Mapping the minimal murine T cell and B cell epitopes within a peptide vaccine candidate from the conserved region of the M protein of group A streptococcus

Abstract

The highly conserved C-terminus of the M protein of group A streptococcus (GAS) is a promising vaccine candidate. An epitope within the conserved C-terminus of the M protein, peptide 145 (a 20-mer with the sequence: LRRDLDASREAKKQVEKALE), has been defined which is the target of opsonic antibodies in both humans and mice, and is recognized by the sera of most adults living in areas of high streptococcal exposure. However, due to potential cross-reactivity between T cells stimulated by this region of the M protein and host cardiac myosin, it is critical to define precisely the minimal protective epitopes within p145. Studies have shown that the immunodominant epitope expressed by p145 is conformational, occurring as an alpha-helical coiled-coil. To enable us to map the murine minimal B cell and T cell epitopes within p145, we have used a novel strategy that allowed us to present shorter sequences of p145 in a native-like conformation. The minimal B cell epitope was found to be contained within residues 7-20 of the p145 sequence, and we have shown that mice immunized with this region are able to generate antibodies that bind to and also opsonize the organism GAS. The T cell epitope is located at the N-terminal region of the p145 sequence, residues 3-14. We have managed, therefore, to define a vaccine candidate--a minimal opsonic B cell epitope within the p145 sequence--that does not incorporate a potentially deleterious T cell epitope.

An efficient, chemically-defined semisynthetic lipid-adjuvanted nanoparticulate vaccine development system

“…PCR-based Gene Synthesis 7 to Generate GAS Polytope Gene. The polytope protein sequence (Table S1) was designed to incorporate: 1) a thrombin cleavable Nterminal hexahistidine tag, 2) seven GAS M protein N-terminal peptide sequences, 8 3) a sequence from the conserved C-terminal GAS M protein segment (p145, underlined) fused with yeast GCN4 sequences to promote helicity (yielding the J14 peptide), 9 and 4) a PADRE1024.03 universal T-helper epitope 10 (sequences in Table S1). A di-lysine spacer was used to separate each component.…”

“…Figure 1, A; called lipid core peptides (LCPs)), has been demonstrated to provide adjuvant activity towards a variety of peptide antigens, 5 leading to antibody--mediated (mucosal and systemic) 6 and cellular immunity 7 following parenteral or mucosal administration. 8 This adjuvant activity at least partially involves signaling through Toll--like receptor 2 (TLR2), 9 through which bacterial lipopeptides also signal. 3 In order to develop the aforementioned lipopeptide vaccines, protective peptide epitopes must be mapped for the disease of interest.…”

“…ELISA was performed essentially as described, using sera from individual mice. 9 Peptide antigen stock solutions (5 µg/mL) were made in carbonate coating buffer (66 mM bicarbonate/carbonate pH 9.6) and used to coat polyvinylchloride (PVC) 96-well microtiter plates (100 µL/well; 0.5 µg/well) at 4 °C for 16 hours. The antigen solutions were then removed, and the wells blocked with 5 % (w/v) skim milk in PBS-T (0.05 % (v/v) tween-20, PBS) (150 µL/well) at 37 °C for 1.5 h. The plates were thoroughly washed (3× ddH 2 O, 2× PBS-T), and twofold serial dilutions of sera in 0.5 % (w/v) skim milk in PBS-T (starting at 1:100; 100 µL/well) produced.…”

Comparison of Fluorinated and Nonfluorinated Lipids in Self-Adjuvanting Delivery Systems for Peptide-Based Vaccines

“…25 The secondary structure of the vaccine candidates 12−15 was measured using the circular dichroism (CD) analysis. Minima at approximately 205 and 222 nm correlate with the formation of α-helicies (Figure 2e).…”

JJo, a Recombinant Dimer of Conformationally Restricted Peptide Elicits Protective Response against Group A Streptococcus (GAS) Isolates from a GAS-Endemic Region

“…The amino terminus is highly polymorphic and is the basis for typing while the carboxyl terminus including the C-repeat region (CRR) is highly conserved among GAS isolates [5]. Comprehensive dissection of the CRR has resulted in the identification of protective opsonic B cell epitopes that are void of cross reactive B-and T-cell epitopes; hence these B cell epitopes are regarded as safe vaccine candidates [6]. These peptides however, are conformationally restricted.…”