Long term immunity is one of the cornerstones of a successful vaccine. Pathogen-specific memory T cells undergo changes in phenotype, function and location over time which would influence their ability to respond to a secondary challenge [36
]. In this study we assessed the impact of increasing age on the frequency, phenotype and recall potential of VACV-specific CD8 T cell memory pools in mice that received a traditional first generation replicating or a third generation non-replicating smallpox vaccine. We demonstrated long term stable CD8 T cell responses in mice immunized with either MVA or NYCBH. Both groups of mice were able to mount robust secondary CD8 T cell responses when challenged with a lethal dose of VACV-WR. We also assessed patterns of activation and memory markers on antigen-specific T cells and found distinct differences in cells that respond at mucosal versus systemic sites. Our studies provide important insights into the impact of aging on VACV-specific CD8 memory T cells established earlier in life and their ability to respond to a lethal poxvirus challenge. Overall, our results indicate that both first and third generation smallpox vaccines were able to induce long-term protection against a lethal VACV-WR challenge.
We compared immune responses in mice that were administered two doses of MVA by the i.m. route to mice that were administered 1 dose of NYCBH by tail scarification specifically because these routes and the number of immunizations have been shown to induce optimal responses for these vaccines in both humans and mice [3
]. We too have noted that mice administered 1 dose of MVA by the tail scarification route were more susceptible to lethal challenge with VACV-WR at early and late time points and had less robust CD8 T cell responses. We administered 10–100 fold less MVA (106
PFU) compared to other immunization protocols in order to be consistent with the dose administered to mice immunized with NYCBH in this study.
To characterize the antibody responses in immunized mice, we assessed both neutralizing antibody titers as well as ELISA titers directed against the challenge virus VACV-WR. We found low levels of neutralizing antibodies in the sera of MVA immune mice at all time points. Administration of 106
PFU of MVA by the i.m. route has previously been shown not to induce VACV-specific neutralizing antibodies [12
]. Surprisingly, while a greater proportion of mice immunized with NYCBH had neutralizing antibodies, the overall titers were also low. Other studies have also reported low neutralizing antibody levels in immune mice that have been scarified with standard vaccines [37
]. As expected ELISA titers were higher than neutralizing antibody titers in both groups with higher levels in the sera of mice immunized with MVA compared to NYCBH at the 6 month time point. We do not think that these ELISA titers however are associated with protection.
In our hands, MVA and NYCBH were comparable in inducing long term protection against a lethal VACV-WR challenge. We used a well accepted intranasal challenge model with VACV-WR, a neurovirulent VACV strain [14
]. Ferrier–Rembert et al found that MVA and NYVAC were markedly less effective than VACV-Lister in protection from a lethal cowpox challenge and Coulibaly et al found that higher doses of MVA were required for protection against ectromelia challenge [9
]. These results may be explained by the pathogenic orthopoxviruses that were used to evaluate cross-protection in VACV-immunized animals. Interestingly, despite the weight loss and low antibody titers in mice immunized with either MVA or NYCBH, both groups were protected from a lethal challenge with VACV-WR. The low levels of neutralizing antibodies induced in these mice may have been sufficient for protection. Alternatively the immunization may have elicited the generation of antibody secreting cells that were triggered to secrete neutralizing antibodies upon VACV challenge in our immune mice. There was a trend towards significant differences in viral titers in naïve mice that received VACV immune sera (from mice immunized 3 months prior with NYCBH) compared to naïve mice that received PBS prior to VACV-WR challenge. We believe that adoptive transfer of only 200 μl of serum diluted the low levels of neutralizing antibodies which led to this borderline significance and that these antibodies do play a role in protection. Transfer of increased amounts of immune serum may have led to significant differences.
Moutafsti et al recently demonstrated that peptide immunization of naïve T cells were able to induce robust B8R-specific T cells that were sufficient for protection from a lethal intranasal challenge [41
]. Cornberg et al demonstrated that E7R-specific memory CD8 T cells were able to reduce viral titers in naïve syngeneic recipient mice administered 1 × 106
PFU of VACV [42
]. Our data using adoptive transfer of virus-specific CD8 T cells showed significant decreases in viral titers compared to titers in the lungs of naïve mice that were challenged. Our data support findings that CD8 T cells may contribute to limit viral replication during VACV-WR challenge by the intranasal route.
The efficacy of viral vaccines depends in part on the generation of a pool of potent, long-lived memory T cells ready to expand rapidly upon re-exposure to antigen. We were interested in knowing whether effective T cell memory was maintained long term in mice immunized with either vaccine and if there were distinct differences in the phenotype of cells generated by immunization with MVA or NYCBH. We used the expression of KLRG1 as an indicator of short lived terminal effector CD8 T cells (SLECs) as well as a marker of immune senescence since long lived protective memory T cells express low levels of KLRG1 [33
]. We too found that a significant portion of TET+
T cells express KLRG1 during acute vaccinia infection in the spleen and the lung (data not shown). However a higher frequency of TET+
T cells remain KLRG1+
(approximately 40–50% ) 3 months after infection with MVA or NYCBH compared to T cells in mice with LCMV or Sendai virus infections [25
]. By 15 months however, a lower frequency of TET+
T cells (15–20%) expressed KLRG1indicating that attrition of KLRG1+
T cells occurred between 3 and 15 months. Interestingly, two months post secondary challenge in 15 mo immune mice, 50% of the TET+
T cells were KLRG1+
suggesting that the recent challenge had increased the frequencies of KLRG1+
T cells. The data suggest that the decrease in number of KLRG1+
memory T cells may be related to the long time from exposure to a primary or secondary VACV infection and that the further away one is from immunization, the fewer KLRG1+
Members of the TNF-receptor family which include CD27 are involved in orchestrating activation, differentiation and death of immune cells [44
]. Studies have shown that the interaction between CD27 and its cellular ligand CD70 regulates formation of effector and memory T cells after antigenic challenge in vivo [44
] and a recent study demonstrated that CD27 signaling directed IL-2 production that is essential for the survival of T cells in nonlymphoid tissue [46
]. Influenza-specific and murine CMV-specific CD8+
T cells are thought to represent a specialized memory T cell subset, which preferentially localize outside the secondary lymphoid organs and may represent memory T cells with high cytolytic potential [45
]. On the other hand, cells with a CD27hi
phenotype have been shown to mediate the strongest recall response in Sendai virus infection while cells with a CD27low
phenotype mediated the weakest response [26
]. Our data indicate that CD27 expression is down regulated on a significant portion of memory T cells in peripheral sites such as the lung while virtually all antigen-specific cells express CD27 in the spleen. The frequency of CD27+
cells did not change in the lung in response to a recent challenge and at two months following secondary challenge when memory T cells were quiescent again. In contrast the frequency of CD27+
T cells decreased in the spleen post secondary challenge. Our data support evidence that repeated exposure to antigen or localization at sites of antigenic challenge may result in the loss of CD27 expression in lung memory T cells. Alternatively, our data may suggest that the quality of virus-specific T cells in the lung which is the first line of defense against virus challenge may be compromised since a smaller proportion of cells express CD27.
The exact mechanisms that contribute to protection from a lethal poxvirus challenge are complex and likely involve multiple components of the immune system. Several factors including the innate immune response, cellular and humoral arms of the immune system, initial viral dose and the kinetics of virus replication in mucosal and systemic sites are likely to contribute to protection. The findings from this study do shed light on the impact of age on established memory CD8 T cell pools, the long term recall efficacy and immunobiology of aging VACV-specific CD8 T cells and will provide insight into future vaccine development and use of third generation smallpox vaccines.