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

Abstract

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.

FIG. 1.

Domains and critical residues of B. anthracis PA important for binding of 1G3 and 14B7. PA consists of domains 1 to 4. Domain 1 is composed of subdomains 1a (residues 1 to 167) and 1b (residues 168 to 258). Residues 1 to 258 contain a furin cleavage site, and upon cleavage by furin, a hydrophobic region of PA is exposed to allow binding by EF and/or LF (6, 32). Domain 2 is composed of residues 259 to 487 and is involved in oligomerization and formation of the channel for LF and EF entry into the host cell cytosol (6, 24, 41, 54). Domain 3 is made up of residues 488 to 595, and the only known function is thought to be oligomerization (32). Domain 4, which consists of residues 596 to 736, is essential for binding of host cell receptors (32). MAb 1G3 recognizes an epitope on a 17-kDa fragment located between residues Ser-168 and Phe-314 that partly overlaps domain 1 (specifically, residues 168 to 258). This epitope is involved in LF binding to PA. 1G3 preferentially binds to PA63 (specifically, between residues Ser-168 and Phe-314, a region which spans domains 1 and 2) and inhibits the binding of LF to PA bound to the cell surface. Removal of the 20-kDa fragment (domain 1a) is required to expose the epitope recognized by 1G3. 1G3 does not inhibit binding of PA83 to the PA cell surface receptor. MAb 14B7 recognizes an epitope on domain 4 between residues Asp-671 and Ile-721 and inhibits binding of PA83 to the cell surface receptor but does not prevent binding of LF to PA. 14B7 does not inhibit LTx activity after PA83 has bound to the PA cell surface receptor (32). Note that this image is for illustration and is not to actual scale.

FIG. 2.

Schematic of the R-, NC-, and RB-TNA assays (Ab represents either antiserum or MAb). The traditional R-TNA assay (A) performed as described by Li et al. (23) and the NC-TNA assay (B) both quantify neutralization from antibodies that recognize unbound PA83 in solution, although some recognition of bound PA63 can occur after the cocktail of PA, LF, and Ab is added to the cells. The NC-TNA assay (B) was developed solely to include cell cooling steps so that the results of the RB-TNA assay may be compared to those of the traditional R-TNA assay. The cooling step performed in the NC-TNA assay slightly affected the potency of Ab neutralization (as evident in Table 2; comparison of R- and NC-TNA assays), although the level of neutralization by the Ab is proportional between the assays. The RB-TNA assay (C) was developed to delineate the level of neutralization from antibodies that complexed with only the RB form of PA (PA63), as unbound PA is removed from the cells prior to the addition of Ab and LF. The cooling steps for this assay served to cool the lipid bilayer cell membrane to slow PA internalization so that after unbound PA was removed, the subsequently added anti-ATx Ab could effectively bind surface-bound PA without the presence of LF. The LF was added after unbound Ab was removed so that the cells could be warmed and intoxication could proceed.

FIG. 3.

Correlation of log₁₀ ED₅₀ titers for week 30 postvaccination samples combined between the NC- and RB-TNA assays and separately based on survivors versus nonsurvivors. Linear regression analysis was performed on the log₁₀ ED₅₀ data from both the NC- and RB-TNA assays to determine if the assays correlated with each other using rhesus macaque sera taken from the AVRP project. Panel A represents the week 30 postvaccination samples combined (n = 101), whereas panel B represents the week 30 postvaccination samples analyzed separately based on survivors versus nonsurvivors. As part of the analysis, the r² and slope were calculated. For the combined analysis, there was a strong positive correlation (r² = 0.9060) between the RB- and NC-TNA assay ED₅₀ titers. In addition, separate regression lines were fitted to the 30-week postvaccination samples from surviving (n = 76) and nonsurviving (n = 25) animals to determine if there was a difference in assay correlation for either cohort of animals. As part of the analysis, the r² and slope were calculated for the ED₅₀ titers. To test whether there was a statistically significant difference between the slopes of the surviving animals and the nonsurviving animals, a t test at a 0.05 level of significance was used. The slopes were not statistically significantly different (P = 0.3389), and there was a strong positive correlation (survivor r² = 0.8969, nonsurvivor r² = 0.8838) between RB- and NC-TNA assay ED₅₀ titers.

FIG. 4.

Logistic regression curves of log₁₀ ED₅₀ titers from week 30 postvaccination samples. Logistic regression curves were fitted to the log₁₀ ED₅₀ data using the 30-week postvaccination samples (n = 101) from both the NC- and RB-TNA assays to determine if the assays were predictive of survival. (A) First, models were fitted to the log₁₀ ED₅₀ RB-TNA assay data by challenge month (12 months, green line, n = 39; 30 months, blue line, n = 33; 52 months, red line, n = 29). It was determined that challenge time was not a significant effect in the RB-TNA assay logistic models (P value of 0.6111), which permitted analysis of the RB-TNA assay data regardless of the challenge month (overall model, black line). (B) Next, the models were fitted to the log₁₀ ED₅₀ NC-TNA assay data by challenge month (12 months, green line, n = 39; 30 months, blue line, n = 33; 52 months, red line, n = 29). It was determined that challenge time was not a significant effect in the NC-TNA assay logistic models (P value of 0.5282), which permitted analysis of the NC-TNA assay data regardless of the challenge month (overall model, black line). (C) Finally, since the challenge times did not significantly affect the RB- and NC-TNA assay models, the overall models from the assays were combined and assessed together regardless of the challenge month (black line, RB-TNA assay; blue line, NC-TNA assay). From this analysis, it was found that the slopes are significant in both models (P value of 0.0015 for the RB-TNA assay logistic model and P value of 0.0020 for the NC-TNA assay logistic model); thus, both the NC- and RB-TNA assays were predictive of survival, regardless of the challenge month.

FIG. 5.

Relationship between NC- and RB-TNA assay ED₅₀ titers in surviving animals (n = 20) at week 30 postvaccination and at day 14 postchallenge (log₁₀ ED₅₀). Linear regression analysis was performed on the log₁₀ ED₅₀ data using the 30-week postvaccination samples and day 14 postchallenge samples from both the NC- and RB-TNA assays to determine if the assays correlated with each other. Separate regression lines were fitted to the 30-week postvaccination titers and the day 14 postchallenge titers to determine if there was a difference in assay correlation for either cohort of animals. As part of the analysis, the r² and slope were calculated for the ED₅₀ values. To test whether there was a statistically significant difference between the slopes of the 30-week postvaccination samples and day 14 postchallenge samples, a t test at a 0.05 level of significance was used. The slopes were found to be not statistically significantly different (P value of 0.7309).