Sheldon L. Morris

Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major.

CD4+ T cells have a crucial role in mediating protection against a variety of pathogens through production of specific cytokines. However, substantial heterogeneity in CD4+ T-cell cytokine responses has limited the ability to define an immune correlate of protection after vaccination. Here, using multiparameter flow cytometry to assess the immune responses after immunization, we show that the degree of protection against Leishmania major infection in mice is predicted by the frequency of CD4+ T cells simultaneously producing interferon-γ, interleukin-2 and tumor necrosis factor. Notably, multifunctional effector cells generated by all vaccines tested are unique in their capacity to produce high amounts of interferon-γ. These data show that the quality of a CD4+ T-cell cytokine response can be a crucial determinant in whether a vaccine is protective, and may provide a new and useful prospective immune correlate of protection for vaccines based on T-helper type 1 (TH1) cells.

Compensatory ahpC gene expression in isoniazid-resistant Mycobacterium tuberculosis.

Mutations that eliminate KatG catalase-peroxidase activity prevent activation of isoniazid and are a major mechanism of resistance to this principal drug for the treatment of Mycobacterium tuberculosis infections. However, the loss of KatG activity in clinical isolates seemed paradoxical because KatG is considered an important factor for the survival of the organism. Expression of either KatG or the recently identified alkyl hydroperoxidase AhpC was sufficient to protect bacilli against the toxic effects of organic peroxides. To survive during infection, isoniazid-resistant KatG mutants have apparently compensated for the loss of KatG catalase-peroxidase activity by a second mutation, resulting in hyperexpression of AhpC.

Expression of katG in Mycobacterium tuberculosis Is Associated with Its Growth and Persistence in Mice and Guinea Pigs

The molecular mechanisms associated with the pathogenesis of tuberculosis are not well understood. The present study evaluated the role of catalase-peroxidase as a potential virulence factor for Mycobacterium tuberculosis. Growth and persistence of M. tuberculosis H37Rv in intravenously infected BALB/ c mice were compared with katG-deleted, isoniazid-resistant M. tuberculosis H37RVINHR. Transformation of M. tuberculosis H37Rv (TBkatG) or Mycobacterium intracellulare (MACkatG) genes into M. tuberculosis H37RvINHR restored its catalase-peroxidase activities and the ability of the recombinants to persist in spleens of mice and guinea pigs. Transformation with the TBkatG gene with the codon 463 R-->L mutation also restored catalase-peroxidase activity and enhanced persistence. However, transformants with the codon 275 T-->P mutant expressed low levels of enzymatic activity and failed to persist in guinea pig spleen, although they did survive in mouse tissues. These results indicate that KatG contributes to the ability of M. tuberculosis to grow and survive within the infected host tissues.

The ESAT6 protein of Mycobacterium tuberculosis induces apoptosis of macrophages by activating caspase expression.

The secreted Mycobacterium tuberculosis protein, ESAT6, has been studied extensively in pathogenicity and vaccine experiments. Despite these studies little is known about the function of this protein. In this report, we demonstrate that ESAT6 induces apoptosis in THP‐1 human macrophages using fluorescein isothiocyanate‐Annexin V and intracellular caspase staining. We show that the induction of apoptosis by ESAT6 is dependent on the dose of the protein and the expression of caspase genes. Using real‐time RT‐PCR, we found that expression of caspase‐1, ‐3, ‐5, ‐7 and ‐8 genes was upregulated in cells treated with ESAT6 relative to untreated cells. Furthermore, we show that while infection of THP‐1 cells with wild‐type M. tuberculosis strain H37Rv resulted in significant apoptosis 48 h post infection, a deletion mutant that does not express ESAT6 failed to induce significant apoptosis. Finally, experimental results using a cell impermeable fluorescent stain suggests that the formation of membrane pores may be a primary mechanism by which ESAT6 evokes an apoptotic response.

Site-directed mutagenesis of the katG gene of Mycobacterium tuberculosis: effects on catalase-peroxidase activities and isoniazid resistance.

Recent studies examining the molecular mechanisms of isoniazid (INH) resistance in Mycobacterium tuberculosis have demonstrated that a significant percentage of drug‐resistant strains are mutated in the katG gene which encodes a catalase–peroxidase, and the majority of these alterations are missense mutations which result in the substitution of a single amino acid. In previous reports, residues which may be critical for enzymatic activity and the drug‐resistant phenotype have been identified by evaluating INH‐resistant clinical isolates and in vitro mutants. In this study, site‐directed mutagenesis techniques were utilized to alter the wild‐type katG gene from M. tuberculosis at 13 of these codons. The effects of these mutations were determined using complementation assays in katG‐defective, INH‐resistant strains of Mycobacterium smegmatis and Mycobacterium bovis BCG. This mutational analysis revealed that point mutations in the katG gene at nine of the 13 codons can cause drug resistance, and that enzymatic activity and resistance to INH are inversely related. In addition, mutations in the mycobacterial catalase–peroxidase which reduce catalase activity also decrease peroxidase activity.

Novel recombinant BCG expressing perfringolysin O and the over-expression of key immunodominant antigens; pre-clinical characterization, safety and protection against challenge with Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has infected approximately two billion individuals worldwide with approximately 9.2 million new cases and 1.6 million deaths annually. Current efforts are focused on making better BCG priming vaccines designed to induce a comprehensive and balanced immunity followed by booster(s) targeting a specific set of relevant antigens in common with the BCG prime. We describe the generation and immunological characterization of recombinant BCG strains with properties associated with lysis of the endosome compartment and over-expression of key Mtb antigens. The endosome lysis strain, a derivative of BCG SSI-1331 (BCG(1331)) expresses a mutant form of perfringolysin O (PfoA(G137Q)), a cytolysin normally secreted by Clostridium perfringens. Integration of the PfoA(G137Q) gene into the BCG genome was accomplished using an allelic exchange plasmid to replace ureC with pfoA(G137Q) under the control of the Ag85B promoter. The resultant BCG construct, designated AERAS-401 (BCG(1331) DeltaureC::OmegapfoA(G137Q)) secreted biologically active Pfo, was well tolerated with a good safety profile in immunocompromised SCID mice. A second rBCG strain, designated AFRO-1, was generated by incorporating an expression plasmid encoding three mycobacterial antigens, Ag85A, Ag85B and TB10.4, into AERAS-401. Compared to the parental BCG strain, vaccination of mice and guinea pigs with AFRO-1 resulted in enhanced immune responses. Mice vaccinated with AFRO-1 and challenged with the hypervirulent Mtb strain HN878 also survived longer than mice vaccinated with the parental BCG. Thus, we have generated improved rBCG vaccine candidates that address many of the shortcomings of the currently licensed BCG vaccine strains.

The immunogenicity of single and combination DNA vaccines against tuberculosis

DNA immunization is a promising new approach for the development of novel tuberculosis vaccines. In this study, the immune responses following the administration of single and combination tuberculosis DNA vaccines were evaluated. Single DNA vaccines encoding the MPT-63 and MPT-83 tuberculosis antigens evoked partial protection against an aerogenic challenge with M. tuberculosis Erdman in the mouse model of pulmonary tuberculosis. Immunization with a multivalent combination DNA vaccine (containing the ESAT-6, MPT-64, MPT-63, and KatG constructs) generated immune responses that indicated an absence of antigenic competition since antigen-specific cell-mediated and humoral responses were detected to each component of the mixture. More importantly, the combination vaccine elicited a strong protective response relative to the protection evoked by live BCG vaccine.

The rpsL gene and streptomycin resistance in single and multiple drug‐resistant strains of Mycobacterium tuberculosis

The recent emergence of indolent and rapidly fatal drug‐resistant strains of Mycobacterium tuberculosis has renewed interest in defining the molecular mechanisms of drug resistance in the tubercle bacilli. In this report, we have examined the mechanism of resistance to streptomycin (Sm) in M. tuberculosis through the cloning and nucleotide sequence analysis of the gene encoding the ribosomal SR protein (rpsL gene) from streptomycin‐resistant strains and their streptomycin‐sensitive parental strains. We have demonstrated that five singly SmRM. tuberculosis strains and an SmR isolate that has reduced sensitivity to multiple antibiotics have identical point mutations at codon 43 of the rpsL gene. Mutations at this same site confer SmR in Escherichia coli. In contrast, two other multiple drug‐resistant M. tuberculosis strains that are resistant to Sm have rpsL genes that have the same nucleotide sequence as their drug‐sensitive parent strains, suggesting that different resistance mechanisms are involved in these strains.

Vaccine-induced anti-tuberculosis protective immunity in mice correlates with the magnitude and quality of multifunctional CD4 T cells.

The development of improved vaccines against Mycobacterium tuberculosis has been hindered by a limited understanding of the immune correlates of anti-tuberculosis protective immunity. In this study, we examined the relationship between long-term anti-tuberculosis protection and the mycobacterial-specific CD4 multifunctional T (MFT) cell responses induced by five different TB vaccines (live-attenuated, subunit, viral vectored, plasmid DNA, and combination vaccines) in a mouse model of pulmonary tuberculosis. In a 14-month experiment, we showed that TB vaccine-induced CD4 T cell responses were heterogenous. Antigen-specific monofunctional CD4 T cells expressing single cytokines and MFT CD4 T cells expressing multiple cytokines (IFN-γ and TNF-α, IFN-γ and IFN-γ, TNF-α, and IL-2, and all three cytokines) were identified after the immunizations. Interestingly, compared to the monofunctional cells, significantly higher median fluorescent intensities (MFIs) for IFN-γ and TNF-α were detected for triple-positive MFT CD4 T cells induced by the most protective vaccines while modest differences in relative MFI values were seen for the less protective preparations. Most importantly during the 14-month study, the levels of vaccine-induced pulmonary and splenic protective immunity correlated with the frequency and the integrated MFI (iMFI, frequency×MFI) values of triple-positive CD4 T cells that were induced by the same vaccines. These data support efforts to use MFT cell analyses as a measure of TB vaccine immunogenicity in human immunization studies.

DNA Immunization in a Mouse Model of Latent Tuberculosis: Effect of DNA Vaccination on Reactivation of Disease and on Reinfection with a Secondary Challenge

ABSTRACT Individuals who are latently infected with Mycobacterium tuberculosis can develop active disease via either endogenous reactivation of the latent bacilli or exogenous reinfection with a second mycobacterial strain. In this study, we investigated whether immunization with a tuberculosis DNA vaccine cocktail that induces significant protective responses in mice could prevent reactivation of disease in a murine latent-tuberculosis model. In addition, we assessed whether DNA vaccination could retard the growth of a secondary aerogenic infection with M. tuberculosis (exogenous reinfection) in latently infected mice. In the reactivation studies, administration of the DNA vaccine combination did not prevent recrudescence of the latent infection after injection of dexamethasone. Moreover, for the reinfection experiments, only a modest decrease in the growth of a secondary M. tuberculosis challenge in DNA-vaccinated animals, compared to controls, was observed 14 and 28 days after the reinfection of previously exposed mice. Interestingly, although proliferation of the secondary challenge was reduced significantly in a nonvaccinated chronic-infection group relative to the naïve controls, the number of bacilli still increased by 500-fold 1 month after the secondary challenge in mice with active tuberculosis. These results indicate that novel immunotherapeutic approaches will be required to prevent reactivation of infection or reinfection of individuals with latent tuberculosis.