DNA vaccine with discontinuous T‐cell epitope insertions into HSP65 scaffold as a potential means to improve immunogenicity of multi‐epitope Mycobacterium tuberculosis vaccine

Abstract

DNA-based vaccine is a promising candidate for immunization and induction of a T-cell-focused protective immune response against infectious pathogens such as Mycobacterium tuberculosis (M. tb). To induce multi-functional T response against multi-TB antigens, a multi-epitope DNA vaccine and a 'protein backbone grafting' design method is adopted to graft five discontinuous T-cell epitopes into HSP65 scaffold protein of M. tb for enhancement of epitope processing and immune presentation. A DNA plasmid with five T-cell epitopes derived from ESAT-6, Ag85B, MTB10.4, PPE25 and PE19 proteins of H37Rv strain of M. tb genetically inserted into HSP65 backbone was constructed and designated as pPES. After confirmation of its in vitro expression efficiency, pPES DNA was i.m. injected into C57BL/6 mice with four doses of 50 µg DNA followed by mycobacterial challenge 4 weeks after the final immunization. It was found that pPES DNA injection maintained the ability of HSP65 backbone to induce specific serum IgG. ELISPOT assay demonstrated that pPES epitope-scaffold construct was significantly more potent to induce IFN-γ(+) T response to five T-cell epitope proteins than other DNA constructs (with epitopes alone or with epitope series connected to HSP65), especially in multi-functional-CD4(+) T response. It also enhanced granzyme B(+) CTL and IL-2(+) CD8(+) T response. Furthermore, significantly improved protection against Mycobacterium bovis BCG challenge was achieved by pPES injection compared to other DNA constructs. Taken together, HSP65 scaffold grafting strategy for multi-epitope DNA vaccine represents a successful example of rational protein backbone engineering design and could prove useful in TB vaccine design.

Protective Vaccine Efficacy of the Complete Form of PPE39 Protein from Mycobacterium tuberculosis Beijing/K Strain in Mice

“…In addition, PE/PPE family proteins have highly immunogenic T-cell epitopes that induce secretion of gamma interferon (IFN-␥) (29,30). A multiepitope DNA vaccine, including peptides derived from PE19 and PPE25, induces potent IFN-␥ responses (31).…”

Vaccination with a Paramyosin-Based Multi-Epitope Vaccine Elicits Significant Protective Immunity against Trichinella spiralis Infection in Mice

“…As CD4 + T cells are crucial in determining the functional status of both innate and adaptive immune responses, it is essential to comprise appropriate CD4 + T cell epitopes to improve the vaccine efficacy ( Rosa et al, 2010 ). Many studies have shown that protective efficacy can be significantly improved by including an array of promiscuous T cell epitopes in vaccine constructs ( Wen et al, 2014 ; Wu et al, 2016 ). Meanwhile, protective B-cell epitopes are also essential for developing epitope-based vaccines ( Zhao et al, 2015 ; Sharma and Dixit, 2016 ).…”

Intranasal Vaccination with Mannosylated Chitosan Formulated DNA Vaccine Enables Robust IgA and Cellular Response Induction in the Lungs of Mice and Improves Protection against Pulmonary Mycobacterial Challenge

“…M. tuberculosis H37Rv strain was provided by Fifth People's Hospital of Suzhou and ELISPOT assay using inactivated H37Rv was conducted in ABSL II facility. Recombinant DNA construct, pPES, with 5 T-cell-epitopes from H37Rv (MTB10.4 3–11 , ESAT-6 1–20 , Ag85B 241–255 , PPE25 241–255 , and PE19 4–18 ) grafted into HSP65 scaffold was prepared by us as previously reported (Wu et al, 2016 ). Peptides with sequences (ESAT-6 1–20 , MTEQQWNFAGIEAAASAIQG; Ag85B 241–255 , QDAYNAAGGHNAVFN; MTB10.4 3–11 , QIMYNYPAM; PPE25 241–255 , AQFFASIAQQLTFGP; PE19 4–18 , VTTQPEALAAAAANL) were synthesized by GL Biochem Corp (Shanghai, China) with purity over 95%.…”

“…Peptides with sequences (ESAT-6 1–20 , MTEQQWNFAGIEAAASAIQG; Ag85B 241–255 , QDAYNAAGGHNAVFN; MTB10.4 3–11 , QIMYNYPAM; PPE25 241–255 , AQFFASIAQQLTFGP; PE19 4–18 , VTTQPEALAAAAANL) were synthesized by GL Biochem Corp (Shanghai, China) with purity over 95%. HSP65 protein was prokaryotically expressed and purified as previously described (Wu et al, 2016 ). Mannosylated chitosan (MCS) was synthesized from chitosan (CS, MW 390,000; Sigma) by Tianjin Chemsyntech Chemical Technology Co. Ltd. according to the reported procedures (Nanda et al, 2014 ).…”

“…Therefore, immunization via mucosal routes to evoke SIgA and a poly-functional M.tb specific T cell immune response in the lung is of great significance for the establishment of protective pulmonary immunity against TB. In this regard, our group has developed a cationic polysaccharides chitosan delivered DNA construct carrying multi-T epitopes grafted into HSP65 scaffold (pPES) which leads to enhanced induction of pulmonary immunity and anti-TB protection (Ai et al, 2013 ; Wu et al, 2016 ).…”