Immunogenicity in mice and non-human primates of the Group A Streptococcal J8 peptide vaccine candidate conjugated to CRM197

Abstract

Vaccine development for Group A streptococcal (GAS) infection has been extensively focused on the N-terminal hypervariable or the C-terminal conserved regions of the M protein, a major virulence factor of GAS. We evaluated the immunogenicity and functional activity of the conserved C-terminal peptide vaccine candidate, J8, conjugated to CRM197, in two mouse strains: C3H (H2(k)) and Balb/c (H2(d)), and in rhesus macaques. Mice were immunized with J8-CRM197 formulated with Amorphous Aluminum Hydroxyphosphate Sulfate Adjuvant (AAHSA), and non-human primates were immunized with J8-CRM197 formulated with AAHSA, ISCOMATRIX (TM) adjuvant, or AAHSA/ISCOMATRIX adjuvant. J8-CRM197 was immunogenic in mice from both H2(k) and H2(d) backgrounds, and the antibodies generated bound to the surface of four different GAS serotypes and had functional bacterial opsonic activity. Mice immunized with J8-CRM197/AAHSA demonstrated varying degrees of protection from lethal challenge. We also demonstrated that J8-CRM197 is immunogenic in non-human primates. Our data confirm the utility of J8 as a potential GAS vaccine candidate and demonstrate that CRM197 is an acceptable protein carrier for this peptide.

Keywords: Group A streptococcus; Streptococcus pyogenes; immunogenicity; mice; non-human primates; vaccine.

Figure 1. Serum IgG antibody responses to J8 in Balb/c and C3H mice over time. Titers from C3H and Balb/c mice immunized with J8 -CRM197/AAHSA (n = 10) at 12.5μg/dose based on specific peptide content (A). Dose titration of J8-CRM197/AAHSA in Balb/c mice (n = 3), mice were immunized with 12.5, 5, 1 and 0.1 μg /dose, based on J8 peptide content (B).

Figure 2. Photomicrographs (representative of the slide) of immunofluorescent (fluorescein isothiocyanate) - stained Group A streptococci (GAS, serotype M3) viewed under the same conditions, using a microscope with a 100X objective. Bacteria were stained with pre-immune mouse sera (A), CRM197 immune serum (B), J8-CRM197 immune serum (C) and type M3 immune serum as positive control (D). All sera were taken after the last boost and diluted 1:50.

Figure 3. Average percent opsonization of GAS M97 strain by sera taken one week after the last boost (Day 50). Filled symbols (●▲) represent two independent experiments using mouse sera taken from C3H immunized mice and open symbols (○∆) represent two independent experiments using sera taken from Balb/c mice. In all four experiments only J8-CRM197/AAHSA immune serum promoted killing of the bacteria (activity ranging from 57–93%) which was statistically significant compared with the negative control (CRM197/AAHSA or AAHSA) immune serum p < 0.001.

Figure 4. Survival of C3H and Balb/c mice after challenge. Balb/c mice challenged intraperitoneally (i.p.) with GAS pM1 SR in mucin (A) 90% of the J8-CRM197/AAHSA immunized group and 100% of the heat-killed M1 immunized group (positive control) survived challenge p < 0.0001 relative to CRM197/AAHSA and AAHSA groups. C3H mice challenged intranasally (i.n.) with GAS M3 SR (B) 70% percent of the J8-CRM197/AAHSA immunized group and 100% of the recombinant M protein immunized group (positive control) were protected from challenge, while 60% of the CRM197/MAA group also survived challenge p < 0.001 relative to the AAHSA group.

Figure 5. J8-specific serum IgG titers in rhesus macaques immunized with J8-CRM197 formulated with three different adjuvants. Antibody titers were evaluated by ELISA. Note that after the second immunization, animals that received J8-CRM197/ISCOMATRIX adjuvant (IMX) had higher anti-J8 IgG titers statistically significant compared with the group that only received J8-CRM197/AAHSAA p = 0.0331*, and the group that received J8-CRM197/ISCOMATRIX adjuvant (IMX) p = 0.0001***.