Epitope-focused peptide immunogens in human use adjuvants protect rabbits from experimental inhalation anthrax

Abstract

Background: Anthrax represents a formidable bioterrorism threat for which new, optimized vaccines are required. We previously demonstrated that epitope-focused multiple antigenic peptides or a recombinant protein in Freund's adjuvant can elicit Ab against the loop neutralizing determinant (LND), a cryptic linear neutralizing epitope in the 2ß2-2ß3 loop of protective antigen from Bacillus anthracis, which mediated protection of rabbits from inhalation challenge with B. anthracis Ames strain. However, demonstration of efficacy using human-use adjuvants is required before proceeding with further development of an LND vaccine for testing in non-human primates and humans.

Methods: To optimize the LND immunogen, we first evaluated the protective efficacy and immune correlates associated with immunization of rabbits with mixtures containing two molecular variants of multiple antigenic peptides in Freunds adjuvant, termed BT-LND(2) and TB-LND(2). TB-LND(2) was then further evaluated for protective efficacy in rabbits employing human-use adjuvants.

Results: Immunization of rabbits with TB-LND(2) in human-use adjuvants elicited protection from Ames strain spore challenge which was statistically indistinguishable from that elicited through immunization with protective antigen. All TB-LND(2) rabbits with any detectable serum neutralization prior to challenge were protected from aerosolized spore exposure. Remarkably, rabbits immunized with TB-LND(2) in Alhydrogel/CpG had significant anamnestic increases in post-challenge LND-specific Ab and neutralization titers despite little evidence of spore germination in these rabbits.

Conclusions: An LND-specific epitope-focused vaccine may complement PA-based vaccines and may represent a complementary stand-alone vaccine for anthrax.

Keywords: Antibody; Epitope; Inhalation anthrax; Neutralization; Peptide; Vaccine.

Fig. 1

Diagrammatic representation of LND MAP immunogens. Shown are diagrammatic representations of the individual MAPs which comprise the respective immunogen mixtures. As shown, BT-LND(2) is comprised of two LND MAPs: one containing the LND linked to the N-terminus of the T* helper T cell epitope and the second containing the LND peptide linked to the C-terminus of the P30 helper T cell epitope. TB-LND(2) contains the identical P30-containing LND MAP along with a second MAP containing the LND peptide linked to the C-terminus of the T* helper T cell epitope. Immunogens are not drawn to scale.

Fig. 2

Immunization with LND MAPs in human use adjuvants protects rabbits from spore challenge with B. anthracis Ames strain. Shown are Kaplan–Meier survival curves from groups of rabbits (n = 9) immunized with either TB-LND(2) formulated in different adjuvants, PA83 in ALOH or an irrelevant MAP in CFA and then challenged with aerosolized spores of B. anthracis Ames strain. An unimmunized naïve group of rabbits (n = 6) was also challenged. All naïve and irrelevant control rabbits died by day 4 and 6, respectively. All groups of rabbits immunized with TB-LND(2) and the PA controls were significantly protected from challenge compared to the naïve and irrelevantly immunized groups (p < 0.0001, Log Rank test). There were no significant differences in survival between the individual groups immunized with TB-LND(2) in the different adjuvant formulations, or between these groups and PA-immunized rabbits.

Fig. 3

Antibody and TNA responses prior to challenge from rabbits immunized with TB-LND(2) or a control MAP. Rabbits were immunized 5 times at two-week intervals with TB-LND(2) or the control MAP in CFA as described in the text. Approximately 10 days after the final immunization and two weeks prior to challenge, rabbits were bled and sera was analyzed by ELISA for immunoreactivity with PA (A) and in the TNA (B) as described in Section 2. Each circle represents the response of a single rabbit and horizontal lines represent geometric means. Open circles represent rabbits that did not survive the spore challenge. The lower limit of quantification for the TNA is a reciprocal dilution of 16. Samples with neutralization below this lower limit were assigned a value of 8.

Fig. 4

Analysis of postchallenge sera for LF-specific IgM and IgG, and for PA-specific Ab and neutralization. Survivor rabbit sera obtained 14 days after spore challenge was analyzed by ELISA for reactivity with immobilized LF (A). The lower limit of quantification for the ELISA is a reciprocal dilution of 16. Samples with titers below this level were assigned a value of 8. Each circle represents a data point from an individual rabbit and horizontal bars represent geometric means. Also shown are pre-and post-challenge antibody and neutralization titers from individual rabbits which survived challenge. Pre-challenge sera obtained 10 days prior to challenge and post-challenge survivor sera obtained 14 days after challenge were analyzed by ELISA (B) and in the TNA (C). Rabbits in the ALOH/QS-21 group had significantly increased antibody and neutralization titers post-challenge (EC₅₀ Ab = 25,094, ED₅₀ neutralization = 7096) compared to pre-challenge (EC₅₀ Ab = 4146, ED₅₀ neutralization = 518, p = 0.008 for Ab, p = 0.023 for TNA, Wilcoxon matched-pairs signed-rank test). Rabbits in the ALOH/CpG group also demonstrated significant increases in both antibody and neutralization titers detectable in post-challenge sera (EC₅₀ Ab = 14,216, ED₅₀ neutralization = 8649) compared to pre-challenge levels (EC₅₀ Ab = 2843, ED₅₀ neutralization = 395, p = 0.039 for Ab and TNA, Wilcoxon matched-pairs signed-rank test). The differences between pre- and post-challenge serum antibody and neutralization titers among survivor rabbits in the ALOH and ALOH/MPL groups and the PA (positive control) group were not significant. There were no significant differences in the post-challenge geometric mean Ab and neutralization titers between the respective adjuvant groups.

Fig. 5

In vitro inhibition of post-challenge Ab and TNA responses with LND peptide. Shown are the % reductions in antibody binding (A) and neutralization (B) for survivor rabbits from the ALOH/QS-21 and ALOH/CpG groups following in vitro incubation with inhibitory concentrations of the LND peptide as described in the Methods. The percent reductions in antibody binding and neutralization detectable in survivor sera of ALOH/CpG rabbits was significantly greater than the reductions determined in the ALOH/QS-21 group (p = 0.001 for Ab and neutralization, Students t test). A positive control LND-specific antisera and negative control PA-specific antisera were inhibited 100% and 0% respectively in both the Ab and neutralization inhibition assays (not shown). Percent reductions in antibody binding were determined through use of the following formula: % Reduction = (EC₅₀ titer with irrelevant peptide-EC₅₀ titer with LND peptide)/EC₅₀ titer with irrelevant peptide × 100. Percent reductions in neutralization were determined through use of the following formula: % Reduction = (ED₅₀ titer with irrelevant peptide-ED₅₀ titer with LND peptide)/ED₅₀ titer with irrelevant peptide × 100.