It is well recognized that interindividual variations exist in immune responses to vaccines and that these variations are in part due to host genetic polymorphisms in HLA and other genes (24
). Each HLA molecule binds a distinct set of self- and antigenic peptides that can trigger CMI responses and influence humoral responses through T helper cells. HLA-homozygous individuals logically present a more limited range of peptides than heterozygous individuals at the same HLA loci. Presentation of a narrower range of peptides may lead to diminished CD4+
T cell recognition and in turn may elicit weaker antigen-specific T cell responses. However, diminished T cell response can also be a result of T cell-mediated defects or allelic polymorphisms in genes other than HLA (25
The goal of this study was to examine associations between LP responses to PA following AVA vaccination and HLA genotypes, haplotypes, and homozygosity. We did not find significant associations of individual HLA alleles or haplotypes with interindividual variations in cellular immune responses to AVA. We did, however, find statistically significant associations with overall homozygosity and homozygosity at one class I locus and at two class II loci whose alleles are in tight linkage disequilibrium (LD). We found that class I (A) and class II (DQA1 and DQB1) homozygosity (as determined by four-digit molecular HLA typing) was significantly associated with an overall decrease in LP response compared with class I (A) and class II (DQA1 and DQB1) heterozygosity. Furthermore, according to the results of the two-digit molecular HLA typing, class I (A and B) and class II (DRB1, DQA1, and DPB1) homozygosity was associated with decreased PA-specific lymphoproliferation. The linkage between decreased lymphocyte proliferation and HLA homozygosity suggests that HLA-heterozygous individuals generate stronger CMI responses to anthrax PA than homozygous subjects who carry these specific alleles. It is possible that differences in the repertoire of PA-derived epitope presentation are likely the basis for these associations between HLA homozygosity and decreased LP response. However, the biological significance of these results and whether these findings can be generalized to other B. anthracis
virulence factors (LF and EF) are unclear. Further, based on comparisons with a subset of European-American subjects in this study with available genome-wide genotyping on the Affymetrix 6.0 array (12
), we did not find any association between heterozygosity at the HLA loci and genome-wide heterozygosity (data not shown).
class II alleles are encoded by DR, DQ, and DP polymorphic genes, and many class II alleles have been found to be important immunogenetic markers for both vaccine-induced viral and bacterial immune responses (14
). A relationship between specific DRB1-DQA1-DQB1 haplotypes and a significantly lower AbPA humoral response to AVA has been previously demonstrated (14
). In particular, that study found an association between the DRB1*01:02-DQA1*01:01-DQB1*05:01 haplotype and significantly lower AbPA levels following AVA (14
). Our study found that A, DQA1, and DQB1 homozygosity (at four-digit molecular resolution) and A, B, DRB1, and DQA1 homozygosity (at two-digit molecular resolution) are significantly associated with diminished PA-specific lymphoproliferation. This suggests that AVA vaccination induces a dominant HLA-restricted immune response to PA antigens and that HLA homozygosity at both class I and class II antigen presentation pathways potentially diminishes an individual's ability to generate strong cellular immune responses to PA antigens. The possible use of PA-derived epitopes for DRB1*04:01 and DRB1*07:01 and class II tetramers as tools to examine PA-specific Th2 CD4+
cellular immune responses in AVA vaccinees was recently illustrated (28
). This is of great interest in light of the potential use of HLA tetramers as tools to monitor T cell responses in vaccinated individuals and for design of subunit and peptide vaccine candidates against B. anthracis
and other pathogens (29
Narrower restriction of immune responses in HLA-homozygous individuals (the long recognized “homozygote disadvantage”) has been reported for hepatitis C virus (31
), human immunodeficiency virus (HIV) (32
), hepatitis B virus (HBV) (34
), and herpes simplex virus type 1 (HSV-1) (35
) infections. These studies clearly demonstrated that HLA homozygosity may be a susceptibility factor for infection (36
). Studies correlating HLA homozygosity with the immune responses to HBV, measles virus, and mumps virus vaccines have demonstrated similar results (22
). For example, HLA homozygosity has been correlated with nonresponse to hepatitis B (HBsAg) vaccine in individuals homozygous for the haplotype HLA-B8, SC01, DR3 (38
). These observations, together with our current findings, indicate that HLA homozygosity may adversely influence immune responses to bacterial as well as viral vaccines.
