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

Abstract

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.

Fig 1

Humoral antibody responses to a 3-IM schedule of AVA. Vaccinated rhesus macaques received AVA (0.5 ml) on a 3-dose (0, 1, and 6 months) i.m. schedule with either HuAVA or a 1:5, 1:10, 1:20, or 1:40 AVA dilution. Vertical arrows indicate injection time points. PP, days after priming at 6 months. Control animals received saline injections. (A) Anti-PA IgG GMC (μg/ml) and 95% CI for each dose group on each study day. (B) Linear regression analysis of TNA (ED₅₀) versus anti-PA IgG (μg/ml) postpriming at 7 months (n = 120 observations). The equation for the best fit line is shown (r² = 0.881). ○, HuAVA; ▼, 1:5 AVA; □, 1:10 AVA; ⧫, 1:20 AVA; △, 1:40 AVA; ×, controls.

Fig 2

Anti-PA IgG subclass distributions and avidity analyses. (A) Relative subclass levels were determined for samples that had total anti-PA IgG levels of ≥10 μg/ml, had all four subclass IgG ELISAs performed, and had at least one IgG subclass detected. The HuAVA and 1:5 AVA subclass profiles were representative of all of the AVA groups. Error bars represent 95% CI for groups/days with at least 3 samples. Vertical arrows indicate injection time points. PP, days after priming at 6 months. □, HuAVA IgG1; ▽, HuAVA IgG2; △, HuAVA IgG3; ○, HuAVA IgG4; ■, 1:5 AVA IgG1; ▼, 1:5 AVA IgG2; ▲, 1:5 AVA IgG3; ×, 1:5 AVA IgG4. (B) AI determined by dissociation of antibody-antigen complexes in the presence of a chaotropic salt are an indirect measure of the affinity of an antibody for its cognate protein antigen and provide an assessment of immune response maturation. A single vaccination with HuAVA or 1:5 diluted AVA stimulated anti-PA IgG antibodies with measurable AI. Affinity maturation reached its maximum level between 2 and 6 months in response to the 2nd vaccination. The circulating high-avidity antibodies persisted through all time points for which samples met the testing criteria (33 months). Error bars represent 95% CI. ○, HuAVA; ▼, 1:5 AVA; □, 1:10 AVA; ⧫, 1:20 AVA; △, 1:40 AVA.

Fig 3

Cellular immune responses to a 3-IM schedule of AVA. (A) T cell SI for each dose group on each study day. Error bars indicate 95% CI. (B) Memory B cells (mean SFU/10⁶ IgG-secreting cells) determined by ELISpot analyses. Error bars indicate 1 SE. (C and D) Frequencies of IFN-γ (C)- and IL-4 (D)-secreting cells (mean SFU/10⁶ PBMC) determined by ELISpot analyses. Error bars indicate 1 SE. Vertical arrows indicate injection time points. ○, HuAVA; ▼, 1:5 AVA; □, 1:10 AVA; ⧫, 1:20 AVA; △, 1:40 AVA; ×, controls.

Fig 4

Logistic regression analysis of probability of survival versus humoral immune response. The logistic regression curves (solid lines) illustrate the changes in predicted survival probability with increasing immune responses (dashed lines [95% CI]). The dashed horizontal line indicates the 80% protection level; dashed vertical lines indicate the associated immune response levels. The point estimate associated with an 80% probability of survival in this study varied depending on the time of measurement. Selection of 7 months as the time point for determining the COP estimate for 80% probability of survival is supported by the relative narrowness of the 95% CI and the P value for the slope. Observations were plotted with a slight vertical y axis displacement so that overlapping points may be seen. (A) The anti-PA IgG level associated with an 80% probability of survival at 7 months was 97.3 μg/ml (95% CI = 49.4 to 329.7 μg/ml). (B) The TNA level associated with an 80% probability of survival at 7 months was an ED₅₀ of 1,243 (95% CI = 621 to 4,479). ○, animals that died; ●, animals that survived; ⊕, percent surviving animals grouped into bins by 0.5-log₁₀ increments of the measured immune response. Bins contained 1 to 34 animals and are for illustration of how the data fit the logistic regression model. Note that in panel A there was only 1 animal in the first bin, and this animal survived inhalation anthrax with a low anti-PA IgG response.

Fig 5

PE serum anti-PA IgG and TNA responses to infection in naïve and AVA-vaccinated rhesus macaques. Rhesus macaques were exposed to target doses of 200 to 400 LD₅₀ equivalents of aerosolized B. anthracis Ames. The onset and magnitude of infection-induced anti-PA IgG responses in all surviving animals were determined independent of the time since last vaccination. Sample analysis was performed at PE days 3, 5, 7, 14, and 30 for groups challenged at 12 months and at PE days 0, 14, and 30 for groups challenged at 30 and 52 months. Error bars indicate 95% CI. (A) Anti-PA IgG levels (μg/ml). (B) TNA levels (ED₅₀). ○, HuAVA; ▼, 1:5 AVA; □, 1:10 AVA; ⧫, 1:20 AVA; △, 1:40 AVA; ×, controls.