Group A Streptococcus T Antigens Have a Highly Conserved Structure Concealed under a Heterogeneous Surface That Has Implications for Vaccine Design

Abstract

Group A Streptococcus (GAS) (Streptococcus pyogenes) is an important human pathogen associated with significant global morbidity and mortality for which there is no safe and efficacious vaccine. The T antigen, a protein that polymerizes to form the backbone of the GAS pilus structure, is a potential vaccine candidate. Previous surveys of the tee gene, which encodes the T antigen, have identified 21 different tee types and subtypes such that any T antigen-based vaccine must be multivalent and carefully designed to provide broad strain coverage. In this study, the crystal structures of three two-domain T antigens (T3.2, T13, and T18.1) were determined and found to have remarkable structural similarity to the previously reported T1 antigen, despite moderate overall sequence similarity. This has enabled reliable modeling of all major two-domain T antigens to reveal that T antigen sequence variation is distributed along the full length of the protein and shields a highly conserved core. Immunoassays performed with sera from immunized animals and commercial T-typing sera identified a significant cross-reactive antibody response between T18.1, T18.2, T3.2, and T13. The existence of shared epitopes between T antigens, combined with the remarkably conserved structure and high level of surface sequence divergence, has important implications for the design of multivalent T antigen-based vaccines.

A multivalent T-antigen-based vaccine for Group A Streptococcus

“…Previous bioinformatic analysis of two-domain T-antigens revealed a highly conserved core decorated by surface variation along the full length of the protein, with no apparent dominant region 31 . We therefore designed a multivalent vaccine that included whole T-antigen domains to maximise both the inclusion of protective epitopes and protein yield.…”

“…TeeVax1 was constructed as a fusion of alternating N- and C-terminal domains from six different two-domain T-antigens to resemble a hybrid pilus fibre. Primers were designed based on the predicted boundaries for each domain generated from these models 31 . The BamHI restriction site was engineered at the 5′ end, and BglII and EcoRI restriction sites were engineered at the 3′ end of each domain, introduced by the PCR primers (Table S2 ).…”

“…The middle domain from T6, T2, T23, and T25, and the N-terminal domain from T4 were cloned successively into pPROEX-HTb. Previous structural analysis of T-antigens suggested that antigenic epitopes are found across entire surface of the T-antigen structure without any apparent dominant domain 27 , 31 . However, the N-terminal domain was chosen for T4, as previous studies on RrgB indicated that the N-terminal domain was as protective as the full-length protein 35 .…”

“…This observation is similar to those seen in the multivalent M-protein vaccines and may potentially be solved by re-iteration of the same domain at the N-terminus or addition of a protective “cap” protein at the C-terminus 41 . Response to the carboxy-terminal positioned T13 in TeeVax2 was not reduced, possibly because of shared cross-reactive epitopes with T3.2 31 ; or because full-length T13 was incorporated in this chimera, rather than a single domain, and thus may serve as its own protective cap.…”

Genetics, Structure, and Function of Group A Streptococcal Pili

“…Unlike a multivalent M protein vaccine, there are fewer T type variations. However, comparative structural analyses of three two-domain T antigens (FctA), including T3, T13, and T18, revealed that the overall core structure is conserved and variations are distributed through the entire region ( Young et al, 2019 ). Ideally, a candidate vaccine antigen would comprise a multivalent linkage of whole T antigens or domains.…”