Neutralizing antibodies have been thought to constitute an important correlate of protective immunity against smallpox (4
). To perform a test on a large number of samples, we developed a vaccinia virus-specific ELISA technique with high processing ability, because neutralizing assays are time and labor intensive and are not adequate for handling a large number of samples. Therefore, we evaluated the correlation between antibodies detected by the neutralization assay and those detected by ELISA, because the antibodies detected by ELISA do not always reflect the presence of neutralizing antibodies.
It has been shown that the majority of the Japanese population who were vaccinated against smallpox prior to the cessation of the routine vaccination program in 1976 still maintain certain levels of ELISA-detectable virus-specific antibodies. More than 98% of subjects in the pre-1969 birth cohorts and 66% of those in the 1969-to-1975 cohort still maintained detectable levels of IgG antibodies (OD405
values ≥ 0.10). One of the reasons why the subjects in the 1969-to-1975 cohort have a lower seropositivity rate than those in the older cohort is that the vaccination rate per se would have been lower, because they were given only one opportunity to receive a smallpox vaccination. In fact, the smallpox vaccination rate at each stage was reported to be around the 80% level, except in 1974 and 1975, when the rate declined markedly (information from the Ministry of Health, Labor, and Welfare of Japan). The mean OD405
value calculated only from the seropositive subjects among the 1969-to-1975 cohort was 1.32, and this value was not significantly different to that of the pre-1962 cohort or the 1962-to-1968 cohort. Although we do not have precise information on how many vaccinations each individual actually received, this result suggests that the additional vaccinations had little influence on the period or degree of IgG retention, as long as the first vaccination had “taken” successfully. Therefore, further investigations are required to determine whether or not multiple smallpox vaccinations are necessary for acquiring significant protection, although it is generally believed that additional vaccinations are likely to confer a stronger and longer-lasting immune response (2
). Hammarlund et al. reported that the mean antibody titer induced by double vaccinations was very slightly but significantly higher than that induced by a single vaccination but that additional (between 3 and 14) vaccinations did not result in any further increase in long-term antibody production (11
In the present study, most of the individuals whose serum samples exhibited OD405 values of ≥0.30 also retained the neutralizing antibodies. Thus, it was demonstrated that a considerable proportion of the previously vaccinated individuals still retained neutralizing antibodies. Although lower ELISA OD405 values tend to associate with lower neutralizing antibody titers and higher OD405 values tend to associate with higher neutralizing antibody titers, the correlation between them was only moderately positive (R2 = 0.450; P < 0.0001). It may be due to a number of epitopes on the viral proteins other than neutralizing epitopes on vaccinia virus.
This study has revealed that many individuals who were vaccinated 27 to 53 years ago retain a significant degree of antiviral humoral immunity. Although this remaining immunity may no longer provide full protection, it is highly likely to afford at least partial protection. Hammarlund et al. showed that virus-specific T-cell immunity could persist for a long time after smallpox vaccination, perhaps as long as 75 years, declining only slowly, with a half-life of 8 to 15 years (11
). Furthermore, it was shown that virus-specific memory B-cells were maintained for more than 50 years after vaccination and correlated positively with circulating antibody levels (6
). In addition, some epidemiological analyses have also indicated that the immunity achieved after smallpox vaccination may remain for several decades (1
). Taking these data together, it appears that the immunity conferred by smallpox vaccination persists for longer than had previously been expected.
In the present study, we found that more than 98% of the Japanese population in the pre-1969 birth cohorts and 66% of those in the 1969-to-1975 cohort still maintain the vaccinia virus-specific IgG, whereas approximately 80 and 50%, respectively, also retain detectable levels of neutralizing antibodies. These long-term persisting immunities may provide some protective benefits in the case of intentional smallpox reemergence. In addition, the present results may also contribute in making policy of vaccination priority, especially if vaccine supplies become limited in the event of a widespread outbreak.