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1.  Human Monoclonal Antibodies Generated Following Vaccination with AVA Provide Neutralization by Blocking Furin Cleavage but not by Preventing Oligomerization 
Vaccine  2012;30(28):4276-4283.
In order to identify the combination of antibody-mediated mechanisms of neutralization that result from vaccination with anthrax vaccine adsorbed (AVA), we isolated antibody secreting cells from a single donor seven days after booster vaccination with AVA and generated nine fully human monoclonal antibodies (hmAb) with high specificity for protective antigen (PA). Two of the antibodies were able to neutralize lethal toxin in vitro at low concentrations (IC50: p6C01, 0.12 µg/ml and p6F01, 0.45 µg/ml). Passive transfer of either of these hmAbs to A/J mice prior to challenge with lethal toxin conferred 80–90% protection. We demonstrate that hmAb p6C01 is neutralizing by preventing furin cleavage of PA in a dose-dependent manner, but the mechanism of p6F01 is unclear. Three additional antibodies were found to bind to domain 3 of PA and prevent oligomerization, although they did not confer significant protection in vivo and showed a significant prozone-like effect in vitro. These fully human antibodies provide insight into the neutralizing response to AVA for future subunit vaccine and passive immunotherapeutic cocktail design.
PMCID: PMC3367042  PMID: 22425791
anthrax; Anthrax Vaccine Adsorbed; human monoclonal antibodies; passive immunotherapeutics; protective antigen
2.  Influenza vaccination can induce new onset anticardiolipins but not β2-glycoprotein-I antibodies among patients with systemic lupus erythematosus 
Lupus  2012;21(2):168-174.
Antiphospholipid syndrome is characterized by autoantibodies against cardiolipins (aCL), lupus anticoagulant, and independent β2-glycoprotein (β2GPI). Controversy exists as to whether vaccination triggers the development of anti-phospholipid antibodies (aPL) in systemic lupus erythematosus (SLE) patients.
SLE patients (101) and matched controls (101) were enrolled from 2005 to 2009 and received seasonal influenza vaccinations. Sera were tested by ELISA for aCL at baseline, 2, 6, and 12 weeks after vaccination. Vaccine responses were ranked according to an overall anti-influenza antibody response index. Individuals with positive aCL were further tested for β2GPI antibodies.
SLE patients and healthy controls developed new onset aCL post-vaccination (12/101 cases and 7/101 controls, OR 1.81, p=0.34). New onset moderate aCL are slightly enriched in African American SLE patients (5/36 cases; p=0.094). The optical density (OD) measurements for aCL reactivity in patients were significantly higher than baseline at 2 weeks (p<0.05), 6 weeks (p<0.05), and 12 weeks (p<0.05) post vaccination. No new β2GPI antibodies were detected among patients with new aCL reactivity. Vaccine response was not different between patients with and without new onset aCL reactivity (p=0.43).
This study shows transient increases in aCL, but not anti-β2GPI responses, after influenza vaccination.
PMCID: PMC3268677  PMID: 22235049
Influenza; vaccine; antiphospholipid antibodies; systemic lupus erythematosus
3.  B Lymphocyte Stimulator Levels in Systemic Lupus Erythematosus: Higher Circulating Levels in African American Patients and Increased Production after Influenza Vaccination in Patients with Low Baseline Levels 
Arthritis and rheumatism  2011;63(12):3931-3941.
Examine the relationship between circulating B lymphocyte stimulator (BLyS) levels and humoral responses to influenza vaccination in systemic lupus erythematosus (SLE) patients, as well as the effect of vaccination on BLyS levels. Clinical and serologic features of SLE that are associated with elevated BLyS levels will also be investigated.
Clinical history, disease activity measurements and blood specimens were collected from sixty SLE patients at baseline and after influenza vaccination. Sera were tested for BLyS levels, lupus-associated autoantibodies, serum IFN-α activity, 25-hydroxyvitamin D, and humoral responses to influenza vaccination.
Thirty percent of SLE patients had elevated BLyS levels, with African American patients having higher BLyS levels than European American patients (p=0.006). Baseline BLyS levels in patients were not correlated with humoral responses to influenza vaccination (p=0.863), and BLyS levels increased post-vaccination only in the subset of patients in the lowest quartile of BLyS levels (p=0.0003). Elevated BLyS levels were associated with increased disease activity as measured by SLEDAI, PGA, and SLAM in European Americans (p=0.035, p=0.016, p=0.018, respectively), but not in African Americans. Elevated BLyS levels were also associated with anti-nRNP (p=0.0003) and decreased 25(OH)D (p=0.018). Serum IFN-α activity was a significant predictor of elevated BLyS in a multivariate analysis (p=0.002).
