B cell memory plays a central role in conferring protective immunity to many infectious diseases for which there are effective vaccines, and yet little is known about the generation of B cell memory in humans. In this longitudinal study we examined the effect of vaccination on MBC generation in naïve individuals and determined the impact of TLR9 activation on this process in vivo. The results presented here offer new insights into the kinetics of this process and provide evidence that the innate immune receptor TLR9 plays a significant role not only in the generation of MBC in naïve individuals but also in controlling the behavior of existing MBC. For the two protein subunit malaria vaccine candidates, AMA1-C1 and MSP142-C1, the inclusion of CPG 7909 had a dramatic effect, resulting in a more rapid acquisition of vaccine-specific MBC, in greater numbers, that persisted for longer.
The longitudinal design of this study permitted a detailed characterization of the kinetics of MBC generation and maintenance in response to primary and secondary vaccination. The capacity for a detailed characterization was most apparent in the analysis of the AMA1-C1 vaccine trial in which PBMC samples were collected at several time points after each vaccination. We observed that AMA1-C1-specific MBC peaked in the peripheral circulation 7 days after the second and third vaccinations, representing approximately 3–4% of the total IgG+
MBC pool. Although it has been reported for diphtheria vaccination that the magnitude of the peak MBC response decreased with each booster immunization (11
), we did not observe a significant difference between peaks in this study [day 35 CPG 7909 group, 2.94% (95% CI, 2.12–3.75) vs. day 63 CPG 7909 group, 3.45% (95% CI, 2.44–4.35); p=0.328]. The differences between the studies may be due to the length of time between vaccination or the efficacy of the vaccines themselves. It is of interest that the second AMA1-C1 vaccination generated AMA1-C1-specific MBC at levels comparable to those observed after influenza (29
) and smallpox (31
) booster vaccination. Irrespective of CPG 7909 status, the rate of decline of AMA1-C1-specific MBC was approximately 0.4% per month. If this rate held steady, within 2 years the level of AMA1-C1-specific MBC would approach pre-immune levels in the CPG 7909 group. However, we do not know whether, or at what level, the antigen-specific MBC pool reaches equilibrium. In a cross-sectional study 18 months after smallpox vaccination, antigen-specific MBC, as a percentage of the total IgG+
MBC, decreased to 0.1% from a peak of 1% 14 days after vaccination (31
). Similarly, in individuals receiving influenza booster vaccinations, influenza-specific MBC increased from low levels before vaccination to 8.2% of IgG+
MBC 14 days after vaccination, and then declined rapidly to <1% 80 days post-vaccination (29
). Based on these observations, AMA1-C1-specific MBC would be expected to reach equilibrium at ~0.3% within a year after the final vaccination.
Although TLR9 expression is known to be low in naïve B cells and constitutively high in MBC (32
), the impact of this differential expression on the in vivo
responsiveness to CpG in humans at the cellular level is not known. As measured by the MBC response, we observed no effect of CPG 7909 on primary immunization with AMA1-C1 or MSP142
-C1, suggesting that CPG 7909 had little effect on naïve B cells directly, or indirectly through TLR9-expressing PDC. However, once generated by primary immunization, TLR9-expressing antigen-specific MBC responded dramatically to secondary immunization in the presence of CPG 7909. Although the relative impact of TLR9 activation in PDC versus MBC on the secondary response in vivo
is not known, it is clear from the results of our in vitro
experiments () that purified MBC differentiate into ASC upon TLR9 activation, as has been shown by others (28
The mechanisms underlying the apparent expansion and contraction of circulating antigen-specific MBC still need to be elucidated. The contraction phase may represent migration of MBC to lymphoid tissue where newly generated MBC compete for limited homeostatic niches in the MBC compartment. Alternatively, in a manner analogous to T cell antigen-driven expansion and contraction, contraction may represent an activation-induced cell death phenomenon (33
) . What remains unknown is which factors control the magnitude of the peak response and the subsequent steady state level.
