The predominant antibody response generated following at least 3 doses of US anthrax vaccine was, as expected, targeted against PA, although a small subset (6.9%) also generated LF-specific antibodies. In contrast to the US vaccine, the UK vaccine contains both a significant amount of PA and LF, but individuals vaccinated with the UK vaccine typically develop an anti-LF response which is at least one log lower than their anti-PA response 34
. In a number of studies, monoclonal antibodies directed against LF have been found to neutralize LT by in vitro 14, 20-24, 35
and in vivo
testing 20, 24
. In addition, several studies have found that when neutralizing antibodies directed against LF are administered with neutralizing antibodies directed against PA in vivo
, they can provide synergistic protection against both toxin and spore challenge 21-22
. Together, these studies suggest the induction of antibodies to LF following vaccination may provide more protection than PA antibodies alone. Identification of protective epitopes of LF will inform the selection of truncated protein fragments or peptides of LF most likely to induce responses leading to enhanced protection.
As described in a previous study, anti-PA titer is not a perfect predictor of neutralization activity. Indeed, a large proportion of AVA vaccinated individuals (43%), many of which possess high anti-PA titers, have very low neutralizing capacity 28
. Elucidating other factors beyond anti-PA titer which correlate with protection would therefore be a more reliable measure of AVA-induced immunity and a more precise measure of vaccine efficacy. The current study further supports the observation that the number of vaccinations an individual has received and how much time has passed since the last vaccination, in addition to PA titer, are associated with the development of a neutralizing response ( and 28
). In contrast, there were no clear predictors of developing a response to LF, and anti-LF titer was not associated with in vitro
neutralization. However, because of the much higher amount of PA in the vaccine, it is possible that anti-LF responses simply could not be isolated from the response to PA. Indeed, we found no individuals with moderate LF responses that did not have antibodies directed against PA as well. Further studies will focus on raising LF-specific antibodies in isolation for this purpose. It should be noted that this study was not designed to address the impact of time interval between doses, which is known to affect at least the short term response in UK vaccination 36
. Future studies will address this phenomenon in individuals vaccinated with AVA.
In addition to confirming previously reported correlates of protection, this more extensive study suggests that ethnicity may play a role in the development of neutralizing antibodies following anthrax vaccination. A previous study of AVA vaccination with a similar cohort size (n=1564) also found a racial/ethnical difference in the AVA response 37
. When anti-PA IgG antibody responses were determined at two time-points during a standard vaccination schedule, antibody levels were significantly higher in European Americans compared with African Americans at week 8 (during the immunization schedule) but not at month 7 (one month after the last vaccination). In a recent study of elderly individuals of European (n=33), African (n=39), and Hispanic (n=41) descent, influenza-induced proliferation of peripheral blood mononuclear cells (PBMCs) was increased post vaccination in European American and Hispanic individuals, but was not increased in African American individuals 38
Lower responses to the AVA vaccine in African American subjects could be due to a variety of genetic effects such as HLA haplotype, polymorphisms in cytokine or cytokine receptor genes, or variations in cell surface molecules. Several HLA alleles have been associated with non-responsiveness to vaccination including HLA-DRB1*07 39
, HLA-B alleles 46, 15, and 08 as well as DRB1*03 40
. In addition to HLA alleles, variations in IL-1 family member genes have been associated with differences in either the magnitude or the kinetics of the antibody response to the hepatitis B vaccine 41-44
. Polymorphisms in cytokine and cytokine receptor genes, as well as in HLA and pattern recognition receptor mannose-binding lectin-2 genes have all been associated with differing response to the yearly influenza vaccine 40, 45
. Finally, variants of TLR-2, 3, 4, 5, and 6, as well as MyD88 and MD2, SLAM, and CD46, are associated with humoral and cellular immunity to measles, including differences in antibody titers, proliferative responses, and cytokine secretion 41-42
. Most of the studies detailed above were done with a limited cohort of one race or ethnicity; however, as genes are often segregated by race or ethnicity, the effect of these polymorphisms on a particular group could be significant.
While anti-rLF titer was not found to be associated with in vitro
neutralization, fine specificity mapping of the LF response demonstrated that individuals with high neutralization activity contained antibodies directed against at least six specific antigenic regions of LF. Plasma from unvaccinated controls and LF-positive individuals with low neutralization activity did not bind these regions, suggesting that specificity of the anti-LF response is more important to neutralization than the overall antibody quantity. While the in vitro
neutralization observed in these samples might be due to antibodies directed against PA, in viv
o data with column purified antibodies demonstrates that antibodies directed against select regions of LF can provide protection against toxin challenge. The locations of the six antigenic regions were equally distributed between Domains I, II, and III. Monoclonal antibodies directed against Domain I, which is responsible for PA binding and is necessary for LF entry into the cell, are historically the most likely to be neutralizing 14, 20-21, 25, 35
. Monoclonal antibodies have also been found to bind Domains II, III, and IV, which together form the catalytic site of MAPKK cleavage. Site-directed mutagenesis of Domain II can decrease the ability of LF to compete with the phosphorylation of MEK, suggesting a role of Domain II in MAPKK binding. Domain III contains residues that make specific contact with the amino termini of most of the members of the MAPKK family, suggesting it is responsible for substrate recognition. Finally, Domain IV of LF binds zinc, and insertional mutagenesis within this domain can eliminate LF toxicity without eliminating PA binding.
Binding of antibodies to Domain I is often experimentally determined by gel mobility shift assays or radiolabeled LF attachment to PA-incubated cells; this technique, however, may be overstating the contribution of the Domain I-specific response to protection. The technique of fine specificity mapping provides a more accurate localization of binding, and therefore of protective epitopes. Several antibodies that were assumed to bind Domain I (LF8, 10G3) as well as at least one antibody that had not been characterized (9A11) bind epitopes in Domains III and IV 23
. Indeed, when the binding sites of previously described LF-specific monoclonal antibodies are determined by indirect or solid-phase peptide ELISAs, highly antigenic regions are most often found in Domains III and IV 23
. The method of fine-specificity mapping detailed in this study is also reproducible; several of the antigenic regions of LF found in this report (using human plasma following vaccination) coincide with those found in previous studies using sera or plasma from LF immunized mice, most notably epitopes 2, 4, and 5 (which overlap with epitopes 2, 3, and 4 of 23
). To further address the neutralization capacity of antibodies directed specifically toward the novel epitopes found here, future studies will isolate monoclonal antibodies specific to these epitopes and determine their neutralization capacity alone and with anti-PA.
In summary, lethal toxin neutralizing responses elicited by the AVA vaccine are specifically directed against select LF peptides. The antibodies generated by AVA vaccination and specific to these peptides can confer in vivo protection from toxin challenge. Indeed, if used in combination with antibodies to PA and antibiotics, these LF specific antibodies could contribute to effective treatment strategies following anthrax infection. In combination with previous findings correlating the anti-PA response with protection, the identification of protective anti-LF responses following AVA vaccination suggests that inclusion of portions of LF should be considered as a component of further anthrax vaccine formulations.