Identification of Mycobacterial Ribosomal Proteins as Targets for CD4+ T Cells That Enhance Protective Immunity in Tuberculosis.

Mycobacterium tuberculosis remains a threat to global health, and a more efficacious vaccine is needed to prevent disease caused by M. tuberculosis We previously reported that the mycobacterial ribosome is a major target of CD4+ T cells in mice immunized with a genetically modified Mycobacterium smegmatis strain (IKEPLUS) but not in mice immunized with Mycobacterium bovis BCG. Two specific ribosomal proteins, RplJ and RpsA, were identified as cross-reactive targets of M. tuberculosis, but the breadth of the CD4+ T cell response to M. tuberculosis ribosomes was not determined. In the present study, a library of M. tuberculosis ribosomal proteins and in silico-predicted peptide libraries were used to screen CD4+ T cell responses in IKEPLUS-immunized mice. This identified 24 out of 57 M. tuberculosis ribosomal proteins distributed over both large and small ribosome subunits as specific CD4+ T cell targets. Although BCG did not induce detectable responses against ribosomal proteins or peptide epitopes, the M. tuberculosis ribosomal protein RplJ produced a robust and multifunctional Th1-like CD4+ T cell population when administered as a booster vaccine to previously BCG-primed mice. Boosting of BCG-primed immunity with the M. tuberculosis RplJ protein led to significantly reduced lung pathology compared to that in BCG-immunized animals and reductions in the bacterial burdens in the mediastinal lymph node compared to those in naive and standard BCG-vaccinated mice. These results identify the mycobacterial ribosome as a potential source of cryptic or subdominant antigenic targets of protective CD4+ T cell responses and suggest that supplementing BCG with ribosomal antigens may enhance protective vaccination against M. tuberculosis.

Identification of Mycobacterial RplJ/L10 and RpsA/S1 Proteins as Novel Targets for CD4+ T Cells.

Tuberculosis (TB) due to Mycobacterium tuberculosis remains a major global infectious disease problem, and a more efficacious vaccine is urgently needed for the control and prevention of disease caused by this organism. We previously reported that a genetically modified strain of Mycobacterium smegmatis called IKEPLUS is a promising TB vaccine candidate. Since protective immunity induced by IKEPLUS is dependent on antigen-specific CD4+ T cell memory, we hypothesized that the specificity of the CD4+ T cell response was a critical feature of this protection. Using in vitro assays of interferon gamma production (enzyme-linked immunosorbent spot [ELISPOT] assays) by splenocytes from IKEPLUS-immunized C57BL/6J mice, we identified an immunogenic peptide within the mycobacterial ribosomal large subunit protein RplJ, encoded by the Rv0651 gene. In a complementary approach, we generated major histocompatibility complex (MHC) class II-restricted T cell hybridomas from IKEPLUS-immunized mice. Screening of these T cell hybridomas against IKEPLUS and ribosomes enriched from IKEPLUS suggested that the CD4+ T cell response in IKEPLUS-immunized mice was dominated by the recognition of multiple components of the mycobacterial ribosome. Importantly, CD4+ T cells specific for mycobacterial ribosomes accumulate to significant levels in the lungs of IKEPLUS-immunized mice following aerosol challenge with virulent M. tuberculosis, consistent with a role for these T cells in protective host immunity in TB. The identification of CD4+ T cell responses to defined ribosomal protein epitopes expands the range of antigenic targets for adaptive immune responses to M. tuberculosis and may help to inform the design of more effective vaccines against tuberculosis.

Identification of Novel Mycobacterial Targets for Murine CD4+ T-Cells by IFNγ ELISPOT.

Enzyme-linked immunospot (ELISPOT) is an assay used to detect secretion of cytokines from immune cells. The resolution and sensitivity of ELISPOT allow for the detection of rare T cell specificities and small quantities of molecules produced by individual cells. In this chapter, we describe an epitope screening method that uses CD4+ T cell ELISPOT assays to identify specific novel mycobacterial antigens as potential vaccine candidates. In order to screen a large number of candidate epitopes simultaneously, pools of predicted MHC class II peptides were used to identify mycobacterial specific CD4+ T cells. Using this method, we identified novel mycobacterial antigens as vaccine candidates.

Suppression of autophagy and antigen presentation by Mycobacterium tuberculosis PE_PGRS47.

Suppression of major histocompatibility complex (MHC) class II antigen presentation is believed to be among the major mechanisms used by Mycobacterium tuberculosis to escape protective host immune responses. Through a genome-wide screen for the genetic loci of M. tuberculosis that inhibit MHC class II-restricted antigen presentation by mycobacteria-infected dendritic cells, we identified the PE_PGRS47 protein as one of the responsible factors. Targeted disruption of the PE_PGRS47 (Rv2741) gene led to attenuated growth of M. tuberculosis in vitro and in vivo, and a PE_PGRS47 mutant showed enhanced MHC class II-restricted antigen presentation during in vivo infection of mice. Analysis of the effects of deletion or over-expression of PE_PGRS47 implicated this protein in the inhibition of autophagy in infected host phagocytes. Our findings identify PE_PGRS47 as a functionally relevant, non-redundant bacterial factor in the modulation of innate and adaptive immunity by M. tuberculosis, suggesting strategies for improving antigen presentation and the generation of protective immunity during vaccination or infection.

Current efforts and future prospects in the development of live mycobacteria as vaccines.

The development of more effective vaccines against Mycobacterium tuberculosis (Mtb) remains a major goal in the effort to reduce the enormous global burden of disease caused by this pathogen. Whole-cell vaccines based on live mycobacteria with attenuated virulence represent an appealing approach, providing broad antigen exposure and intrinsic adjuvant properties to prime durable immune responses. However, designing vaccine strains with an optimal balance between attenuation and immunogenicity has proven to be extremely challenging. Recent basic and clinical research efforts have broadened our understanding of Mtb pathogenesis and created numerous new vaccine candidates that have been designed to overcome different aspects of immune evasion by Mtb. In this review, we provide an overview of the current efforts to create improved vaccines against tuberculosis based on modifications of live attenuated mycobacteria. In addition, we discuss the use of such vaccine strains as vectors for stimulating protective immunity against other infectious diseases and cancers.

Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays.

The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs.