 5.6 X 105 PFU, compared with less than 50 PFU for the wild type in that circumstance—and is safe in the aotis monkey at up to 106 PFU. In terms of efficacy, the vaccine appears to protect guinea pigs from disease at dosages in the range of 104 to 105 PFU. Immunized aotis monkeys survive challenges with wild-type virus at up to 105 PFU when given intravaginally. These results suggests that this is, at least potentially, a genetically engineered vaccine that will provide a broader immune response than is encountered with subunit vaccines.
Both the method of attenuation and the route of administration seem to influence the efficacy of these candidate vaccines. When mice were inoculated intranasally and then challenged intranasally with either wild virus or HSV-2, there was a reduction of mortality of 20 percent for those inoculated with 134.5-deletion mutants, and 13 percent when a stop code is place at the carboxy terminus. When mice are immunized intranasally and challenged intravaginally, however, the effect is not as great. Researchers are currently looking for IgA and IgG2a in the vaginal secretions of these mice to learn more about these immune responses.
Animal Models. Two new animal models have emerged from this work. The first is a test of the virulence of the virus; the aotis monkey is exquisitely sensitive for the evaluation of genetically engineered viruses, which are inoculated directly into the eye. The second is a rodent model to establish the genetic stability of attenuated vaccines. It involves inoculating virus into the mouse brain, harvesting tissue, raising the virus again, and reinoculating virus into mouse brains on subsequent occasions; nine passages will usually select unstable variants or a reversion to wild type. Inoculation into mouse brain can also be used to evaluate the virulence of attenuated virus.
In response to questions from the audience, Dr. Whitley added the following: