Our study markedly extends the knowledge and solidifies the concept that there is a population of persistently HSV-seronegative adults who demonstrate consistently detectable T cell responses to HSV-2. Although we did not undertake a population-based approach, we did identify 22 subjects, or 29% of all subjects screened, who were seronegative for HSV-1 and HSV-2 by repeated WB analyses but possessed HSV-specific LP and IFN-γ ELISPOT responses. Over 1/3 of HSV-seronegative subjects enrolled into 2 different HSV-2 discordant couples studies were identified as IS suggesting that the IS phenotype is common among HSV-seronegative partners of HSV-2 infected persons.
We identified CD4 and CD8 T cell epitopes to multiple HSV-2 proteins that were present at similar frequencies in PBMC obtained from sequential blood draws spanning several years. Some IS subjects recognized a broad range of HSV-2 epitopes and some peptide-specific responses were present at high frequencies. Of the 16 HSV-2 ORFs used to screen for IFN-γ ELISPOT responses in the IS subjects, UL39 (ICP6) was the most commonly recognized HSV-2 protein and T cells directed at ICP6 were present in 55% of IS subjects followed by ICP4 (35%), ICP0 (25%), UL19 (15%) and UL29 (15%). No responses were measured to UL25, UL35, UL46 and UL11 and single positive responses in individual IS subjects were seen to gD-2, ICP22, ICP27, UL47, UL49, UL27 (gB-2) and US5. These data are in contrast to what we observed in 40 HSV-2+ subjects (including 14 HSV-2+ partners of the IS subjects); while responses were frequent to UL39 (ICP6), ICP4 and ICP0, responses were also frequent to HSV-2 glycoproteins (gB-2 and gD-2) and tegument proteins (UL46 and UL49). This pattern of T cell reactivity in HSV-2+ subjects is consistent a recent study from our group, the most comprehensive study of immunodominant CD8 epitopes to HSV-2 reported to date, which demonstrated that the highest frequencies of CD8 responses in HSV-2+ subjects (n=21) were directed at UL39, UL25, UL27, ICP0, UL46 and UL47 in decreasing order using a CD8 IFN-γ ELISPOT assay and peptide pools representing 48 HSV-2 ORFs (17
). In a study characterizing T cell responses by IFN-γ ELISPOT responses to IE proteins in HSV-2+ subjects, CD8 responses were found to UL49, ICP0 and ICP4 but not to ICP27, ICP22 or gD-2, while CD4 responses were mostly directed at UL49, gD-2, ICP4 and ICP0 (18
). While T cell epitopes were frequent to UL39 in the current IS study, a relative lack of T cell responses to gB-2, gD-2, UL46 and UL49 in IS subjects suggests that the antigenic repertoire of T cells in IS subjects is skewed compared to HSV-2+ subjects. Differential recognition of CD8 T cell epitopes has been described in HIV-exposed persistently seronegative subjects compared to HIV-infected subjects (22
) although how these T cells provide enhanced resistance to these subjects is not clear. The skewing of the T cell response to HSV-2 in IS subjects compared to HSV-2+ subjects may be related to the differences in exposure to HSV antigens in the 2 different cohorts. The preponderance of T cell responses directed at IE proteins in IS subjects suggests that IS subjects have been exposed to replicating virus since IE proteins are the first proteins made during the virus infectious life cycle and are not present in infectious virions. T cells directed at IE proteins would be engaged early in the infectious life cycle and may be able to kill the virally-infected cell before the production of infectious progeny and thus advantageous to the host. If some of the IS subjects are infected with HSV-2 in the absence of seroconversion, the presence of T cells directed at IE proteins at the neural-epidermal junction would provide the quickest defense against the virus spreading to the periphery and may explain why we did not detect any HSV DNA at mucosal sites in IS subjects (4
). Although HSV-2+ subjects possess T cells directed at IE proteins, it is possible that these T cells do not localize to the sites of HSV-2 reactivation and thus cannot contain the spread of infectious virus to the periphery. Studies to assess whether T cells directed at IE proteins are present at local sites of HSV-2 exposure in IS and HSV-2+ subjects are underway and may shed light on the role of these T cells in protection from HSV-2 .
