Producing effective vaccines against conventional and biothreat pathogens is an important goal of biomedical research. Towards that end, immunostimulatory adjuvants such as CpG ODN are being evaluated for their ability to improve the immunogenicity of vaccines targeting anthrax, smallpox, HIV and other infectious agents (reviewed in (32
). Previous research established that CpG ODN accelerated and magnified AVA induced immunity in mice, macaques and humans (13
). Current results confirm and extend these findings by demonstrating that CpG ODN extend the duration of protective immunity through 1 year. They further document that vaccination with CpG-adjuvanted AVA generates a large and long-lived population of high affinity memory B cells that respond so rapidly to challenge that they protect otherwise susceptible animals.
AVA requires six immunizations delivered over 18 months to induce and maintain protective Ab titers in humans, a regimen associated with deleterious side effects including joint pain, gastrointestinal disorders and pneumonia (9
). Pre-clinical and phase I clinical studies show that adding CpG ODN to AVA increases serum IgG anti-PA titers by 6 – 20 fold (26
). As seen in , the serum half-life of anti-PA Abs was similar in mice vaccinated with AVA vs CpG-adjuvanted AVA. However, protection persisted significantly longer in recipients of CpG-adjuvanted vaccine due to their initially higher anti-PA titers ().
Multiple mechanisms have been proposed to explain the ability of CpG ODN to improve AVA immunogencity. Unmethylated CpG DNA directly trigger immune cells that express TLR 9, initiating an innate immune response characterized by the production of pro-inflammatory and Th1 cytokines/chemokines capable of promoting the development of adaptive humoral responses (17
). CpG ODN also induce the functional maturation of professional APCs (34
). The increased availability of such “help” may explain why CpG-adjuvanted AVA induces protection more rapidly than AVA alone, and generates a larger and more avid population of memory B cells. The presence of such help may also facilitate the development of a protective secondary response.
It is well established that serum anti-PA Abs protect against infection, and that resistance is maintained by repeated re-immunization (36
). However, the literature provides examples of animals remaining resistant to infection after their serum Ab response has waned (39
). We therefore examined the susceptibility of mice to challenge after their anti-PA titers fell into the sub-protective range. Surprisingly, half of the mice immunized with CpG-adjuvanted AVA with anti-PA titers 10-fold below the protective baseline survived a 100 LD50
Sterne strain spore challenge (,). This contrasted with only 1/35 mice with the same Ab titer that had been immunized with AVA alone (p. <.01). The survival of mice with sub-protective titers did not correlate with the maximal Ab titer achieved following vaccination, time post vaccination, or dose of AVA (p>.45 for each parameter). Rather, protection correlated with how rapidly the host mounted a humoral response following pathogen challenge (). Specifically, IgG anti-PA titers rose rapidly in mice that survived, but were unchanged in animals that succumbed.
Among survivors, serum anti-PA titers did not reach protective levels (>1:16,000) until >10 days post challenge (by which time virtually all susceptible mice had died). This suggests that the cumulative Ab response over the course of infection, rather than solely at the time of challenge, determines host survival. This interpretation is consistent with results from an earlier study showing that mice challenged shortly after vaccination (when serum anti-PA titers were low) survived infection if their anti-PA response was rising towards protective levels (14
We hypothesize that mice with low serum Ab levels can survive infection if their high-affinity memory B cells respond rapidly to the Ag released following challenge. This possibility could not be evaluated in vivo
due to the rapid demise of AVA-vaccinated animals. Instead, the frequency and speed with which PA-specific memory B cells responded to Ag stimulation ex vivo
was examined using the splenic fragment technique (SFT). The SFT maintains the splenic micro-environment, thereby facilitating the detection of Ag-specific memory B cells (27
). Splenic fragments from all vaccinated mice secreted anti-PA Abs within 6 days of culture with rPA, unlike those from naive mice (). However, three important differences were noted between the response of splenic fragments from mice immunized with AVA alone vs AVA plus CpG ODN. First, significantly more memory B cells were present in the spleens of mice vaccinated with the CpG-adjuvanted vaccine (p. <.05, ). Second, these cells responded more rapidly to Ag stimulation, producing anti-PA Abs by day 3 post stimulation vs day 6 in mice vaccinated only with AVA (). Finally, these B cells responded to lower concentrations of rPA, and produced Ab of higher affinity, that those from mice vaccinated with AVA alone (). These results are consistent with the in vivo
observation that mice immunized with CpG-adjuvanted AVA responded rapidly to anthrax challenge by producing protective anti-PA Abs (–). While several mechanism(s) might contribute to the rapid activation of memory B cells from mice immunized with the CpG-adjuvanted vaccine, data suggest that a critical factor is their high affinity for PA. As seen in , significantly more memory B cells from CpG-adjuvanted animals i) responded to low concentrations of rPA and ii) produced high affinity Abs, than those from mice immunized with AVA alone.
A detailed analysis of the SFT results showed marked intra-animal variability in the response of CpG-adjuvanted mice. Specifically, splenic fragments from approximately one-third of donor mice behaved like those from AVA vaccinated animals: they contained relatively few anti-PA secreting B cells and these were primarily of low affinity. By comparison, splenic fragments from the majority of mice vaccinated with CpG-adjuvanted AVA contained large numbers of memory B cells, many of which secreted Abs of such high affinity that they remained bound to their target Ag despite treatment with 6 M urea (which strips low affinity Abs from rPA (25
)). We speculate that the latter group of mice are those destined to survive challenge. Thus, while other immune elements (such as Ag-responsive T cells) might also contribute to protection, current findings suggest that an important but previously unrecognized goal of anthrax vaccine development should be the generation long-lasting high-affinity memory B cells.
Conventional phase III clinical trials are designed to test whether a vaccine reduces the risk of human infection. Serious technical and ethical limitations prevent this conduct of such studies for vaccines targeting biothreat pathogens. Recognizing this problem, the FDA developed an “animal rule” that allows surrogate markers of protection derived from animal challenge studies to be substituted for evidence of clinical efficacy in human licensure decisions. Vaccine-induced anti-PA Abs correlate with survival from anthrax challenge in multiple animal models, and thus represent one such marker (13
). However, current results indicate that high affinity memory B cells also reduce host susceptibility to infection. Thus, relying on serum anti-PA Ab levels alone for licensure decisions could underestimate the protection conferred by novel vaccines. This leads us to suggest that second and third generation anthrax vaccines should also be evaluated for their ability to generate a durable pool of high-affinity memory B cells.