Chunwei Shi

Enhanced protection against tuberculosis by vaccination with recombinant BCG over-expressing HspX protein.

Immunization with Mycobacterium bovis Bacille Calmette-Guerin (BCG) did not induce adequate Th1 responses to the latency antigen, HspX of M. tuberculosis. To increase the immunogenicity and protective efficacy of BCG, a recombinant BCG strain over-expressing antigen HspX (rBCG::X) was constructed. The recombinant strain rBCG::X expressed high levels of both HspX protein in the cytosol and Ag85B protein in the cytosol and supernatant. Mice vaccinated with rBCG::X produced a more consistent and enduring protective effect against infection with M. tuberculosis, showing lower bacterial load in lung and less severe lung pathology, than the control mice vaccinated with BCG strain containing the vector pMV261. The long-term protection induced by rBCG::X was associated with significant increases in antigen-specific IFN-gamma to both HspX and Ag85B proteins, while PPD-specific IFN-gamma responses declined. Our results suggest that latency antigens of M. tuberculosis may be promising targets for developing more effective recombinant BCG strains to protect against TB.

Immunogenicity and Protective Efficacy against Murine Tuberculosis of a Prime-Boost Regimen with BCG and a DNA Vaccine Expressing ESAT-6 and Ag85A Fusion Protein

Heterologous prime-boost regimens utilizing BCG as a prime vaccine probably represent the best hope for the development of novel tuberculosis (TB) vaccines. In this study, we examined the immunogenicity and protective efficacy of DNA vaccine (pcD685A) expressing the fusion protein of Ag85A and ESAT-6 (r685A) and its booster effects in BCG-immunized mice. The recombinant r685A fusion protein stimulated higher level of antigen-specific IFN-γ release in tuberculin skin test- (TST-) positive healthy household contacts of active pulmonary TB patients than that in TST-negative population. Vaccination of C57BL/6 mice with pcD685A resulted in significant protection against challenge with virulent Mycobacterium tuberculosis H37Rv when compared with the control group. Most importantly, pcD685A could act as a BCG booster and amplify Th1-type cell-mediated immunity in the lung of BCG-vaccinated mice as shown the increased expression of IFN-γ. The most significant reduction in bacterial load of both spleen and lung was obtained in mice vaccinated with BCG prime and pcD685A DNA booster when compared with BCG or pcD685A alone. Thus, our study indicates that pcD685A may be an efficient booster vaccine against TB with a strong ability to enhance prior BCG immunity.

An improved whole-blood gamma interferon assay based on the CFP21-MPT64 fusion protein.

ABSTRACT Differentiation of latent tuberculosis infection (LTBI) from a healthy, unexposed population plays a vital role in the strategy of controlling and eliminating tuberculosis (TB). Both CFP21 and MPT64, antigens encoded by the RD2 region which are restricted in the Mycobacterium tuberculosis complex, are TB-specific diagnostic candidate antigens. In this study, we designed a fusion protein by linking both CFP21 and MPT64 with a 15-amino-acid peptide, (G4S1)3, and overexpressed the fusion protein in Escherichia coli. A new whole-blood gamma interferon assay based on the recombinant fusion protein, CFP21-MPT64 (rCM-WBIA), was developed and compared with the tuberculin skin test (TST) for screening of LTBI in household contacts of patients with sputum-positive TB. rCM-WBIA had a slightly higher sensitivity (66.7%; 24/36 contacts) than that of the TST (61.1%; 22/36 contacts) for household contacts. We found that rCM-WBIA had a very high sensitivity (90.9%) and specificity (71.4%) for LTBI detection compared with TST. The overall agreement between rCM-WBIA and TST was 83.3% (k = 0.64); rCM-WBIA positivity was associated with a larger TST induration. These results suggest that rCM-WBIA, based on the recombinant fusion protein CFP21-MPT64, is a promising alternative diagnostic tool for detection of LTBI.

Immunogenicity and protective efficacy of a novel recombinant BCG strain overexpressing antigens Ag85A and Ag85B.

Recombinant Bacillus Calmette-Guérin (rBCG) strain is the promising vaccine candidate for tuberculosis (TB) prevention, which aims at providing more enduring and enhanced protection than the parental BCG vaccine. In this study, three rBCG strains overexpressing immunodominant antigens Ag85B (rBCG::85B), Ag85A (rBCG::85A), or both (rBCG::AB) of Mycobacterium tuberculosis were constructed, respectively. rBCG strains showed higher level of overexpression of Ag85A and/or Ag85B proteins than BCG containing empty vector pMV261(rBCG::261), which had low levels of endogenous expression of both proteins as expected. rBCG::AB strain could provide the strongest short-term and long-term protection in the lung against intravenous infection with virulent M. tuberculosis than rBCG::261 control and other two rBCG strains overexpressing single antigen. The stronger and longer-lasting protection provided by rBCG::AB than rBCG::261 was correlated with systemic in vitro antigen-specific IFN-γ responses. Therefore, our results indicate that rBCG::AB could be a very promising TB vaccine candidate and should be further evaluated for the preclinical test.