Although HSV-specific LP responses, indicative of CD4 T cell responses, were detected in 77% of the IS subjects, we identified and confirmed only 2 CD4 T cell epitopes in a single IS subject (; ). In our original description of IS subjects, we identified several HSV-specific CD4 T cell clones in an IS subject that were directed at multiple epitopes including ones contained in UL21, UL29, UL46 and UL47 (4
). Additionally, CD4 T cell responses to whole HSV-2 antigen were detectable by ICS in several IS subjects (4
). These results suggest that unlike CD4 T cell responses to whole HSV-antigen, CD4 T cell responses to individual HSV-2 peptides in IS subjects were beneath of level of detection of the ICS assay and that T cell cloning will be required to expand the cells in order to characterize them. In support of this, analysis of CD4 T cell clones generated from PBMC from the 3 IS subjects with positive LP/negative IFN-γ ELISPOT responses to HSV-2 suggests that CD4 T cell responses directed at multiple T cell proteins (gB-2, gD-2, UL19, ICP0, ICP4 and UL39) were present in these subjects. These data also suggest that the frequencies and magnitudes of HSV-specific CD4 T cells are lower than observed in HSV-2 infected subjects where 84% of subjects possessed HSV-2 peptide-specific CD4 T cell responses as measured by ICS and flow cytometry (Laing and Corey, unpublished observations). In contrast, most HSV-2 T cell epitopes that we detected by ICS and flow cytometry in the IS subjects were recognized by CD8 T cells and in many cases, the frequencies of these individual peptide responses were of high magnitude (up to 0.78% of all gated CD8 T cells, ).
While differences in antigenic recognition of T cells in resistant versus infected HIV populations have been observed as mentioned above, differences in other aspects of T cell immunity to HIV in exposed seronegative (ESN) subjects versus HIV-infected subjects have been reported. These include but are not limited to (1) a skewing of naïve and central memory cells (23
), (2) secretion of mainly IL-2 from gag-specific T cells in ESN compared to mainly IFN-γ from gag-specific T cells in HIV-infected subjects (23
), (3) an increase in late effectors and natural killer cells in ESN compared to HIV-infected subjects (23
), (4) greater proliferative activity of CD4 T cells to p24 in ESN compared to HIV-infected subjects (24
), (5) elevated levels of CD4 T cells and RANTES expression in the genital mucosa of HIV-resistant Kenyan commercial sex workers compared to HIV-infected CSW (25
), to name a few. The impact of these differences on preventing HIV infection in the ESN however is not known and different mechanisms have been linked to different ESN cohorts. The IS cohort we have described represents a unique and a novel population in the HSV disease setting to assess potential immune mechanisms involved in HSV resistance; human clinical studies have previously focused on various innate and adaptive immune mechanisms related to the variability in the clinical course of HSV disease expression in HSV-infected subjects (16
) as opposed to mechanisms related to the resistance of HSV infection in HSV-seronegative subjects at risk of infection.
A possible explanation for the presence of HSV-specific T cell responses in the IS subjects is that these responses were primed by related or unrelated cross-reactive T cells such as those that have been observed between different strains of influenza virus or between unrelated viruses such as influenza virus and hepatitis C virus (HCV), EBV or HIV (reviewed in (31
)). We feel that this mechanism is an unlikely explanation for most of the IS subjects we identified since the majority possessed T cells directed at multiple epitopes within HSV-2. This mechanism seems more probable in subjects with single HSV-2 epitope responses as we observed in 4 of the IS subjects and experiments to determine potential cross-reactive epitopes may shed light on a potential immune mechanism of resistance in these subjects.
In summary, IS subjects are common among HSV-seronegative sexual partners of HSV-infected individuals and most IS subjects possess frequent and persistent T cell responses to multiple HSV-2 antigens. This suggests that HSV-2 exposure at mucosal sites can result in the exclusive priming of HSV-2 specific T cell responses. Studying the quantitative and qualitative aspects of T cell immunity in these subjects may invoke new concepts for the correlates of protection against genital HSV-2 infection and the rational design of protective HSV-2 vaccines.