Sushila D'Souza

Improved Immunogenicity and Protective Efficacy of a Tuberculosis DNA Vaccine Encoding Ag85 by Protein Boosting

ABSTRACT C57BL/6 mice were vaccinated with plasmid DNA encoding Ag85 fromMycobacterium tuberculosis, with Ag85 protein in adjuvant, or with a combined DNA prime-protein boost regimen. While DNA immunization, as previously described, induced robust Th1-type cytokine responses, protein-in-adjuvant vaccination elicited very poor cytokine responses, which were 10-fold lower than those observed with DNA immunization alone. Injection of Ag85 DNA-primed mice with 30 to 100 μg of purified Ag85 protein in adjuvant increased the interleukin-2 and gamma interferon (IFN-γ) response in spleen two- to fourfold. Further, intracellular cytokine analysis by flow cytometry also showed an increase in IFN-γ-producing CD4+ T cells in DNA-primed–protein-boosted animals, compared to those that received only the DNA vaccination. Moreover, these responses appeared to be better sustained over time. Antibodies were readily produced by all three methods of immunization but were exclusively of the immunoglobulin G1 (IgG1) isotype following protein immunization in adjuvant and preferentially of the IgG2a isotype following DNA and DNA prime-protein boost vaccination. Finally, protein boosting increased the protective efficacy of the DNA vaccine against an intravenousM. tuberculosis H37Rv challenge infection, as measured by CFU or relative light unit counts in lungs 1 and 2 months after infection. The capacity of exogenously given protein to boost the DNA-primed vaccination effect underlines the dominant role of Th1-type CD4+ helper T cells in mediating protection.

Mapping of murine Th1 helper T-cell epitopes of mycolyl transferases Ag85A, Ag85B, and Ag85C from Mycobacterium tuberculosis

ABSTRACT BALB/c (H-2d) and C57BL/6 (H-2b) mice were infected intravenously with Mycobacterium tuberculosis H37Rv or vaccinated intramuscularly with plasmid DNA encoding each of the three mycolyl transferases Ag85A, Ag85B, and Ag85C from M. tuberculosis. Th1-type spleen cell cytokine secretion of interleukin-2 (IL-2) and gamma interferon (IFN-γ) was analyzed in response to purified Ag85 components and synthetic overlapping peptides covering the three mature sequences. Tuberculosis-infected C57BL/6 mice reacted strongly to some peptides from Ag85A and Ag85B but not from Ag85C, whereas tuberculosis-infected BALB/c mice reacted only to peptides from Ag85A. In contrast, spleen cells from both mouse strains produced elevated levels of IL-2 and IFN-γ following vaccination with Ag85A, Ag85B, and Ag85C DNA in response to peptides of the three Ag85 proteins, and the epitope repertoire was broader than in infected mice. Despite pronounced sequence homology, a number of immunodominant regions contained component specific epitopes. Thus, BALB/c mice vaccinated with all three Ag85 genes reacted against the same amino acid region, 101 to 120, that was also immunodominant for Ag85A in M. bovis BCG-vaccinated and tuberculosis-infected H-2d haplotype mice, but responses were completely component specific. In C57BL/6 mice, a cross-reactive T-cell response was detected against two carboxy-terminal peptides spanning amino acids 241 to 260 and 261 to 280 of Ag85A and Ag85B. These regions were not recognized at all in C57BL/6 mice vaccinated with Ag85C DNA. Our results underline the need for comparative analysis of all three Ag85 components in future vaccination studies.

Lack of Protection in Mice and Necrotizing Bronchointerstitial Pneumonia with Bronchiolitis in Guinea Pigs Immunized with Vaccines Directed against the hsp60 Molecule of Mycobacterium tuberculosis

ABSTRACT In this study, the hsp60 and hsp70 heat shock protein antigens ofMycobacterium tuberculosis were tested as potential vaccine candidates, using purified recombinant protein antigens or antigens encoded in the form of a DNA plasmid vaccine. Guinea pigs vaccinated with a mixture of the two proteins showed no evidence of resistance to low-dose aerosol challenge infection and quickly developed severe lung damage characterized by necrotizing bronchointerstitial pneumonia and bronchiolitis. As a result, we turned instead to a DNA vaccination approach using a plasmid encoding the hsp60 antigen of M. tuberculosis. Although immunogenic in mice, vaccination with plasmid DNA encoding hsp60 was not protective in that model or in the guinea pig model and again gave rise to similar severe lung damage. This study seriously questions the safety of vaccines against tuberculosis that target highly conserved heat shock proteins.

