StreptInCor: a candidate vaccine epitope against S. pyogenes infections induces protection in outbred mice

Abstract

Infection with Streptococcus pyogenes (S. pyogenes) can result in several diseases, particularly in children. S. pyogenes M protein is the major virulence factor, and certain regions of its N-terminus can trigger autoimmune sequelae such as rheumatic fever in susceptible individuals with untreated group A streptococcal pharyngitis. In a previous study, we utilized a large panel of human peripheral blood cells to define the C-terminal protective epitope StreptInCor (medical identity), which does not induce autoimmune reactions. We recently confirmed the results in HLA-transgenic mice. In the present study, we extended the experimental assays to outbred animals (Swiss mice). Herein, we demonstrate high titers of StreptInCor-specific antibodies, as well as appropriate T-cell immune responses. No cross-reaction to cardiac myosin was detected. Additionally, immunized Swiss mice exhibited 87% survival one month after challenge with S. pyogenes. In conclusion, the data presented herein reinforce previous results in humans and animals and further emphasize that StreptInCor could be an effective and safe vaccine for the prevention of S. pyogenes infections.

Figure 1. StreptInCor did not induce cross-reactive antibodies against cardiac myosin.

Each data point represents the titer for each sample, and the bar represents the mean. Specific anti-StreptInCor IgG antibodies from immunized mice (black circles) also recognized rM1 protein (black triangles) but did not react with cardiac myosin (black diamonds). Control animals received only aluminum hydroxide and did not react against StreptInCor (open circles), rM1 protein (open triangles) or cardiac myosin (open diamonds). Endpoint titers were defined as the reciprocal of the highest dilution giving an absorbance higher than two standard deviations above the mean background obtained with non-immune mice sera diluted at 1∶100. P values: *** (≤0.001); ** (≤0.01) and * (≤0.05).

Figure 2. Anti-StreptInCor specific IgG are mainly of IgG1 isotype.

Sera from Swiss mice (n = 6) immunized with 10 µg of StreptInCor adsorbed onto 60 µg of aluminum hydroxide were tested to evaluate the production of IgG isotypes against StreptInCor: IgG1 (black circles); IgG2a (black squares); IgG2b (black triangles); IgG3 (black diamonds). Each data point represents the titer for each sample, and the bar represents the mean. Negative samples: (titers <100). Endpoint titers were defined as the reciprocal of the highest dilution giving an absorbance higher than two standard deviations above the mean background obtained with non-immune mice sera diluted at 1∶100. P values: *** (≤0.001); * (≤0.05).

Figure 3. StreptInCor did not induce T-cell cross reactivity against cardiac myosin.

Splenocytes from Swiss mice (n = 6) immunized with 10 µg of StreptInCor adsorbed onto 60 µg of aluminum hydroxide (black symbols) or injected with aluminum hydroxide alone (controls, open symbols) were incubated with StreptInCor at 1 µg/mL (circles), 10 µg/mL (triangles) or cardiac myosin (diamonds). Stimulation indices ≥2.0 (dotted line) were considered positive. Each data point represents a sample, and the line represents the mean. P values: ** (≤0.01).

Figure 4. StreptInCor promotes long-lasting survival.

Swiss mice (n = 15) from three independent experiments immunized with 10 µg of StreptInCor adsorbed onto 60 µg of aluminum hydroxide (black circles) were protected against S. pyogenes infection in comparison to controls (n = 15) that received only the adjuvant (open circles). The log rank test showed that differences between StreptInCor-immunized Swiss and control groups were statistically significant (P<0.05).