The results presented here also address the controversy surrounding the relationship between MBC, LLPC, and serum Ab levels. In general, we observed a positive correlation between the magnitude of the vaccine-specific MBC response and Ab titers. We also observed that the percentage of vaccine-specific MBC present at the time of the second and third vaccinations predicted Ab titers 2 weeks later. The majority of this Ab was likely produced by short-lived PC given the rapid decline in titers that followed. Similar results were recently reported for infants immunized with the serogroup C meningococcal conjugate vaccine in which the frequency of specific MBC at the time of boosting correlated with post-vaccination titers (15
). However, the Ab titers we observed closer to steady state (approximately 3 and 6 months after the last MSP142
-C1 and AMA1-C1 vaccination, respectively) were likely produced by LLPC, and thus the correlation between MBC and Ab titers at steady state suggests that the maintenance of LLPC may be linked to MBC. The cellular and molecular nature of this relationship remains poorly understood.
In addition to the antigen-specific induction of MBC, we observed an approximately two fold antigen-independent decrease in the frequencies of total IgG+
MBC in circulation 3 days after the majority of vaccinations. This drop may reflect the migration of MBC into lymphoid tissues, apoptosis of MBC, differentiation of MBC into ASC, or a combination thereof. The concurrent increase in CD27+
PC we observed 3 days after the second and third vaccinations in the AMA1-C1/Alhydrogel study suggests that the decline in MBC is due in part to their differentiation into PC. In a separate phase I study of AMA1-C1/Alhydrogel without CPG 7909, we observed a similar increase in PC 3 days after vaccination (unpublished). That polyclonal activation can drive the differentiation of MBC into PC is supported by other studies that have examined the antigen-independent effects of vaccination on PC. Bernasconi et al
. observed an increase in ASC directed against Toxoplasma gondii
and measles 6 days after vaccination with tetanus toxoid (12
). These authors attributed this to the polyclonal activation and differentiation of all MBC into PC. Odendahl et al
. also observed an increase in circulating ASC of unknown specificity 6 days after vaccination with tetanus toxoid, but interpreted the ASC to be LLPC displaced from the bone marrow by newly generated tetanus-specific PC that better competed for bone marrow PC niches (34
). However, a recent study showed that up to one third of circulating ASC appearing after influenza vaccination were not vaccine-specific, and had recently divided, ruling out the possibility that these were LLPC displaced from the bone marrow (10
). Collectively, these findings are consistent with a model in which the decrease in total IgG+
MBC (which we observed after vaccination) is due in part to their polyclonal activation and differentiation into PC (3
An important question raised by these observations is the nature of the polyclonal activation of MBC in vivo
. For the AMA1-C1 vaccine, CPG 7909 was required to induce a consistent decrease in total IgG+
MBC, presumably as a result of direct and/or indirect TLR9 signaling in MBC or PDC, respectively. However, for the MSP142
-C1 vaccine, a decrease in total IgG+
MBC was observed in the absence of CPG 7909, presumably due in large part to the alum adjuvant. Recently, alum has been shown to activate an intracellular innate immune response through the Nalp3 inflammasome and to direct antibody responses by mechanisms that, although incompletely understood, likely involve activation of Th2 cells (35
). Why AMA1-C1/Alhydrogel without CPG 7909 was not associated with a decrease in total IgG+
MBC is unclear, but may reflect a complex interplay between antigen-specific and polyclonal responses. Future studies might explore how activation of TLRs or inflammasomes influences MBC behavior.
The results of this study also provide an important baseline for further investigation of the B cell response to malaria vaccine candidates and natural infection with P. falciparum
, the most lethal of human malaria species. Results of sero-epidemiological studies in malaria endemic areas indicate that humoral immunity to malaria in response to P. falciparum
infection is slow to develop, incomplete, and short lived, all of which suggests that P. falciparum
may have evolved mechanisms to subvert the generation and/or maintenance of immunological memory (4
). It will therefore be of interest to compare the B cell response at the cellular level to candidate malaria vaccines in those with no exposure to P. falciparum
, as in this study, to those living in a malaria endemic area. To that end we are performing similar analyses in individuals enrolled in Phase 1 clinical trials of candidate malaria vaccines in Mali. We are also conducting longitudinal studies in Mali (36
) to understand the mechanisms that underlie protective immunity to malaria and how P. falciparum
may modulate this response. An improved understanding of the B-cell response to malaria vaccine candidates and natural P. falciparum
infection can help guide the development of vaccines that provide sustained protection against this important pathogen.