Serum Samples and Characteristics of the Subjects

Table 1. Table 1. Characteristics of the Subjects.

Table 2. Table 2. Duration of Antigen-Specific Serum Antibody Production.

A total of 630 serum samples from 45 subjects were obtained for this study or were obtained from the Oregon National Primate Research Center serum bank (Table 1). The majority of these serum samples were obtained from scheduled collections, with 50 samples (7.9%) obtained after an unscheduled event (e.g., exposure to an animal). Each subject provided, on average, 14.0 serum samples (median, 11) during an average period of 15.2 years (median, 15.6). The majority of subjects had received smallpox vaccination during childhood and had recovered from viral infections, including measles, mumps, rubella, Epstein–Barr virus, and varicella–zoster infections. The subjects in this cohort had common coexisting conditions but no specific immune deficiencies (Table 1 of the Supplementary Appendix). The duration of serum antibody production was determined with the use of a mixed-effects model of longitudinal analysis. Overall, antigen-specific antibody responses were long-lived, and we found no significant differences in antibody-maintenance patterns according to sex (Table 2).

Vaccinia

Figure 1. Figure 1. Antibody Responses after Viral Infection or Vaccination with Nonreplicating Protein Antigens. Results of longitudinal analyses of serum titers of antibodies against eight antigens in 45 subjects are shown: vaccinia (Panel A), measles (Panel B), mumps (Panel C), rubella (Panel D), Epstein–Barr virus (EBV) (Panel E), varicella–zoster virus (VZV) (Panel F), tetanus (Panel G), and diphtheria (Panel H). To determine the antibody half-life during the maintenance phase of the immune response, data were censored by removing subjects with seronegative and equivocal samples. For seropositive subjects, time points at or up to 3 years after an antibody spike were removed before analysis. The estimated antibody half-lives, 95% confidence intervals (CI), interquartile ranges, and associated P values were obtained with the use of a mixed-effects model of longitudinal analysis. The shaded regions represent the cutoff between seropositive and seronegative serum titers as determined by enzyme-linked immunosorbent assay (ELISA), and the dotted lines indicate the putative protective levels of antibodies, if known. EU denotes ELISA units and IU international units. IU standards were not available for mumps, EBV, and VZV.

One potential mechanism for maintaining long-term immunity to a nonpersisting pathogen is through intermittent reexposure. To examine this possibility, we evaluated antibody responses after smallpox vaccination (Figure 1A). A total of 39 of 43 subjects born before 1972 (91%) were seropositive for vaccinia (Fig. 1A in the Supplementary Appendix), which is consistent with the findings of a previous study.11 Before 2003, when eight subjects received booster vaccination, there were only two instances of an antibody spike that was indicative of vaccination or exposure (0.3 event per 100 person-years). This finding is consistent with the discontinuation of routine vaccination in 1972 and the absence of endemic orthopoxviruses in North America that are known to infect humans. Excluding serum samples obtained after smallpox vaccination in 2003, a putative protective level of antiviral antibody11,21 was present in 28 of the 45 subjects (62%), and the level of vaccinia-specific antibodies decreased slowly, with an estimated half-life of 92 years (95% confidence interval [CI], 46 to infinity; P=0.049).

Measles

Measles is rare in the United States, but it remains a serious threat through importation.22 An analysis of measles-specific antibodies revealed that five subjects had spikes in serum antibody levels (Fig. 1B in the Supplementary Appendix). Two subjects received documented measles–mumps–rubella (MMR) vaccinations; one seroconverted, whereas the other seropositive subject had antibody titers that did not change after vaccination. Four subjects unknowingly contracted a cross-reactive but uncharacterized paramyxovirus infection from exposure to diseased nonhuman primates during a 1999 outbreak. On the basis of the antibody titers from the last available blood sample drawn before a recent MMR vaccination or serologic boosting during the 1999 outbreak of primate paramyxovirus, 43 of the 45 subjects (96%) were seropositive, with a putative protective level of at least 0.2 IU of antimeasles antibodies per milliliter23 in 41 subjects (91%) (Fig. 1B in the Supplementary Appendix). The decrease in measles-specific antibodies (Figure 1B) was not significant (P=0.94) and is likely to be maintained for life (estimated half-life, 3014 years; 95% CI, 104 to infinity).

