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1.  Protective Antigen-Specific Memory B Cells Persist Years after Anthrax Vaccination and Correlate with Humoral Immunity 
Toxins  2014;6(8):2424-2431.
Anthrax Vaccine Adsorbed (AVA) generates short-lived protective antigen (PA) specific IgG that correlates with in vitro toxin neutralization and protection from Bacillus anthracis challenge. Animal studies suggest that when PA-specific IgG has waned, survival after spore challenge correlates with an activation of PA-specific memory B cells. Here, we characterize the quantity and the longevity of AVA-induced memory B cell responses in humans. Peripheral blood mononuclear cells (PBMCs) from individuals vaccinated ≥3 times with AVA (n = 50) were collected early (3–6 months, n = 27) or late after their last vaccination (2–5 years, n = 23), pan-stimulated, and assayed by ELISPOT for total and PA-specific memory B cells differentiated into antibody secreting cells (ASCs). PA-specific ASC percentages ranged from 0.02% to 6.25% (median: 1.57%) and did not differ between early and late post-vaccination individuals. PA-specific ASC percentages correlated with plasma PA-specific IgG (r = 0.42, p = 0.03) and toxin neutralization (r = 0.52, p = 0.003) early post vaccination. PA-specific ASC percentages correlated with supernatant anti-PA both early (r = 0.60, p = 0.001) and late post vaccination (r = 0.71, p < 0.0001). These data suggest PA-specific memory B cell responses are long-lived and can be estimated after recent vaccination by the magnitude and neutralization capacity of the humoral response.
PMCID: PMC4147590  PMID: 25123559
Anthrax Vaccine Adsorbed; cellular immunity; lethal toxin neutralization; protective antigen
2.  Anthrax Vaccination Induced Anti-Lethal Factor IgG: Fine Specificity and Neutralizing Capacity 
Vaccine  2011;29(20):3670-3678.
The efficacy biomarker of the currently licensed anthrax vaccine (AVA) is based on quantity and neutralizing capacity of anti-Protective Antigen (anti-PA) antibodies. However, animal studies have demonstrated that antibodies to Lethal Factor (LF) can provide protection against in vivo bacterial spore challenges. Improved understanding of the fine specificities of humoral immune responses that provide optimum neutralization capacity may enhance the efficacy of future passive immune globulin preparations to treat and prevent inhalation anthrax morbidity and mortality. This study (n = 1000) was designed to identify AVA vaccinated individuals who generate neutralizing antibodies and to determine what specificities correlate with protection. The number of vaccine doses, years post vaccination, and PA titer were associated with in vitro neutralization, reinforcing previous reports. In addition, African American individuals had lower serologic neutralizing activity than European Americans, suggesting a genetic role in the generation of these neutralizing antibodies. Of the vaccinated individuals, only 69 (6.9%) had moderate levels of anti-LF IgG compared to 244 (24.4%) with low and 687 (68.7%) with extremely low levels of IgG antibodies to LF. Using overlapping decapeptide analysis, we identified six common LF antigenic regions targeted by those individuals with moderate levels of antibodies to LF and high in vitro toxin neutralizing activity. Affinity purified antibodies directed against antigenic epitopes within the PA binding and ADP-ribotransferase-like domains of LF were able to protect mice against lethal toxin challenge. Findings from these studies have important implications for vaccine design and immunotherapeutic development.
PMCID: PMC3233230  PMID: 21420416
Bacillus anthracis; Anthrax; Anthrax Vaccine Adsorbed; Lethal Factor; Protective Antigen; correlate of protection
3.  Select human anthrax protective antigen (PA) epitope-specific antibodies provide protection from lethal toxin challenge 
The Journal of infectious diseases  2010;202(2):251-260.
Bacillus anthracis remains a serious bioterrorism concern, and the currently licensed vaccine remains an incomplete solution for population protection from inhalation anthrax and has been associated with concerns regarding efficacy and safety. Thus, understanding how to generate long lasting protective immunity with reduced immunizations or providing protection through post exposure immunotherapeutics are long sought goals. Through evaluation of a large military cohort, we characterized the levels of antibodies against protective antigen and found that over half of anthrax vaccinees had low levels of in vitro toxin neutralization capacity in their sera. Using solid phase epitope mapping and confirmatory assays, we identified several neutralization-associated humoral epitopes and demonstrated that select anti-peptide responses mediated protection in vitro. Finally, passively transferred antibodies specific for select epitopes provided protection in an in vivo lethal toxin mouse model. Identification of these antigenic regions has important implications for vaccine design and the development of directed immunotherapeutics.
PMCID: PMC2891133  PMID: 20533877
anthrax; vaccination; antibodies; protective antigen
4.  Cell-Mediated Immune Response to Tuberculosis Antigens: Comparison of Skin Testing and Measurement of In Vitro Gamma Interferon Production in Whole-Blood Culture 
Although delayed-type hypersensitivity skin testing with tuberculin purified protein derivative (PPD) is the standard for tuberculosis screening, its variability suggests the need for a more sensitive, noninvasive test. An in vitro whole-blood assay has been proposed as an alternative. Using health care worker volunteers, we confirmed the correlation between PPD skin test (PPD-ST) results (positive, induration of >15 mm) and a standardized gamma interferon (IFN-γ) assay, QuantiFERON-TB (Q-IFN), manufactured by CSL Biosciences in Australia, and we evaluated Mycobacterium tuberculosis culture subfractions as potential substitutes for PPD. Twenty healthy volunteers with positive PPD-ST results and 20 PPD-ST-negative controls were enrolled. Whole blood was cultured with human PPD antigens (HuPPD), Mycobacterium avium complex (MAC) PPD, phytohemagglutinin (PHA), and four M. tuberculosis culture subfractions: low-molecular-weight culture, filtrate, culture filtrate without lipoarabinomannan, soluble cell wall proteins, and cytosolic proteins, all developed from M. tuberculosis strain H37RV. Secretion of IFN-γ (expressed as international units per milliliter) was measured by an enzyme immunoassay. The PPD or subculture fraction response as a percentage of the PHA response was used to determine positivity. Sixteen of 20 PPD-ST-positive individuals were classified as M. tuberculosis positive by Q-IFN, and 1 was classified as MAC positive. Sixteen of 20 PPD-ST-negative individuals were M. tuberculosis negative by Q-IFN, 2 were MAC positive, and 2 were M. tuberculosis positive. The tuberculosis culture subfractions stimulated IFN-γ production in PPD-ST-positive volunteers, and significant differences could be seen between the two PPD-ST groups with all subfractions except soluble cell wall protein; however, the response was variable and no better than the Q-IFN PPD. The agreement between the Q-IFN test and the PPD-ST was good (Cohen's kappa = 0.73). The Q-IFN assay can be a useful tool in further studies of immune responses to M. tuberculosis antigens.
PMCID: PMC96059  PMID: 11238218

Results 1-4 (4)