Vaccine based on a ubiquitous cysteinyl protease and streptococcal pyrogenic exotoxin A protects against Streptococcus pyogenes sepsis and toxic shock

Abstract

Background: The gram-positive bacterium Streptococcus pyogenes is a common pathogen of humans that causes invasive infections, toxic-shock syndrome, rheumatic fever, necrotizing fasciitis and other diseases. Detection of antibiotic resistance in clinical isolates has renewed interest in development of new vaccine approaches for control S. pyogenes sepsis. In the study presented, a novel protein vaccine was examined. The vaccine was based on a recombinant protein fusion between streptococcal pyrogenic exotoxin B (SpeB), a cysteinyl protease expressed by all clinical isolates, and streptococcal pyrogenic exotoxin A (SpeA), a superantigen produced by a large subset of isolates.

Results: A novel protein was produced by mutating the catalytic site of SpeB and the receptor binding surface of SpeA in a fusion of the two polypeptides. Vaccination of HLA-DQ8 transgenic mice with the SpeA-SpeB fusion protein protected against a challenge with the wild-type SpeA that was lethal to naïve controls, and vaccinated mice were protected from an otherwise lethal S. pyogenes infection.

Conclusion: These results suggest that the genetically attenuated SpeA-SpeB fusion protein may be useful for controlling S. pyogenes infections. Vaccination with the SpeA-SpeB fusion protein described in this study may potentially result in protective immunity against multiple isolates of S. pyogenes due to the extensive antibody cross-reactivity previously observed among all sequence variants of SpeB and the high frequency of SpeA-producing strains.

Figure 1

Biological activity of SpeA mutants. A. Mutations of amino acid position leucine 42 of SpeA to arginine or alanine resulted in greatly diminished interactions with cell surface MHC class II molecules, measured by laser fluorescence-activated flow cytometry and FITC-labeled rabbit anti-SpeA antibody. B. Mutations of amino acid position leucine 42 of SpeA to arginine or alanine resulted in greatly diminished activation of human lymphocytes. Human T-cell proliferation was assessed by [³H]thymidine incorporation (12 h pulse) after 60 h of culture. Each data point represents the mean of triplicate determinations; SEM ≤ 5%.

Figure 2

Antibody recognition of SpeA (L42R)-SpeB (C47S) fusion protein. A. Antibody recognition in vitro. Coomassie Blue stain of isolated SpeA (L42R)-SpeB (C47S), lane 1; Western blot using-affinity purified, rabbit anti-SpeB (lane 2) or anti-SpeA antibody (lane 3). B. Antibody response and recognition in vivo. Mice (BALB/c) were vaccinated three times with 10 μg of each protein and adjuvant (MPL), allowing two weeks between injections. Sera from each experimental group (n = 5) were pooled for measurement of specific antibodies. Data shown are antigen-specific antibodies (ELISA units) present in a 1:100,000 dilution of pooled sera from mice vaccinated with SpeA (L42R), SpeA (L42R)-SpeB (C47S) fusion or adjuvant only.

Figure 3

Protection of transgenic HLA-DQ8 mice from Streptococcus pyogenes sepsis following vaccination with SpeA (L42R)-SpeB (C47S) fusion protein. Mice (5 per group) were vaccinated three times with 10 μg of each protein with adjuvant (MPL), allowing two weeks between injections. Three weeks after the last vaccination the mice were injected (i.v) with 10 LD₅₀ of S. pyogenes and survival was monitored for 10 days.