Successful vaccination against smallpox is conventionally determined by the presence of a take at the site of inoculation. In primary vaccinations, the take involves a 2- to 3-week progression from papule to vesicle, to pustule, to scab (27
) but is more rapid, around 8 days, in revaccinations (23
). The lesion is thought to be a result of viral damage and inflammation at the site of inoculation (26
). In this study, we were able to compare the vaccinia virus-specific antibody profiles of 23 no-takes with naïve and previously vaccinated individuals with a take, both before and after vaccination. We used objective criteria to define preexisting immunity and response to vaccine based on six signature vaccinia virus antigens on microarrays (three membrane proteins and three other structural proteins), shown previously to dominate the response to DryVax in humans (6
). The membrane proteins comprised WR150/A27L, WR101/H3L, and WR113/D8L. These are the membrane targets of antibodies that neutralize intracellular mature virions (IMVs) and contribute to protection (7
). The remaining three signature antigens comprise the WR070/I1L core protein (20
), WR118/D13L, which is a membrane scaffold protein involved in intracellular morphogenesis (37
), and WR148/-, an A-type inclusion protein homolog that is strongly recognized by most vaccinated subjects (9
The no-takes studied here were a heterogeneous population, receiving lyophilized (DryVax) or frozen (APSV) vaccine at doses ranging from undiluted to 1/100, and with differing antibody titers, both before and after vaccination. We pooled data from DryVax and APSV vaccinees to increase the sample size. This was done on the basis that we did not see obvious differences between the DryVax and APSV responder profiles (see B). Moreover, both are derived from the NYCBOH parent strain and retain high take rates after dilution to 1/10 (11
). However, we are aware that while diluted APSV (1/10) induces comparable antibody and T cell responses to undiluted vaccine (32
), dilution of DryVax has dose-dependent effects on both (4
). The studies described here are presented with this caveat. No-takes that failed to respond are true cases of vaccination failure, for which the skin reaction is a good indicator. This could be due to insufficient dose, problems with vaccine delivery, etc. In contrast, no-take responders are likely to have other underlying reasons for the lack of a take. When these were segregated and compared with the takes, the analyses consistently showed that the profiles after vaccination are essentially identical. This suggests that in no-take responders, the presence or absence of particular antibody responses is not a major determinant in whether a skin take develops.
Unlike the response component, significant differences in preexisting antibodies were found to exist, with the no-take responders having the highest level of preexisting antibody. Thus, 53% of no-takes and 75% of no-take responders had preexisting antibody by arrays, compared to 6% of takes (). The elevated preexisting antibody in the no-takes was also supported by ELISAs using purified proteins. Currently, the origin of these preexisting antibodies is not known. Several lines of evidence argue in favor of exposure to a related orthopoxvirus as the origin, rather than an unrelated but antigenically cross-reactive source. First, the preexisting antibodies are known hallmarks of a vaccinia infection, particularly of previously vaccinated individuals, for which there is no significant difference (C). The preexisting antibody profile is dominated by late structural proteins and includes antibodies to IMV membrane proteins WR101/H3L, WR113/D8L, WR149/A26L, and WR150/A27L and core proteins WR129/A10L and WR070/I1L. Second, these preexisting antibodies are not against novel targets, ruling out an unrelated but cross-reactive etiological agent. Third, the lack of response to WR132/A13L in ELISA is also consistent with preexisting immunity to orthopox (15
). Finally, none of the vaccinees had exclusion criteria indicating previous smallpox vaccination. Overall, the data are consistent with previous exposure to a related orthopoxvirus. We have no information about exposure to other environmental orthopoxviruses by the individuals studied here. However, natural exposure to such viruses may have the same effect as previous smallpox vaccination and contribute to the attenuation of the take.
Attenuation of the take may be antibody or T cell mediated. Although we have not measured T cell responses in the this study, we favor the notion that preexisting T cell memory leads to rapid clearance of infection at the site of inoculation and the attenuation of the take without interfering with the boosting effect on the antibody arm of the response. In another study, numbers of residual vaccinia virus-specific CD4+
T cells in individuals vaccinated against smallpox 30 years previously are inversely associated with the size of the skin take upon revaccination (30
). Thus, memory T cells remaining after the primary vaccination are likely to contribute to attenuation of the take after revaccination. A similar process may be operating in the vaccinia-naïve no-takes studied here if they were already exposed to a vaccinia virus-like orthopox virus. The same study (30
) also found no relationship between neutralizing antibody titers after revaccination and lesion size. This is consistent with the findings presented here in which we saw no difference in antibody profiles between takes and no-takes after vaccination. Interestingly, the authors found no association between residual (day 0) vaccinia virus-neutralizing antibody titers and lesion size after revaccination, whereas our data are consistent with a role of d0 titers and lack of a take. The discrepancy does highlight that there are likely to be subtle differences between bona fide revaccinations and the no-take population studied here. For example, the etiological agent postulated here, or when our no-take population may have been exposed, may differ from that of the other study. Moreover, the different serological assays used and the different sample sizes may also be influential.
In summary, we have profiled the antibody responses of vaccinia takes and no-takes. The main findings are that the no-takes can be classified into responder and nonresponder populations. In this study, nonresponsiveness was a vaccine dose-related phenomenon, and the skin test provides an accurate indicator of vaccination failure in these individuals. In no-takes that responded to the vaccine, the skin reaction is not an accurate indicator of vaccination failure. No-take responders do not differ significantly from takes after vaccination, suggesting that the failure to take is not related to the antibody response to vaccination. In contrast, no-takes tend to have higher preexisting antibody titers before vaccination than takes, with profiles not dissimilar to those of previously vaccinated individuals. The origin of these preexisting antibodies is not thought to be vaccinia, but they nevertheless appear to be having an attenuating effect on the take, similar to previous vaccination.