Comparative investigation of the pathogenicity of three Mycobacterium tuberculosis mutants defective in the synthesis of p-hydroxybenzoic acid derivatives.

p-Hydroxybenzoic acid derivatives (p-HBADs) are glycoconjugates secreted by all Mycobacterium tuberculosis isolates whose contribution to pathogenicity remains to be determined. The pathogenicity of three transposon mutants of M. tuberculosis deficient in the biosynthesis of some or all forms of p-HBADs was studied. Whilst the mutants grew similarly to the wild-type strain in macrophages and C57BL/6 mice, two of the mutants induced a more severe and diffuse inflammation in the lungs. The lack of production of some or all forms of p-HBADs in these two mutants also correlated with an increased secretion of the pro-inflammatory cytokines tumour-necrosis factor alpha, interleukin 6 and interleukin 12 in vivo. We propose that the loss of production of p-HBADs by tubercle bacilli results in their diminished ability to suppress the pro-inflammatory response to infection and that this ultimately provokes extensive pulmonary lesions in the C57BL/6 model of tuberculosis infection.

Induction of high levels of protective immunity in mice after vaccination using dendritic cells infected with auxotrophic mutants of Mycobacterium tuberculosis.

Adoptively transferred dendritic cells presenting antigens derived from different pathogens have been shown to elicit specific T cell responses and to induce protective antibacterial immunity. We describe here the induction of high levels of protective immunity in mice using dendritic cells infected with auxotrophic mutants of Mycobacterium tuberculosis. We provide evidence that protection is superior to BCG and that it is associated with increased priming of CD4+ and CD8+ T cells specific for mycobacterial antigens. This method for generating high levels of anti-bacterial protective immunity could be helpful in the design of novel vaccines against tuberculosis and other intracellular pathogens.

Anti-mycobacterial immunity induced by a single injection of M. leprae Hsp65-encoding plasmid DNA in biodegradable microparticles.

A single sub-cutaneous injection of a plasmid DNA encoding a mycobacterial heat shock protein 65 (Hsp65) entrapped in biodegradable poly(lactic-co-glycolic acid) microspheres produced high titers of antibodies, measured 5 months after the injection in BALB/c mice. Splenocytes secreted IFN-gamma and exerted an anti-bacterial effect on macrophages infected in vitro with Mycobacterium tuberculosis. The results are encouraging with regard to obtaining good compliance and vaccination coverage with candidate plasmid DNA vaccines, especially in developing countries.

Therapeutic efficacy of high-dose intravenous immunoglobulin in Mycobacterium tuberculosis infection in mice.

Intravenous immunoglobulin (IVIg) is used to treat patients with primary antibody deficiencies and, at high doses, to treat a range of autoimmune and inflammatory disorders. With high-dose IVIg (hdIVIg), immunomodulatory mechanisms act on a range of cells, including T cells, B cells, and dendritic cells. Here, we demonstrate that the treatment of M. tuberculosis-infected mice with a single cycle of hdIVIg resulted in substantially reduced bacterial loads in the spleen and lungs when administered at either an early or late stage of infection. Titration of the IVIg showed a clear dose-response effect. There was no reduction in bacterial load when mice were given equimolar doses of another human protein, human serum albumin, or maltose, the stabilizing agent in the IVIg preparation. HdIVIg in vitro had no inhibitory effect on the growth of M. tuberculosis in murine bone marrow-derived macrophages. In addition, the effect of hdIVIg on bacterial loads was not observed in nude mice, suggesting the involvement of conventional T cells. Analysis of T cells infiltrating the lungs revealed only small increases in CD8(+) but not CD4(+) T-cell numbers in hdIVIg-treated mice. The mechanism of action of hdIVIg against tuberculosis in mice remains to be determined. Nevertheless, since hdIVIg is already widely used clinically, the magnitude and long duration of the therapeutic effect seen here suggest that IVIg, or components of it, may find ready application as an adjunct to therapy of human tuberculosis.

The order of prime-boost vaccination of neonatal calves with Mycobacterium bovis BCG and a DNA vaccine encoding mycobacterial proteins Hsp65, Hsp70, and Apa is not critical for enhancing protection against bovine tuberculosis.