Comparative Analysis of Different Vaccine Constructs Expressing Defined Antigens from Mycobacterium tuberculosis

BACKGROUND Studies of different vaccine constructs have demonstrated variable efficacy against Mycobacterium tuberculosis in animal models. Despite the fact that these vaccines have used one or another of a very small number of immunodominant antigens, a direct comparison of the relative efficacy of the antigens and delivery systems has been difficult, because the studies have used different parameters for assessment. METHODS We compared the efficacies of the most commonly used vaccine constructs--adjuvanted protein, plasmid DNA, and live bacterial vectors--bearing the immunodominant secreted antigens early secreted antigen target-6 and antigen 85B, either alone or as a fusion protein. Mice were vaccinated with these constructs, and the effects of different delivery systems on protective efficacy (as assessed by survival studies and by monitoring bacterial load) and antigen-specific responses (including the contribution of CD4 and CD8 T cells to these responses) were assayed by various methods. RESULTS The relative efficacy of different vaccines is dependent on the delivery system, the antigen, and the animal model. Likewise, the relative immunodominance of individual antigens in the fusion molecule is altered by the choice of delivery system. CONCLUSION These results clearly demonstrate the importance of assessing vaccine function by use of multiple parameters and indicate which parameters are most reliable for assessing vaccine efficacy.

CD4+ T cells contain Mycobacterium tuberculosis infection in the absence of CD8+ T cells in mice vaccinated with DNA encoding Ag85A.

The contribution of CD8+ and CD4+ T cell‐mediated effector functions against Mycobacterium tuberculosis infection elicited by i.m. vaccination with plasmid DNA encoding the immunodominant Ag85A antigen of M. tuberculosis was studied. Ag85A DNA‐vaccinated β2‐microglobulin gene‐deficient (β2m–/–) mice, which lack CD8+ T cells, produced Ag85‐specific antibodies and Th1 type cytokines similar to wild‐type mice. Although β2m–/– mice were more susceptible to M. tuberculosis infection, following vaccination they efficiently controlled bacterial replication in spleen and lungs 4 weeks post‐infection. In contrast, mice lacking CD4+ T cells were neither sensitized by the Ag85A DNA vaccine to produce Ag85‐specific antibodies or Th1 type cytokines nor did they contain a M. tuberculosis challenge infection. In addition, Ag85A DNA‐vaccinated IFN‐γ gene knockout mice produced Ag85‐specific antibodies and IL‐2 but died rapidly following a M. tuberculosis challenge infection. Collectively, these data support the view that IFN‐γ‐producing CD4+ T cells, independently of CD8+ T cells, may mediate the protective effect of the Ag85A DNA vaccine.

Functional characterization of in vivo effector CD4+ and CD8+ T cell responses in acute Toxoplasmosis: An interplay of IFN-γ and cytolytic T cells

Development of prophylactic vaccines against Toxoplasma gondii is based on the observation that latently infected subjects are protected against secondary infection during pregnancy. Cocktail DNA vaccines have been shown to provide high resistance to parasite challenge, and latently infected mice are protected against acute disease. In order to characterize the associated Th1 cellular immune responses in vivo, we used H2-K(k) bone marrow macrophage cell lines constitutively expressing T. gondii GRA1, GRA7 or ROP2 antigens, for the in vivo characterization of antigen-specific T cells in an antigenic challenge model, and as target cells in an in vivo CTL assay. In latently infected C3H/HeN mice, CD4(+) and CD8(+) T cells were recruited to the peritoneal cavity after i.p. challenge with these syngeneic cell lines. GRA1 and GRA7-specific T cells from infected mice were IFN-gamma(+) FasL(-) CD107(-). No IFN-gamma or lytic markers were observed against ROP2. In cocktail DNA vaccinated C3H/HeN mice, the response was restricted to GRA1-specific CD8(+) IFN-gamma(-) FasL(-) CD107(+) T cells. Target cells expressing GRA1 and GRA7, but not ROP2, were efficiently killed in an in vivo CTL assay in latently infected mice, while in DNA vaccinated mice only lysis of GRA1 expressing target cells was observed. Both forms of immunization, DNA vaccination and latent infection, completely protected mice against acute Toxoplasmosis. The results obtained in this work suggest that distinct in vivo cytolytic effector mechanisms are at work in DNA vaccinated and latently infected mice, but both converge to protect against acute toxoplasmosis.

Induction of in vivo functional Db-restricted cytolytic T cell activity against a putative phosphate transport receptor of Mycobacterium tuberculosis.

Using plasmid vaccination with DNA encoding the putative phosphate transport receptor PstS-3 from Mycobacterium tuberculosis and 36 overlapping 20-mer peptides spanning the entire PstS-3 sequence, we determined the immunodominant Th1-type CD4+ T cell epitopes in C57BL/10 mice, as measured by spleen cell IL-2 and IFN-γ production. Furthermore, a potent IFN-γ-inducing, Db-restricted CD8+ epitope was identified using MHC class I mutant B6.C-H-2bm13 mice and intracellular IFN-γ and whole blood CD8+ T cell tetramer staining. Using adoptive transfer of CFSE-labeled, peptide-pulsed syngeneic spleen cells from naive animals into DNA vaccinated or M. tuberculosis-infected recipients, we demonstrated a functional in vivo CTL activity against this Db-restricted PstS-3 epitope. IFN-γ ELISPOT responses to this epitope were also detected in tuberculosis-infected mice. The CD4+ and CD8+ T cell epitopes defined for PstS-3 were completely specific and not recognized in mice vaccinated with either PstS-1 or PstS-2 DNA. The H-2 haplotype exerted a strong influence on immune reactivity to the PstS-3 Ag, and mice of the H-2b, p, and f haplotype produced significant Ab and Th1-type cytokine levels, whereas mice of H-2d, k, r, s, and q haplotype were completely unreactive. Low responsiveness against PstS-3 in MHC class II mutant B6.C-H-2bm12 mice could be overcome by DNA vaccination. IFN-γ-producing CD8+ T cells could also be detected against the Db-restricted epitope in H-2p haplotype mice. These results highlight the potential of DNA vaccination for the induction and characterization of CD4+ and particularly CD8+ T cell responses against mycobacterial Ags.