Likewise, our previous work with measles virus vaccine demonstrated significant associations of overall and specific homozygosity at HLA loci with lower levels of measles virus IgG antibodies after one dose of vaccine (37
). Homozygosity at an increased number of HLA loci, as well as homozygosity at the class I A locus, has been correlated with both decreased mumps virus vaccine-specific antibody levels and lymphoproliferation (22
). However, two doses of measles virus vaccination appear to diminish this “homozygote disadvantage” despite HLA homozygosity status at least for measles virus humoral Ab responses (22
), suggesting that additional vaccine doses may overcome this genetic disadvantage (39
). Similarly, homozygosity within the DPB1 locus showed no disadvantage for both rubella virus-induced IgG antibody levels after two doses of rubella vaccine (40
). This dampening phenomenon may imply that additional anthrax immunizations may be necessary to induce higher levels of immunity in individuals who are homozygous for specific HLA alleles.
This is the first report of the effect of HLA genotypes and HLA homozygosity on cell-mediated (lymphoproliferative) immune responses following AVA vaccination. The strengths of our study included the use of subjects selected from a multicenter, randomized clinical trial that tested multiple schedules of the licensed AVA vaccine. Our study also used high-resolution HLA class I and class II genotyping using PCR-based technologies, including automated RSCA and sequence-based typing. Limitations of the study included a somewhat small sample size and limited racial diversity of our study sample, since 80% of the study subjects were Caucasian. Replication of our findings in African-Americans and other racial groups will be required to clarify the role of homozygosity of HLA alleles in AVA-induced immunity in different races and ethnicities. Additionally, lymphocyte proliferation testing was used as a proxy for PA-specific CMI. Due to the long (43-month) follow-up of the AVA000 study, a traditional, accurate, and sensitive in vitro
assay was utilized to assess the functional capacity of T lymphocytes to respond to the major component of the AVA vaccine. However, these data on recall lymphoproliferative response to rPA in our AVA-vaccinated subjects do not represent a true “CMI” correlate of protection induced by vaccination (41
The central role of cellular immune responses in postvaccination protection from anthrax was recently demonstrated in a study of AVA-induced long-term protection in rhesus macaques (15
). A striking aspect of those data was that a three-dose intramuscular AVA priming series elicited persistent production of functional PA-specific gamma interferon (IFN-γ)- and interleukin-4 (IL-4)-producing T cells and of memory B cells as long as 50.5 months postvaccination when serum AbPA titers were low or undetectable. Irrespective of the humoral antibody titers at the time of infection, nonhuman primates were able to mount a robust and protective anamnestic response after aerosol exposure to B. anthracis
. Analogous CMI profiles and anamnestic anti-PA IgG responses were also evident in human AVA vaccinees (42
While we found a strong recall lymphoproliferative response to rPA in our AVA-vaccinated subjects, Ingram et al. found significant elevations of T cell IFN-γ release in response to B. anthracis
LF (domain IV) but not PA as measured by enzyme-linked immunosorbent spot (ELISpot) assays in United Kingdom-licensed Anthrax Vaccine Precipitated (AVP)-vaccinated subjects (29
). Naturally infected subjects demonstrate strong CD4+
T cell responses to both PA and LF (29
). Those authors theorized that vaccination skewed the immune response toward a Th2 response, an idea that was supported by the limited response in IL-5 and IL-13 and contrasted with the response seen in the cutaneous anthrax patients (29
). It is not known if AVA vaccination similarly biases toward a Th2 response or what the lymphoproliferative response would be to LF.
In conclusion, this report illustrates HLA gene contribution to host immunity associated with variable cellular immune responses to AVA. This information is likely to help to identify antigenic protective peptides within the PA of B. anthracis and influence future anthrax vaccine design. New anthrax vaccine candidates that offer protection and result in long-lasting immunity are needed. Additional studies are also necessary to replicate these findings and determine whether HLA-heterozygous individuals generate a stronger cellular immune response to other virulence factors (B. anthracis LF and EF) than HLA-homozygous subjects.