Mumps

Although typically less severe than measles, mumps is another childhood disease with the potential for serious complications.18 In this cohort, 41 of 45 subjects (91%) were seropositive for mumps (Fig. 1C in the Supplementary Appendix). Two subjects received MMR vaccinations during the period of observation, and two other subjects had spikes in antibody levels, one in 1998 and the other in 2004. These spikes may have been the consequence of exposure to naturally occurring mumps rather than MMR vaccination. This incidence rate of 0.3 event per 100 person-years is consistent with a steady decrease in prevalence18 since routine vaccination began in 1977. Antibody responses to mumps (Figure 1C), like those to measles, were long-lived (estimated half-life, 542 years; 95% CI, 90 to infinity) and showed no significant decrease (P=0.69).

Rubella

Rubella was a leading cause of birth defects in the United States before immunization programs were implemented in the 1970s.18 A total of 40 of the 45 subjects in our cohort (89%) were seropositive for rubella (Fig. 1D in the Supplementary Appendix). We identified two subjects with documented MMR vaccinations and one subject with a spike in preexisting rubella-specific titers in 2003. Because of an 8-year gap between contiguous serum samples, it is unclear whether the change in this latter subject was due to a natural case of rubella or variability in serum titers. In any case, a spike in rubella-specific antibodies is uncommon, with a low overall incidence rate of 0.15 event per 100 person-years. A putative protective level of at least 10 IU of antirubella antibodies per milliliter24 was reached in 39 of the 45 subjects (87%). Rubella-specific immunity (Figure 1D) was maintained, with an estimated half-life of 114 years (95% CI, 48 to infinity) and no significant rate of decrease (P=0.15).

After reviewing the medical histories of the subjects, we identified six subjects in whom only vaccine-induced immunity against measles had developed, four subjects in whom vaccine-induced immunity against mumps had developed, and seven subjects in whom vaccine-induced immunity against rubella had developed. Exclusion of these subjects from the longitudinal analysis shown in Figure 1 had no substantial effect on the calculated duration of serum antibody responses (Table 2 of the Supplementary Appendix). Moreover, although reexposure to the same or a serologically cross-reactive virus may boost antibody titers, these data with regard to vaccinia, measles, mumps, and rubella indicate that repetitive environmental exposures and infections are not absolutely required for maintaining long-term antiviral antibody responses.

Epstein–Barr Virus and Varicella–Zoster Virus

In contrast to acute viral infections, chronic and latent viral infections may either persist or be reactivated from latency, thereby “boosting” immune responses in the infected person. Antibody titers were determined for two latent herpesviruses, Epstein–Barr virus and varicella–zoster virus. A total of 37 of the 45 subjects (82%) were seropositive for Epstein–Barr virus, with seroconversion in 1 subject occurring during the observation period (Fig. 1E in the Supplementary Appendix). Four seropositive subjects had antibody titers that spiked during observation, indicating that reactivation or reexposure events had occurred, but at a relatively low rate (0.6 event per 100 person-years). Humoral immunity against Epstein–Barr virus (Figure 1E) showed no significant decrease (P=0.99) and is likely to be maintained for life (estimated half-life, 11,552 years; 95% CI, 63 to infinity).

Unlike antibody responses to Epstein–Barr virus, antibody responses to varicella–zoster virus showed frequent fluctuations (Fig. 1F in the Supplementary Appendix). All 45 subjects were seropositive for varicella–zoster virus, and 10 of the 45 subjects (22%) had antibody spikes (1.6 events per 100 person-years). Two subjects described an episode of shingles at or near the time of the observed spike in antibody responses to varicella–zoster virus, one subject may have been exposed to recently vaccinated children, six subjects do not recall having shingles or any known exposure to patients with varicella–zoster virus, and information was not available for one subject. Immunity (Figure 1F) decreased slowly, with an estimated half-life of 50 years (95% CI, 30 to 153; P=0.005). Thus, although the infection is latent with evidence of the most frequent reexposure and reactivation events, varicella–zoster virus induced the most short-lived antibody response of the viruses we examined.