Evaluation of the immune response induced by DNA vaccine cocktail expressing complete SAG1 and ROP2 genes against toxoplasmosis

Toxoplasma gondii, the pathogen of toxoplasmosis, can infect most mammals and birds. The high incidence and severe or lethal damages of toxoplasmosis clearly indicate the need for the development of a more effective vaccine. We constructed a DNA cocktail, containing plasmids encoding the full-length SAG1 and ROP2 genes of T. gondii and evaluated its immune response and protective efficacy in comparison with single-gene vaccines and control groups. We immunized BALB/c mice intramuscularly three times. DNA cocktail elicited IgG and IFN-γ, TNF-α and IL-2 greater than single-gene plasmids and increased survival time against a lethal challenge with the highly virulent T. gondii RH strain. The current study shows that pc-SAG1+ pc-ROP2 as a cocktail DNA vaccine produces higher Th1 immune response than single-gene plasmids and cocktail DNA is effective to prime an enhanced and balanced specific immunity.

Partial Reconstitution of the CD4+-T-Cell Compartment in CD4 Gene Knockout Mice Restores Responses to Tuberculosis DNA Vaccines

ABSTRACT Reactivation tuberculosis (TB) is a serious problem in immunocompromised individuals, especially those with human immunodeficiency virus (HIV) coinfection. The adaptive immune response mediated by CD4+ and CD8+ T cells is known to confer protection against TB. Hence, vaccines against TB are designed to activate these two components of the immune system. Anti-TB DNA vaccines encoding the immunodominant proteins Ag85A, Ag85B, and PstS-3 from Mycobacterium tuberculosis are ineffective in mice lacking CD4+ T cells (CD4−/− mice). In this study, we demonstrate that reconstitution of the T-cell compartment in CD4−/− mice restores vaccine-specific antibody and gamma interferon (IFN-γ) responses to these DNA vaccines. The magnitude of the immune responses correlated with the extent of reconstitution of the CD4+-T-cell compartment. Reconstituted mice vaccinated with DNA encoding PstS-3, known to encode a dominant Db-restricted CD8+-T-cell epitope, displayed CD8+-T-cell responses not observed in CD4−/− mice. M. tuberculosis challenge in reconstituted mice led to the extravasation of IFN-γ-producing CD4+ and CD8+ T cells into lungs, the primary site of bacterial replication. Importantly, a reconstitution of 12 to 15% of the CD4+-T-cell compartment resulted in Ag85B plasmid DNA-mediated protection against a challenge M. tuberculosis infection. Our findings provide evidence that anti-TB DNA vaccines could be effective in immunodeficient individuals after CD4+-T-lymphocyte reconstitution, as may occur following antiretroviral therapy in HIV+ patients.

Culture filtrate specific H-2b restricted CD8+ T cells activated in vivo by Mycobacterium tuberculosis or bovis BCG recognize a restricted number of immunodominant peptides

We previously demonstrated that Bacillus Calmette-Guerin (BCG) immunization activated D(b) restricted CD8+ cytolytic T lymphocyte (CTL) recognizing target cells incubated with mycobacterial culture filtrate. Here, we show that in vitro restimulation of spleen cells from BCG vaccinated or Mycobacterium tuberculosis infected mice with culture filtrate antigens leads to the appearance of a high percentage of D(b) restricted IFNgamma synthesizing CD8+ T cell blasts. Transporter associated protein-2 mutated RMA-S cells incubated with soluble culture filtrate proteins had their MHC class I D(b) but not K(b) molecules stabilized at the surface indicating that only D(b) ligands might be generated by antigen presenting cells. MHC class I bound peptides were acid eluted from the surface of RMA-S cells incubated with M. tuberculosis culture filtrate proteins. The crude peptide preparation was able to sensitize RMA-S cells for recognition by culture filtrate-specific cytolytic T cells. Peptides were subsequently fractionnated by reverse-phase high performance liquid chromatography and the main biological activity was identified in two fractions. These results provide a further evidence that the processing of exogenous culture filtrate proteins in vitro leads to the presentation of a restricted number or even a single immunodominant peptide to culture filtrate-specific CD8+ T cells.