Sherry R. Crowe

Vitamin D deficiency is associated with an increased autoimmune response in healthy individuals and in patients with systemic lupus erythematosus

Objectives 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). Methods 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. Results 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). Conclusions 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.

Influenza vaccination responses in human systemic lupus erythematosus: Impact of clinical and demographic features

OBJECTIVE Vaccination against common pathogens, such as influenza, is recommended for patients with systemic lupus erythematosus (SLE) to decrease infections and improve health. However, most reports describing the vaccination response are limited to evaluations of SLE patients with quiescent disease. This study focuses on understanding the clinical, serologic, therapeutic, and demographic factors that influence the response to influenza vaccination in SLE patients with a broad range of disease activity. METHODS Blood specimens and information on disease activity were collected from 72 patients with SLE, at baseline and at 2, 6, and 12 weeks after influenza vaccination. Influenza-specific antibody responses were assessed by determining the total serum antibody concentration (B(max)), relative affinity (K(a)), and level of hemagglutination inhibition in the plasma. Using a cumulative score, the patients were evenly divided into groups of high or low vaccine responders. Autoantibody levels were evaluated at each time point using immunofluorescence tests and standard enzyme-linked immunosorbent assays. RESULTS Compared to high responders, low responders to the vaccine were more likely to have hematologic criteria (P = 0.009), to have more American College of Rheumatology classification criteria for SLE (P = 0.05), and to be receiving concurrent prednisone treatment (P = 0.04). Interestingly, European American patients were more likely to be low responders than were African American patients (P = 0.03). Following vaccination, low responders were more likely to experience disease flares (P = 0.01) and to have increased titers of antinuclear antibodies (P = 0.04). Serum interferon-α activity at baseline was significantly higher in patients in whom a flare occurred after vaccination compared to a matched group of patients who did not experience a disease flare (P = 0.04). CONCLUSION Ancestral background, prednisone treatment, hematologic criteria, and evidence of increased likelihood of disease flares were associated with low antibody responses to influenza vaccination in SLE patients.

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.

OBJECTIVE To 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, and to investigate clinical and serologic features of SLE that are associated with elevated BLyS levels. METHODS Clinical history, disease activity measurements, and blood specimens were collected from 60 SLE patients at baseline and after influenza vaccination. Sera were tested for BLyS levels, lupus-associated autoantibodies, serum interferon-α (IFNα) activity, 25-hydroxyvitamin D (25[OH]D), and humoral responses to influenza vaccination. RESULTS Thirty percent of the SLE patients had elevated BLyS levels, with African American patients having higher BLyS levels than white 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 postvaccination only in the subset of patients with BLyS levels in the lowest quartile (P = 0.0003). Elevated BLyS levels were associated with increased disease activity, as measured by the SLE Disease Activity Index, physicians global assessment, and Systemic Lupus Activity Measure in white patients (P = 0.035, P = 0.016, and P = 0.018, respectively), but not in African Americans. Elevated BLyS levels were also associated with anti-nuclear RNP (P = 0.0003) and decreased 25(OH)D (P = 0.018). Serum IFNα activity was a significant predictor of elevated BLyS in a multivariate analysis (P = 0.002). CONCLUSION Our findings indicate that African American patients with SLE 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.

Select Human Anthrax Protective Antigen Epitope-Specific Antibodies Provide Protection from Lethal Toxin Challenge

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 provide protection through postexposure 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 serum levels of in vitro toxin neutralization capacity. Using solid-phase epitope mapping and confirmatory assays, we identified several neutralization-associated humoral epitopes and demonstrated that select antipeptide 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.

Anthrax vaccination induced anti-lethal factor IgG: Fine specificity and neutralizing capacity

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.

Human Monoclonal Antibodies Generated Following Vaccination with AVA Provide Neutralization by Blocking Furin Cleavage but not by Preventing Oligomerization

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 (IC(50): 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.

Humoral responses to independent vaccinations are correlated in healthy boosted adults

BACKGROUND Roughly half of U.S. adults do not receive recommended booster vaccinations, but protective antibody levels are rarely measured in adults. Demographic factors, vaccination history, and responses to other vaccinations could help identify at-risk individuals. We sought to characterize rates of seroconversion and determine associations of humoral responses to multiple vaccinations in healthy adults. METHODS Humoral responses toward measles, mumps, tetanus toxoid, pertussis, hepatitis B surface antigen, and anthrax protective antigen were measured by ELISA in post-immunization samples from 1465 healthy U.S. military members. We examined the effects of demographic and clinical factors on immunization responses, as well as assessed correlations between vaccination responses. RESULTS Subsets of boosted adults did not have seroprotective levels of antibodies toward measles (10.4%), mumps (9.4%), pertussis (4.7%), hepatitis B (8.6%) or protective antigen (14.4%) detected. Half-lives of antibody responses were generally long (>30 years). Measles and mumps antibody levels were correlated (r=0.31, p<0.001), but not associated with select demographic features or vaccination history. Measles and mumps antibody levels also correlated with tetanus antibody response (r=0.11, p<0.001). CONCLUSIONS Vaccination responses are predominantly robust and vaccine specific. However, a small but significant portion of the vaccinated adult population may not have quantitative seroprotective antibody to common vaccine-preventable infections.

Influenza A (H1N1) virus infection triggers severe pulmonary inflammation in lupus-prone mice following viral clearance.

Each year, up to one fifth of the United States population is infected with influenza virus. Although mortality rates are low, hundreds of thousands are hospitalized each year in the United States. Specific high risk groups, such as those with suppressed or dysregulated immune systems, are at greater danger for influenza complications. Respiratory infections are a common cause of hospitalizations and early mortality in patients with systemic lupus erythematosus (SLE); however, whether this increased infection risk is a consequence of the underlying dysregulated immune background and/or immunosuppressing drugs is unknown. To evaluate the influenza immune response in the context of lupus, as well as assess the effect of infection on autoimmune disease in a controlled setting, we infected lupus-prone MRL/MpJ-Fas(lpr) mice with influenza virus A PR/8/34 H1N1. Interestingly, we found that Fas(lpr) mice generated more influenza A virus specific T cells with less neutrophil accumulation in the lung during acute infection. Moreover, Fas(lpr) mice produced fewer flu-specific IgG and IgM antibodies, but effectively cleared the virus. Further, increased extrinsic apoptosis during influenza infection led to a delay in autoimmune disease pathology with decreased severity of splenomegaly and kidney disease. Following primary influenza A infection, Fas(lpr) mice had severe complications during the contraction and resolution phase with widespread severe pulmonary inflammation. Our findings suggest that influenza infection may not exacerbate autoimmune pathology in mice during acute infection as a direct result of virus induced apoptosis. Additionally, autoimmunity drives an enhanced antigen-specific T cell response to clear the virus, but persisting pulmonary inflammation following viral clearance may cause complications in this lupus animal model.