Mechanism of Protection Induced by Group A Streptococcus Vaccine Candidate J8-DT: Contribution of B and T-Cells Towards Protection

Abstract

Vaccination with J8-DT, a leading GAS vaccine candidate, results in protective immunity in mice. Analysis of immunologic correlates of protection indicated a role of J8-specific antibodies that were induced post-immunization. In the present study, several independent experimental approaches were employed to investigate the protective immunological mechanisms involved in J8-DT-mediated immunity. These approaches included the passive transfer of mouse or rabbit immune serum/antibodies in addition to selective depletion of T-cell subsets prior to bacterial challenge. Passive transfer of J8-DT antiserum/antibodies from mice and rabbits conferred significant resistance against challenge to mice. To exclude the possibility of involvement of other host immune factors, the studies were repeated in SCID mice, which highlighted the need for an ongoing immune response for long-lived protection. Depletion of CD4+ and CD8+ T-cell subsets confirmed that an active de novo immune response, involving CD4+ T-helper cells, is required for continued synthesis of antibodies resulting in protection against GAS infection. Taken together these results indicate an involvement of CD4+ T-cells in J8-DT-mediated protection possibly via an ability to maintain antibody levels. These results have considerable relevance to the development of a broad spectrum passive immunotherapy for GAS disease.

Peptide Amphiphile Micelles Self-Adjuvant Group A Streptococcal Vaccination

“…High-pressure liquid chromatography with mass spectrometry controlled fraction collection (LCMS; Shimadzu Corp.) utilizing a reversed-phase C8 column (Waters) with a gradient of acetonitrile in Milli-Q water containing 0.1% formic acid was employed to purify J8 peptide. For J8 peptide amphiphiles, the hydrophobic moiety dipalmitoylglutamic acid (diC 16 ) was synthesized by a previously established method (34). J8 peptide was synthesized similarly to above except the C-terminal lysine was protected with DDE instead of Boc which was used for the other lysines.…”

“…J8 peptide was synthesized similarly to above except the C-terminal lysine was protected with DDE instead of Boc which was used for the other lysines. The peptide was treated with 2% hydrazine in DMF to orthogonally deprotect the Cterminal lysine amine group which was covalently coupled to diC 16 by an amidation reaction yielding J8-diC 16 peptide amphiphiles. J8-diC 16 was further processed and purified by the same methods as the J8 peptide.…”

“…To fabricate micelles, J8-diC 16 peptide amphiphiles were film cast by dissolving them in methanol and evaporating the solvent using nitrogen as a drying gas. Hydration of the films with water or phosphate-buffered saline (PBS) followed by thorough vortexing induced spontaneous micelle formation.…”

“…J8-diC 16 micelles were characterized by previously defined methodologies (33,35,36) including critical micelle concentration (CMC) analysis, transmission electron microscopy (TEM), and circular dichroism (CD). CMC was measured by fluorescent sequestration where varying concentrations of J8-diC 16 were exposed to 1 mM 1,6-diphenyl-1,3,5-hexatriene (DPH) which greatly increases in fluorescence intensity when trapped within the micelle core. Solutions were prepared and allowed to equilibrate for 1 h prior to fluorescent measurement utilizing a TECAN Infinite M200 plate reader (ex.…”

“…To enhance the corresponding host immune response, peptide vaccines are often delivered by aid of a delivery vehicle or with immune boosting substances termed adjuvants. While promising experiments have been published that employ J8 peptide delivery vehicles (14,15) or adjuvanted J8 peptide (10,16), this research has yet to lead to a commercially viable GAS vaccine, so novel systems need to be explored. An effective construct should concentrate the peptide antigen, protect it from degradation, enhance its cellular uptake, and adjuvant its immunogenicity in order to induce a robust immune response (17,18).…”