Although we have previously shown that HSV-1 gB498-505-specific CTL can be induced by a variety of methods (5
), we had yet to determine the protective nature of CTL directed toward this single, immunodominant epitope against HSV pathogenesis. The data presented here demonstrate that i.n. immunization of C57BL/6 mice with a recombinant vaccinia or influenza virus expressing the single CTL recognition epitope HSV gB498-505 induced both primary and memory CTL responses directed against this epitope. Mice immunized with the rVV showed a reduction in HSV colonization of the CNS and exhibited protection from a lethal challenge of HSV-2. These results indicate that the induction of a single epitope-specific CTL response is sufficient to confer a significant level of protection from lethal HSV-2 infection.
The relative contribution of CD8+
T cells and other components of the adaptive immune response to the control of HSV infection has been controversial. Both T-cell depletion and adoptive transfer studies in mice have supported a role for CD8+
T cells in the control of HSV infections (6
). However, using a murine zosteriform model of HSV infection, the CD4+
T-cell subset has been demonstrated to be primarily responsible for control of HSV infection (37
). The apparent discrepancies in the relative importance of each of these subsets may be a function of the particular model of HSV infection used in each of these studies. HSV infection in humans is known to elicit the generation of HSV-specific CD8+
T cells (56
), although CD4+
T cells have also been readily detected in individuals seropositive for HSV (74
). Recently, Posavad et al. demonstrated a correlation between low HSV-specific precursor CD8+
CTL frequencies and severe HSV recurrences in HIV-infected individuals (58
). These findings further indicate the importance of CD8+
T cells in the control of HSV infection of humans.
This study used multiple recombinant viral vectors for the induction of HSV-specific CTL with a single specificity. An rVV (rVV-ES-gB498-505) encoding the gB498-505 CTL recognition epitope fused to the adenovirus E3/19K ES (2
) was generated. The expression of a gB498-505 CTL epitope peptide fused to this sequence allowed the peptide to be inserted directly into the lumen of the endoplasmic reticulum, thus bypassing the requirement for TAP-dependent peptide transport (Fig. C). Our laboratory has previously demonstrated that the fusion of an SV40 T-antigen immunorecessive epitope sequence to the ES can confer increased immunogenicity (20
). An rVV expressing gB498-505 without the fused ES and the recombinant influenza virus also served as effective immunogens.
Immunization with rVV-ES-gB498-505 provided protection from i.n. challenge with a lethal dose of HSV-2 (Fig. ). Although only 29% of immunized mice survived the challenge with 5 × 105
PFU, there was a clear delay in the death of those mice that ultimately succumbed to the lethal HSV-2 infection. However, 69% of rVV-ES-gB498-505-immunized mice survived HSV-2 challenge with a fivefold-lower dose (105
PFU). The lower level of protection seen in mice challenged with the higher dose suggests that there is a critical threshold of infection which the gB498-505-specific CTL can adequately control and that higher levels of viral challenge may overwhelm the capacity of these HSV-specific CTL to control the infection. It is important to note that the observed protection was afforded by a single immunization with rVV-ES-gB498-505. Therefore, it is possible that an increased level of protection is provided by an additional immunization with this or other vectors expressing the gB498-505 epitope. Protection from lethal infection was also observed upon immunization with two other recombinant viral vectors, rVV-gB498-505 (Fig. C) and WSN/NA/gB (Fig. ), confirming that a CTL response directed against the single gB498-505 CTL epitope can confer resistance. The observed protection afforded by immunization with rVV-gB498-505 (Fig. C) demonstrated that the gB498-505-specific protection was not dependent on or enhanced by the presence of an ES fused to the gB498-505 epitope. The observed protection was also not dependent on the use of an i.n. route of immunization, since B6 mice immunized intraperitoneally with rVV-ES-gB498-505 were protected from lethal i.n. HSV-2 challenge (data not shown). Overall, these findings demonstrate that the presence of HSV-specific CTLm prior to infection can control a lethal HSV challenge without the additional presence of a preexisting HSV-specific humoral or CD4+
T-cell response. CD4+
T cells have been shown to be required in a primary HSV-specific CTL response but not in a secondary CTL response (26
). The CD4+
T-cell help needed for the generation of CTL in B6 mice immunized with the recombinant viruses expressing the gB498-505 epitope may be provided by vector-specific CD4+
Our results suggest the possibility that the epitope-specific CTL act in the CNS to control viral infection. However, the possibility also exists that gB498-505-specific CTL limit HSV-2 replication at the initial site of infection, the nasal mucosa, and thus prevent the infection of innervating neurons. Previous studies in mice have demonstrated the sequential course of i.n. HSV infection initiating in the nasal mucosa followed by the dissemination of the infection to the brain and trigeminal ganglia (4
). Immunization with rVV-ES-gB498-505 significantly reduced the extent of viral colonization of the CNS tissues (Fig. ). The mechanisms underlying this protection may include direct CTL-mediated lysis of HSV-2-infected cells or the production of cytokines such as gamma interferon which directly or indirectly serve to limit the establishment and/or spread of the viral infection (46
Since both HSV-1 and HSV-2 are able to establish infections at mucosal surfaces, the use of a mucosal route of immunization may be most effective in the development of an effective human vaccine. Previous studies have demonstrated that anti-HSV immunity may be induced by mucosal immunization with vectors which express HSV proteins (22
). For example, immunization with a recombinant adenovirus expressing full-length HSV gB has been demonstrated to confer protection from HSV infection. Moreover, this protection correlated with the induction of an HSV-specific neutralizing antibody and CTL response (21
). Other approaches used for the induction of protective HSV-specific immune responses include immunization with attenuated HSV or replication-defective HSV mutants (9
). In addition, vectors expressing HSV-encoded proteins (10
) have been shown to be effective in mediating protection against HSV infection.
In summary, our results suggest that the induction of HSV-specific CD8+ T cells provides an alternative approach for the generation of acquired immunity to HSV. This study not only confirms the significance of CD8+ CTL in the control of HSV infection but also illustrates the potential importance of designing mucosal HSV vaccines which target specific CTL recognition epitopes encoded within HSV.