Humoral and Cell-Mediated Immune Responses to Alternate Booster Schedules of Anthrax Vaccine Adsorbed in Humans.

Protective antigen (PA)-specific antibody and cell-mediated immune (CMI) responses to annual and alternate booster schedules of anthrax vaccine adsorbed (AVA; BioThrax) were characterized in humans over 43 months. Study participants received 1 of 6 vaccination schedules: a 3-dose intramuscular (IM) priming series (0, 1, and 6 months) with a single booster at 42 months (4-IM); 3-dose IM priming with boosters at 18 and 42 months (5-IM); 3-dose IM priming with boosters at 12, 18, 30, and 42 months (7-IM); the 1970 licensed priming series of 6 doses (0, 0.5, 1, 6, 12, and 18 months) and two annual boosters (30 and 42 months) administered either subcutaneously (SQ) (8-SQ) or IM (8-IM); or saline placebo control at all eight time points. Antibody response profiles included serum anti-PA IgG levels, subclass distributions, avidity, and lethal toxin neutralization activity (TNA). CMI profiles included frequencies of gamma interferon (IFN-γ)- and interleukin 4 (IL-4)-secreting cells and memory B cells (MBCs), lymphocyte stimulation indices (SI), and induction of IFN-γ, IL-2, IL-4, IL-6, IL-1ß, and tumor necrosis factor alpha (TNF-α) mRNA. All active schedules elicited high-avidity PA-specific IgG, TNA, MBCs, and T cell responses with a mixed Th1-Th2 profile and Th2 dominance. Anti-PA IgG and TNA were highly correlated (e.g., month 7,r(2)= 0.86,P< 0.0001, log10 transformed) and declined in the absence of boosters. Boosters administered IM generated the highest antibody responses. Increasing time intervals between boosters generated antibody responses that were faster than and superior to those obtained with the final month 42 vaccination. CMI responses to the 3-dose IM priming remained elevated up to 43 months. (This study has been registered at ClinicalTrials.gov under registration no. NCT00119067.).

A three-dose intramuscular injection schedule of anthrax vaccine adsorbed generates sustained humoral and cellular immune responses to protective antigen and provides long-term protection against inhalation anthrax in rhesus macaques.

A 3-dose (0, 1, and 6 months) intramuscular (3-IM) priming series of a human dose (HuAVA) and dilutions of up to 1:10 of anthrax vaccine adsorbed (AVA) provided statistically significant levels of protection (60 to 100%) against inhalation anthrax for up to 4 years in rhesus macaques. Serum anti-protective antigen (anti-PA) IgG and lethal toxin neutralization activity (TNA) were detectable following a single injection of HuAVA or 1:5 AVA or following two injections of diluted vaccine (1:10, 1:20, or 1:40 AVA). Anti-PA and TNA were highly correlated (overall r(2) = 0.89 for log(10)-transformed data). Peak responses were seen at 6.5 months. In general, with the exception of animals receiving 1:40 AVA, serum anti-PA and TNA responses remained significantly above control levels at 28.5 months (the last time point measured for 1:20 AVA), and through 50.5 months for the HuAVA and 1:5 and 1:10 AVA groups (P < 0.05). PA-specific gamma interferon (IFN-γ) and interleukin-4 (IL-4) CD4(+) cell frequencies and T cell stimulation indices were sustained through 50.5 months (the last time point measured). PA-specific memory B cell frequencies were highly variable but, in general, were detectable in peripheral blood mononuclear cells (PBMC) by 2 months, were significantly above control levels by 7 months, and remained detectable in the HuAVA and 1:5 and 1:20 AVA groups through 42 months (the last time point measured). HuAVA and diluted AVA elicited a combined Th1/Th2 response and robust immunological priming, with sustained production of high-avidity PA-specific functional antibody, long-term immune cell competence, and immunological memory (30 months for 1:20 AVA and 52 months for 1:10 AVA). Vaccinated animals surviving inhalation anthrax developed high-magnitude anamnestic anti-PA IgG and TNA responses.

Vaccination of rhesus macaques with the anthrax vaccine adsorbed vaccine produces a serum antibody response that effectively neutralizes receptor-bound protective antigen in vitro.

Anthrax toxin (ATx) is composed of the binary exotoxins lethal toxin (LTx) and edema toxin (ETx). They have separate effector proteins (edema factor and lethal factor) but have the same binding protein, protective antigen (PA). PA is the primary immunogen in the current licensed vaccine anthrax vaccine adsorbed (AVA [BioThrax]). AVA confers protective immunity by stimulating production of ATx-neutralizing antibodies, which could block the intoxication process at several steps (binding of PA to the target cell surface, furin cleavage, toxin complex formation, and binding/translocation of ATx into the cell). To evaluate ATx neutralization by anti-AVA antibodies, we developed two low-temperature LTx neutralization activity (TNA) assays that distinguish antibody blocking before and after binding of PA to target cells (noncomplexed [NC] and receptor-bound [RB] TNA assays). These assays were used to investigate anti-PA antibody responses in AVA-vaccinated rhesus macaques (Macaca mulatta) that survived an aerosol challenge with Bacillus anthracis Ames spores. Results showed that macaque anti-AVA sera neutralized LTx in vitro, even when PA was prebound to cells. Neutralization titers in surviving versus nonsurviving animals and between prechallenge and postchallenge activities were highly correlated. These data demonstrate that AVA stimulates a myriad of antibodies that recognize multiple neutralizing epitopes and confirm that change, loss, or occlusion of epitopes after PA is processed from PA83 to PA63 at the cell surface does not significantly affect in vitro neutralizing efficacy. Furthermore, these data support the idea that the full-length PA83 monomer is an appropriate immunogen for inclusion in next-generation anthrax vaccines.

Transcriptional analysis of protective antigen-stimulated PBMC from non-human primates vaccinated with the anthrax vaccine absorbed.

The transcriptional responses in recombinant protective antigen (PA)-stimulated peripheral blood mononuclear cells (PBMCs) from Anthrax Vaccine Absorbed (AVA)-vaccinated rhesus macaques were evaluated using Affymetrix HGU133 Plus 2.0 GeneChips. PBMCs from animals vaccinated at 0, 4, and 26 weeks were harvested at week 30, stimulated with PA, and RNA isolated. The expression of 295 unigenes was significantly increased in PA-stimulated compared to non-stimulated PBMCs; no significant decrease in gene expression was observed. These upregulated transcripts encoded for proteins functioning in both innate and adaptive immunity. Results were corroborated for several genes by real-time RT-PCR. This study provides information on the potential underlying transcriptional mechanisms in the immune response to PA in AVA-vaccinated rhesus macaques.

Variation in the hepatic gene expression in individual male Fischer rats.

A new tool beginning to have wider application in toxicology studies is transcript profiling using microarrays. Microarrays provide an opportunity to directly compare transcript populations in the tissues of chemical-exposed and unexposed animals. While several studies have addressed variation between microarray platforms and between different laboratories, much less effort has been directed toward individual animal differences especially among control animals where RNA samples are usually pooled. Estimation of the variation in gene expression in tissues from untreated animals is essential for the recognition and interpretation of subtle changes associated with chemical exposure. In this study hepatic gene expression as well as standard toxicological parameters were evaluated in 24 rats receiving vehicle only in 2 independent experiments. Unsupervised clustering demonstrated some individual variation but supervised clustering suggested that differentially expressed genes were generally random. The level of hepatic gene expression under carefully controlled study conditions is less than 1.5-fold for most genes. The impact of individual animal variability on microarray data can be minimized through experimental design.