During herpes simplex virus type 1 (HSV-1) neuronal latency, the only viral RNA detected is from the latency-associated transcript (LAT) gene. We have made a LAT deletion mutant of McKrae, an HSV-1 strain with a very high in vivo spontaneous reactivation rate. This mutant (dLAT2903) lacks the LAT promoter and the first 1.6 kb of the 5' end of LAT. dLAT2903 was compared with its parental virus and with a rescued virus containing a restored LAT gene (dLAT2903R). Replication of the LAT mutant in tissue culture, rabbit eyes, and rabbit trigeminal ganglia was similar to that of the rescued and parental viruses. On the basis of semiquantitative PCR analysis of the amount of HSV-1 DNA in trigeminal ganglia, the LAT mutant was unimpaired in its ability to establish latency. In contrast, spontaneous reactivation of dLAT2903 in the rabbit ocular model of HSV-1 latency and reactivation was decreased to approximately 33% of normal. This decrease was highly significant (P < 0.0001) and demonstrates that in an HSV-1 strain with a high spontaneous reactivation rate, deletion of LAT can dramatically decrease in vivo spontaneous reactivation. We also report here that deletion of LAT appeared to eliminate rather than just reduce in vivo induced reactivation.

We have constructed recombinant baculoviruses individually expressing seven of the herpes simplex virus type 1 (HSV-1) glycoproteins (gB, gC, gD, gE, gG, gH, and gI). Vaccination of mice with gB, gC, gD, gE, or gI resulted in production of high neutralizing antibody titers to HSV-1 and protection against intraperitoneal and ocular challenge with lethal doses of HSV-1. This protection was statistically significant and similar to the protection provided by vaccination with live nonvirulent HSV-1 (90 to 100%o survival). In contrast, vaccination with gH produced low neutralizing antibody titers and no protection against lethal HSV-1 challenge. Vaccination with gG produced no significant neutralizing antibody titer and no protection against ocular challenge. However, gG did provide modest, but statistically significant, protection against lethal intraperitoneal challenge (75% protection). Compared with the other glycoproteins, gG and gH were also inefficient in preventing the establishment of latency. Delayed-type hypersensitivity responses to HSV-1 at day 3 were highest in gG-, gH-, and gE-vaccinated mice, while on day 6 mice vaccinated with gC, gE, and gI had the highest delayed-type hypersensitivity responses. All seven glycoproteins produced lymphocyte proliferation responses, with the highest response being seen with gG. The same five glycoproteins (gB, gC, gD, gE, and gI) that induced the highest neutralization titers and protection against lethal challenge also induced some killer cell activity. The results reported here therefore suggest that in the mouse protection against lethal HSV-1 challenge and the establishment of latency correlate best with high preexisting neutralizing antibody titers, although there may also be a correlation with killer cell activity.

We have constructed a recombinant baculovirus expressing high levels of the herpes simplex virus type 1 (HSV-1) glycoprotein L (gL) in Sf9 cells. Sf9 cells infected with this recombinant virus synthesized three polypeptides of 26-27 kDa 28 kDa, and 31 kDa. The 28 and 31 kDa species were sensitive to tunicamycin and N-glycosidase F (PNGase F) treatment, suggesting that they were glycosylated. As shown by both indirect immunofluorescence and Western blot analysis, using polyclonal antibodies to synthetic gL peptides indicated that the baculovirus expressed gL was abundant on the surface of baculovirus gL infected Sf9 cells. A small fraction of the 31 kDa polypeptide was secreted into the extracellular medium as judged by Western blot analysis. The secreted form of gL was completely resistant to Endoglycosidase H (Endo-H), while the membrane associated form of gL was only partially resistant to Endo-H treatment, suggesting that the secreted gL represented a subpopulation of the membrane bound gL. Mice vaccinated with baculovirus expressed gL produced serum antibodies that reacted with authentic HSV-1 gL. However, these mice produced no HSV-1 neutralizing antibody (titer < 1:10) and they were not protected from lethal intraperitoneal or lethal ocular challenge with HSV-1. Thus, when used as a vaccine in the mouse model, gL, similar to our findings with HSV-1 gH, but unlike our results with the other 6 HSV-1 glycoproteins that we have expressed in this baculovirus system, did not provide any protection against HSV-1 challenge.

The herpes simplex virus type 1 (HSV-1) ICP34.5 gene is a neurovirulence gene in mice. In addition, some ICP34.5 mutants have been reported to have a reduced efficiency of induced reactivation as measured by in vitro explantation of latently infected mouse ganglia. However, since spontaneous reactivation is almost nonexistent in mice, nothing has been reported on the effect of ICP34.5 mutants on spontaneous reactivation in vivo. To examine this, we have deleted both copies of the ICP34.5 neurovirulence gene from a strain of HSV-1 (McKrae) that has a high spontaneous reactivation rate in rabbits and used this mutant to infect rabbit eyes. All rabbits infected with the ICP34.5 mutant virus (d34.5) survived, even at challenge doses greater than 4 ؋ 10 7 PFU per eye. In contrast, a 200-fold-lower challenge dose of 2 ؋ 10 5 PFU per eye was lethal for approximately 50% of rabbits infected with either the wild-type McKrae parental virus or a rescued ICP34.5 mutant in which both copies of the ICP34.5 gene were restored. In mice, the 50% lethal dose of the ICP34.5 mutant was over 10 6 PFU, compared with a value of less than 10 PFU for the rescued virus. The ICP34.5 mutant was restricted for replication in rabbit and mouse eyes and mouse trigeminal ganglia in vivo. The spontaneous reactivation rate in rabbits for the mutant was 1.4% as determined by culturing tear films for the presence of reactivated virus. This was more than 10-fold lower than the spontaneous reactivation rate determined for the rescued virus (19.6%) and was highly significant (P < 0.0001, Fisher exact test). Southern analysis confirmed that the reactivated virus retained both copies of the ICP34.5 deletion. Thus, this report demonstrates that (i) the ICP34.5 gene, known to be a neurovirulence gene in mice, is also important for virulence in rabbits and (ii) in vivo spontaneous reactivation of HSV-1 in the rabbit ocular model, although reduced, can occur in the absence of the ICP34.5 gene.

Recombinant virus RAV 9395 was constructed by deleting both copies of the gamma(1)34.5 gene, and the UL55 and UL56 open reading frames from herpes simplex virus type 2 (HSV-2) strain G. The potential use of RAV 9395 as an HSV-2 vaccine was investigated by evaluating the ability of RAV 9395 to protect guinea pigs from severe disease by HSV-2(G) challenge. RAV 9395 administered intramuscularly reduced both lesion development and severity in a dose-dependent manner in guinea pigs challenged with HSV-2(G). The frequency of reactivation of RAV 9395 from explanted dorsal root ganglia was low compared with that of HSV-2(G). Immunization with RAV 9395 at doses of 1 x 10(5) pfu and above generally precluded the establishment of latency by the challenge virus. The results presented in this report lend support for the development of genetically engineered live HSV vaccines.