Tetanus and Diphtheria

Tetanus and diphtheria vaccination has been recommended since the 1940s, primarily with the combined tetanus–diphtheria toxoid vaccine for adults. This recommendation has resulted in a sharp decrease in the incidence of both diseases18 and prolonged maintenance of antibody titers (Figure 1G and 1H). A titer of more than 0.01 IU of antitetanus antibodies per milliliter is considered to be protective.25 ELISA titers of more than 0.16 IU per milliliter correlate well with neutralizing activity; a titer of 0.16 IU per milliliter is the lowest level reliably detected by means of ELISA26 and is similar to our detection limit of 0.15 IU per milliliter (200 ELISA units). Protective antitetanus responses (Fig. 1G in the Supplementary Appendix) were clearly identified in 42 of the 45 subjects (93%), but this response rate may not reflect the true number of protected persons, since 0.01 IU is below the limit of detection by ELISA. Frequent tetanus boosters resulted in 31 instances of an antibody spike in 27 subjects (4.9 events per 100 person-years) (Fig. 1G in the Supplementary Appendix). Tetanus-specific antibodies decreased rapidly, with an estimated half-life of 11 years (95% CI, 10 to 14; P<0.001), which is similar to the decrease shown in a model reported more than 40 years ago.27

To determine whether the rapid antibody decay observed with tetanus held true for other protein antigens, we measured immunity against diphtheria (Figure 1H). Antidiphtheria antibody titers of more than 0.01 IU per milliliter are considered to be protective,28 and in 40 of the 45 subjects (89%) diphtheria-specific antibodies remained above our detection limit (200 ELISA units, or 0.04 IU per milliliter) (Fig. 1H in the Supplementary Appendix). It is possible that the remaining five subjects also had sustained protective antidiphtheria immunity but that it could not be definitively measured in these studies. Diphtheria-specific antibody spikes were observed, typically in parallel to responses against tetanus, as expected because of the combined tetanus and diphtheria formulation recommended for adult vaccination. An analysis of diphtheria-specific antibodies indicated an estimated half-life of 19 years (95% CI, 14 to 33; P<0.001). These results suggest that antibody maintenance is greatly influenced by the nature of the antigen, with these proteins eliciting quantitatively shorter antibody responses than those observed after viral infection.

Antibody Maintenance in Individual Subjects

Figure 2. Figure 2. Longitudinal Analysis of Serum Antibody Titers in Representative Subjects. Serum antibody responses against eight antigens were followed in four subjects with serum samples for a period of more than 25 years. Each subject received a booster smallpox vaccination in 2003, and serum antibody levels were monitored at close intervals after immunization (days 7, 14, 21, 30, and 60 after vaccination) to determine whether a defined viral infection would result in increased antibody production against other nonspecific antigens. Subject 1 (Panel A) was seronegative for Epstein–Barr virus (EBV), as indicated by a single data point of less than 200 ELISA units (EU). Subjects 2, 3, and 4 (Panels B, C, and D) were seropositive for EBV. VZV denotes varicella–zoster virus.

One model of antibody maintenance predicts that long-term responses are maintained by non–antigen-specific stimulation, also referred to as “bystander activation” of memory B cells during antigenically unrelated infections.3,8,29 To determine the potential effects of heterologous infection and vaccination, we measured humoral responses to eight antigens in four subjects followed longitudinally for more than 25 years and at weekly intervals after smallpox vaccination (Figure 2). Common immunologic events, including tetanus and diphtheria booster immunization (in Subjects 1 through 4), Epstein–Barr virus seroconversion (in Subject 2), and varicella–zoster virus reexposure or reactivation (in Subject 4), occurred during the period of observation but showed little effect on other antigen-specific antibody responses. After a defined infection with vaccinia (i.e., booster smallpox vaccination), there was little or no alteration in antibody responses to seven other antigens (<6% average change) (Figure 2), despite vaccinia-specific antibody responses that increased by approximately 4000% at the peak of the response. These results are consistent with those in six subjects who received booster smallpox vaccination and two subjects who received primary smallpox vaccination and later received either live yellow-fever vaccination or MMR vaccination (data not shown). Together, these findings suggest that nonspecific bystander activation is an unlikely mechanism in the maintenance of long-term antibody responses.