African American SLE patients have higher BLyS levels regardless of disease activity. Humoral response to influenza vaccination is not correlated with baseline BLyS levels in SLE patients and only those patients with low baseline BLyS levels demonstrate an increased BLyS response after vaccination.
PMCID: PMC3234134  PMID: 22127709
Systemic lupus erythematosus (SLE); Cytokines
4.  MHC Class II and Non-MHC Class II Genes Differentially Influence Humoral Immunity to Bacillus anthracis Lethal Factor and Protective Antigen 
Toxins  2012;4(12):1451-1467.
Anthrax Lethal Toxin consists of Protective Antigen (PA) and Lethal Factor (LF), and current vaccination strategies focus on eliciting antibodies to PA. In human vaccination, the response to PA can vary greatly, and the response is often directed toward non-neutralizing epitopes. Variable vaccine responses have been shown to be due in part to genetic differences in individuals, with both MHC class II and other genes playing roles. Here, we investigated the relative contribution of MHC class II versus non-MHC class II genes in the humoral response to PA and LF immunization using three immunized strains of inbred mice: A/J (H-2k at the MHC class II locus), B6 (H-2b), and B6.H2k (H-2k). IgG antibody titers to LF were controlled primarily by the MHC class II locus, whereas IgG titers to PA were strongly influenced by the non-MHC class II genetic background. Conversely, the humoral fine specificity of reactivity to LF appeared to be controlled primarily through non-MHC class II genes, while the specificity of reactivity to PA was more dependent on MHC class II. Common epitopes, reactive in all strains, occurred in both LF and PA responses. These results demonstrate that MHC class II differentially influences humoral immune responses to LF and PA.
PMCID: PMC3528256  PMID: 23342680
Bacillus anthracis; protective antigen; lethal factor; vaccine; antibody response; MHC class II; mouse; genetic background
5.  Vitamin D deficiency is associated with an increased autoimmune response in healthy individuals and in patients with systemic lupus erythematosus 
Annals of the rheumatic diseases  2011;70(9):1569-1574.
Vitamin D deficiency is widespread and has been associated with many chronic diseases, including autoimmune disorders. A study was undertaken to explore the impact of low vitamin D levels on autoantibody production in healthy individuals, as well as B cell hyperactivity and interferon α (IFNα) activity in patients with systemic lupus erythematosus (SLE).
Serum samples from 32 European American female patients with SLE and 32 matched controls were tested for 25-hydroxyvitamin D (25(OH)D) levels, lupus-associated autoantibodies and serum IFNα activity. Isolated peripheral blood mononuclear cells were tested for intracellular phospho-ERK 1/2 as a measure of B cell activation status.
Vitamin D deficiency (25(OH)D <20 ng/ml) was significantly more frequent among patients with SLE (n=32, 69%) and antinuclear antibody (ANA)-positive controls (n=14, 71%) compared with ANA-negative controls (n=18, 22%) (OR 7.7, 95% CI 2.0 to 29.4, p=0.003 and OR 8.8, 95% CI 1.8 to 43.6, p=0.011, respectively). Patients with high B cell activation had lower mean (SD) 25(OH)D levels than patients with low B cell activation (17.2 (5.1) vs 24.2 (3.9) ng/ml; p=0.009). Patients with vitamin D deficiency also had higher mean (SD) serum IFNα activity than patients without vitamin D deficiency (3.5 (6.6) vs 0.3 (0.3); p=0.02).
The observation that ANA-positive healthy controls are significantly more likely to be deficient in vitamin D than ANA-negative healthy controls, together with the finding that vitamin D deficiency is associated with certain immune abnormalities in SLE, suggests that vitamin D plays an important role in autoantibody production and SLE pathogenesis.
PMCID: PMC3149865  PMID: 21586442
6.  Influenza vaccination responses in human systemic lupus erythematosus: impact of clinical and demographic features 
Arthritis and rheumatism  2011;63(8):2396-2406.
Vaccination against common pathogens, such as influenza, is recommended for SLE patients to decrease infections and improve health. However, most vaccination response reports are limited to evaluation of SLE patients with quiescent disease. This study focuses on understanding the clinical, serological, therapeutic, and demographic factors which influence the response to influenza vaccination in patients with a range of disease activities.