A DNA vaccine expressing CFP21 and MPT64 fusion protein enhances BCG-induced protective immunity against Mycobacterium tuberculosis infection in mice

The efficacy of Bacillus Calmette–Guérin (BCG) vaccine in preventing adult tuberculosis (TB) is highly variable. Genetic differences between BCG vaccine substrains, which can be divided into early strains and late strains based on the loss of region of difference two (RD2), may result in the variability and BCG substrains. The effect of lack of RD2 on the protective efficacy of BCG substrains against TB remains unknown. In this study, we demonstrated that CFP21 and MPT64(rCM) fusion protein, encoded by RD2 of Mycobacterium tuberculosis, could stimulate higher level of interferon (IFN)-γ in tuberculin skin test (TST)-positive healthy population than in TST-negative healthy population. Compared with naive mice challenged with virulent M. tuberculosis H37Rv, C57BL/6 mice vaccinated with pcD2164 DNA expressing rCM protein resulted in a greater decrease in the bacterial load of lung. Moreover, pcD2164 could boost the protective immunity in mice primed by BCG than BCG alone or DNA vaccination alone, as evidenced by lower bacterial load in the lung tissue and reduced lung pathology. The protection induced by BCG prime-DNA vaccine boost strategy was associated with significant increases in rCM protein-specific IFN-γ. Therefore, our results clearly indicate that the loss of RD2 has an important influence on the protective efficacy of different BCG substrains. These findings will benefit the optimal choice of BCG substrain for neonatal immunization and rational design of new vaccines for the prevention of TB.

Enhanced and durable protective immune responses induced by a cocktail of recombinant BCG strains expressing antigens of multistage of Mycobacterium tuberculosis.

Although Bacillus Calmette-Guérin (BCG) vaccine confers protection from Mycobacterium tuberculosis infection in children, its immune protection gradually wanes over time, and consequently leads to an inability to prevent the reactivation of latent infection of M. tuberculosis. Therefore, improving BCG for better control of tuberculosis (TB) is urgently needed. We thus hypothesized that recombinant BCG overexpressing immunodominant antigens expressed at different growth stages of M. tuberculosis could provide a more comprehensive protection against primary and latent M. tuberculosis infection. Here, a novel cocktail of recombinant BCG (rBCG) strains, namely ABX, was produced by combining rBCG::85A, rBCG::85B, and rBCG::X, which overexpressed respective multistage antigens Ag85A, Ag85B, and HspX of M. tuberculosis. Our results showed that ABX was able to induce a stronger immune protection than individual rBCGs or BCG against primary TB infection in C57BL/6 mice. Mechanistically, the immune protection was attributed to stronger antigen-specific CD4(+) Th1 responses, higher numbers of IFN-γ(+) CD4(+) TEM and IL-2(+) CD8(+) TCM cells elicited by ABX. These findings thus provide a novel strategy for the improvement of BCG efficacy and potentially a promising prophylactic TB vaccine candidate, warranting further investigation.

Differential Immune Responses and Protective Effects in Avirulent Mycobacterial Strains Vaccinated BALB/c Mice

Screening live mycobacterial vaccine candidates is the important strategy to develop new vaccines against adult tuberculosis (TB). In this study, the immunogenicity and protective efficacy of several avirulent mycobacterial strains including Mycobacterium smegmatis, M. vaccae, M. terrae, M. phlei, M. trivial, and M. tuberculosis H37Ra were compared with M. bovis BCG in BALB/c mice. Our results demonstrated that differential immune responses were induced in different mycobacterial species vaccinated mice. As BCG-vaccinated mice did, M. terrae immunization resulted in Th1-type responses in the lung, as well as splenocytes secreting IFN-γ against a highly conserved mycobacterial antigen Ag85A. M. smegmatis also induced the same splenocytes secreting IFN-γ as BCG and M. terrae did. In addition, M. terrae and M. smegmatis-immunized mice predominantly increased expression of IL-10 and TGF-β in the lung. Most importantly, mice vaccinated with H37Ra and M. vaccae could provide the same protection in the lung against virulent M. tuberculosis challenge as BCG. The result may have important implications in developing adult TB vaccine.

LSECs express functional NOD1 receptors: A role for NOD1 in LSEC maturation-induced T cell immunity in vitro

HighlightsLSECs stimulated with MDP only can induce the upregulation of the co‐inhibitory molecule PD‐L1.DAP stimulation in vitro could promote LSEC maturation and activate HBV‐specific T cell responses.T cells pre‐primed by DAP‐treated LSECs can inhibit HBV expression and replication in vivo.These results are of particular relevance for the regulation of the local innate immune response against HBV infections. &NA; Liver sinusoidal endothelial cells (LSECs) are organ resident APCs capable of antigen presentation and subsequent tolerization of T cells under physiological conditions. In this study, we investigated whether LSEC pretreatment with NOD‐like receptor (NLR) agonists can switch the cells from a tolerogenic to an immunogenic state and promote the development of T cell immunity. LSECs constitutively express NOD1, NOD2 and RIPK2. Stimulation of LSECs with DAP induced the activation of NF‐&kgr;B and MAP kinases and upregulated the expression of chemokines (CXCL2/9, CCL2/7/8) and cytokines (IFN‐&ggr;, TNF‐&agr; and IL‐2). Pretreatment of LSECs with DAP induced significantly increased IFN‐&ggr; and IL‐2‐production by HBV‐stimulated CD8+ T cells primed by DAP‐treated LSECs. Consistently, a significant reduction in the HBV DNA and HBsAg level occurred in mice receiving T cells primed by DAP‐treated LSECs. MDP stimulation had no impact on LSECs or HBV‐stimulated CD8+ T cells primed with MDP‐treated LSECs except for the upregulation of PD‐L1. DAP stimulation in vitro could promote LSEC maturation and activate HBV‐specific T cell responses. These results are of particular relevance for the regulation of the local innate immune response against HBV infections.