Individual variations in antibody maintenance (Figure 1) indicated that although the antigen itself plays an important role, it is not the sole factor determining the longevity of antibody responses. The findings in Subject 1 (Figure 2A) were largely representative of those in the overall cohort, with no measurable decrease in measles-specific antibodies and short-lived tetanus-specific and diphtheria-specific responses (estimated half-life, 14 and 12 years, respectively). Subject 2 (Figure 2B) had a similar pattern, with no decrease in measles-specific antibodies, whereas tetanus and diphtheria responses had half-lives estimated at 13 and 11 years, respectively. Subject 3 (Figure 2C) had a measles-specific antibody response that underwent a slow but measurable decrease (estimated half-life, 68 years), with tetanus antibodies showing the most rapid rate of decrease (estimated half-life, 8 years). Diphtheria-specific antibody titers did not decrease over the course of two decades, in sharp contrast to tetanus-specific antibody titers. The findings in Subject 4 (Figure 2D) are particularly intriguing because, with the exception of antibodies against Epstein–Barr virus (which showed no evidence of a decrease), all antibody responses decreased at relatively similar rates (estimated half-life, 14 to 31 years). Thus, the antigen, as well as one or more currently unknown host-specific factors, has a role in determining the duration of antibody-response patterns.

B-Cell Memory and Association with Antibody Levels

Figure 3. Figure 3. Relationship between the Number of Memory B Cells, Age, and Serum Antibody Levels. Panel A shows the results of an antigen-specific limiting-dilution assay performed for single time points for 40 subjects with available peripheral-blood mononuclear cells to determine memory B-cell frequencies for vaccinia, measles, mumps, rubella, varicella–zoster virus, Epstein–Barr virus, tetanus, and diphtheria. Solid circles represent samples with positive antibody titers (>200 ELISA units [EU]) or B-cell memory (>5 memory B cells per 106 B cells). Samples that scored below the level of detection for both antibody titer and B-cell memory are shown as open circles. Samples obtained during the acute phase (<1 year after exposure or a spike in the antibody titer) of an antigen-specific immune response are indicated as triangles. Dashed lines indicate the limit of detection. Panel B shows memory B-cell frequencies as compared with serum antibody titers from the corresponding blood sample. Seronegative and equivocal samples (<200 EU and <5 memory B cells per 106 B cells) and samples from subjects undergoing an acute immune response (<1 year after an antibody spike) were excluded before data analysis in order to focus on conditions involving long-term immunologic memory. The results of linear regression analysis are shown with the associated correlation coefficients for each comparison. EBV denotes Epstein–Barr virus, and VZV varicella–zoster virus.

Plasma cells either maintain antibody levels independently30-32 or may require replenishment by the proliferation and differentiation of memory B cells. A requirement for all memory B-cell–dependent theories of antibody maintenance is that a correlation must exist between memory B-cell levels and antibody levels.3,8,29 With the use of a limiting-dilution analysis,13 we found that memory B cells in the circulation were remarkably long-lived (Figure 3A). Previous studies have shown that within 1 month after vaccination, memory B-cell numbers in the circulation are representative of the memory B-cell frequencies observed in other lymphoid compartments such as the spleen.33

Table 3. Table 3. Comparison of Memory B-Cell Frequencies and Serum Antibody Titers.

We next compared memory B-cell frequencies with their corresponding serum antibody titers for eight antigens. A significant correlation between memory B-cell levels and antibody levels was observed after acute infection with measles, mumps, and rubella but not vaccinia (Table 3). There was also no significant correlation between memory B cells and antibodies against varicella–zoster virus or Epstein–Barr virus (viruses that maintain latent reservoirs) or for the tetanus–diphtheria vaccine antigens (Table 3). The strength of the correlation varied widely among antigens, suggesting that memory B-cell frequencies were a poor predictor of serum antibody levels (Figure 3B). For example, only 3% of the variability in antibody levels against tetanus could be explained by memory B-cell frequencies (R2=0.03). This finding suggests that memory B-cell–dependent replenishment of short-lived plasma cells is not likely to be a global mechanism for antibody maintenance.