Blood specimens and disease activity information were collected from seventy-two SLE patients at baseline and 2, 6 and 12 weeks after influenza vaccination. Influenza-specific antibody responses were assessed for antibody concentration (Bmax), relative affinity (Ka), and hemagglutination inhibition (HAI). Using a cumulative score, the subjects were evenly divided into high and low responders. Autoantibody levels were evaluated at each time-point by immunofluorescence and standard ELISAs.
Low responders to the vaccine were more likely to have hematologic criteria (p=0.009), exhibit more ACR criteria (p=0.05), and be on concurrent prednisone treatment (p=0.04). Interestingly, European American patients were more likely to be low responders than African Americans (p = 0.03). Following vaccination, low responders were more likely to experience disease flares (p=0.01) and to have increased ANA titers (p = 0.04). Baseline serum interferon alpha activity was significantly higher in patients that experienced a flare after vaccination compared to a matched group of patients that did not flare (p= 0.04).
Ancestral background, prednisone treatment, hematological criteria and evidence of increased disease flares were associated with low antibody responses to influenza vaccination in SLE patients.
PMCID: PMC3149742  PMID: 21598235
7.  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
8.  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
9.  Sequential B-Cell Epitopes of Bacillus anthracis Lethal Factor Bind Lethal Toxin-Neutralizing Antibodies▿  
Infection and Immunity  2008;77(1):162-169.
The bipartite anthrax lethal toxin (LeTx) consisting of protective antigen (PA) and lethal factor (LF) is a major virulence factor contributing to death from systemic Bacillus anthracis infection. The current vaccine elicits antibodies directed primarily to PA; however, in experimental settings serologic responses to LF can neutralize LeTx and contribute to protection against infection. The goals of the present study were to identify sequential B-cell epitopes of LF and to determine the capacity of these determinants to bind neutralizing antibodies. Sera of recombinant LF-immunized A/J mice exhibited high titers of immunoglobulin G anti-LF reactivity that neutralized LeTx in vitro 78 days after the final booster immunization and protected the mice from in vivo challenge with 3 50% lethal doses of LeTx. These sera bound multiple discontinuous epitopes, and there were major clusters of reactivity on native LF. Strikingly, all three neutralizing, LF-specific monoclonal antibodies tested bound specific peptide sequences that coincided with sequential epitopes identified in polyclonal antisera from recombinant LF-immunized mice. This study confirms that LF induces high-titer protective antibodies in vitro and in vivo. Moreover, the binding of short LF peptides by LF-specific neutralizing monoclonal antibodies suggests that generation of protective antibodies by peptide vaccination may be feasible for this antigen. This study paves the way for a more effective anthrax vaccine by identifying discontinuous peptide epitopes of LF.
PMCID: PMC2612257  PMID: 18981257
10.  Differential Antigen Presentation Regulates the Changing Patterns of CD8+ T Cell Immunodominance in Primary and Secondary Influenza Virus Infections 
The specificity of CD8+ T cell responses can vary dramatically between primary and secondary infections. For example, NP366–374/Db- and PA224–233/Db-specific CD8+ T cells respond in approximately equal numbers to a primary influenza virus infection in C57BL/6 mice, whereas NP366–374/Db-specific CD8+ T cells dominate the secondary response. To investigate the mechanisms underlying this changing pattern of immunodominance, we analyzed the role of antigen presentation in regulating the specificity of the T cell response. The data show that both dendritic and nondendritic cells are able to present the NP366–374/Db epitope, whereas only dendritic cells effectively present the PA224–233/Db epitope after influenza virus infection, both in vitro and in vivo. This difference in epitope expression favored the activation and expansion of NP366–374/Db-specific CD8+ memory T cells during secondary infection. The data also show that the immune response to influenza virus infection may involve T cells specific for epitopes, such as PA224–233/Db, that are poorly expressed at the site of infection. In this regard, vaccination with the PA224–233 peptide actually had a detrimental effect on the clearance of a subsequent influenza virus infection. Thus, differential antigen presentation impacts both the specificity of the T cell response and the efficacy of peptide-based vaccination strategies.
PMCID: PMC2194086  PMID: 12885871
antigen-presenting cells; antigen presentation; CD8-positive T lymphocytes; influenza; immunologic memory

Results 1-10 (10)