Priming neonatal calves at birth with a Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine and boosting with a DNA vaccine consisting of plasmids encoding mycobacterial antigens Hsp65, Hsp70, and Apa or the reverse prime-boost sequence induced similar levels of protection against experimental challenge with Mycobacterium bovis. When M. bovis was isolated from a thoracic lymph node following challenge, the two groups of calves given the prime-boost regimen had significantly lower numbers of M. bovis isolates than those vaccinated with BCG alone. These observations suggest that the exact sequence of administration of a prime-boost vaccination regimen in a neonatal animal model is not critical to the development of immunity.

CD4 T cells guarantee optimal competitive fitness of CD8 memory T cells.

We studied the contribution of CD4 T cell help to survival and competitive fitness of CD8 memory T cells specific for influenza virus nucleoprotein. In agreement with recent studies, the optimal generation of functional memory CD8 T cells required CD4 help, although long-term maintenance of resting CD8 memory T cells did not absolutely depend on the presence of CD4 T cells. Nonetheless, CD4 T cells were essential during differentiation of CD8 memory T cells to imprint on them the capacity to compete effectively with other memory T cells. CD8 memory cells generated with help survived better in secondary polyclonal hosts, and co-transfer into lymphopenic hosts together with "un-helped" CD8 memory cells showed improved homeostatic expansion of CD8 memory cells that had been generated with CD4 help. Therefore, the requirement for CD4 help in CD8 T cell memory extends to homeostatic parameters that ensure the maintenance and competitive fitness of memory clones.

A heterologous DNA priming-Mycobacterium bovis BCG boosting immunization strategy using mycobacterial Hsp70, Hsp65, and Apa antigens improves protection against tuberculosis in mice.

Tuberculosis is responsible for >2 million deaths a year, and the number of new cases is rising worldwide. DNA vaccination combined with Mycobacterium bovis bacillus Calmette Guerin (BCG) represents a potential strategy for prevention of this disease. Here, we used a heterologous prime-boost immunization approach using a combination of DNA plasmids and BCG in order to improve the efficacy of vaccination against Mycobacterium tuberculosis infection in mice. As model antigens, we selected the M. tuberculosis Apa (for alanine-proline-rich antigen) and the immunodominant Hsp65 and Hsp70 mycobacterial antigens combined with BCG. We demonstrated that animals injected with a combination of DNA vectors expressing these antigens, when boosted with BCG, showed increased specific antimycobacterial immune responses compared to animals vaccinated with BCG alone. More importantly, the protection achieved with this regimen was also significantly better than with BCG alone.

RNA encoding the MPT83 antigen induces protective immune responses against Mycobacterium tuberculosis infection.

We have previously demonstrated that vaccination of mice with plasmid DNA vectors expressing immunodominant mycobacterial genes induced cellular immune responses and significant protection against challenge with Mycobacterium tuberculosis. We demonstrate here, using in vitro-synthesized RNA, that vaccination with DNA or RNA constructs expressing the M. tuberculosis MPT83 antigen are capable of inducing specific humoral and T-cell immune responses and confer modest but significant protection against M. tuberculosis challenge in mice. This is the first report of protective immunity conferred against intracellular bacteria by an RNA vaccine. This novel approach avoids some of the drawbacks of DNA vaccines and illustrates the potential for developing new antimycobacterial immunization strategies.

Antimycobacterial calixarenes enhance innate defense mechanisms in murine macrophages and induce control of Mycobacterium tuberculosis infection in mice.

Tuberculosis remains the leading cause of death among infectious diseases, accounting for more than two million deaths annually. The incidence of the disease is increasing globally, partially because of the resurgence of drug-resistant strains of Mycobacterium tuberculosis. Calixarenes are macrocyclic oligomers, some of which are able to modify the growth of M. tuberculosis in infected cells. Most experimental work has been carried out with Macrocyclon, also known as HOC 12.5EO. In this study, we demonstrate that Macrocyclon is effective in controlling M. tuberculosis infections, and we provide evidence that its effect is partially mediated by an l-arginine-dependent mechanism of macrophage activation that involves the activity of the inducible nitric oxide synthase. We also show that Macrocyclon is effective in athymic and major histocompatibility complex class II-/- mice and synthesized a number of structurally related calixarenes expressing significant antimycobacterial activity.

A DNA prime-Mycobacterium bovis BCG boost vaccination strategy for cattle induces protection against bovine tuberculosis.

The variable efficacy of bacillus Calmette-Guérin (Mycobacterium bovis BCG) in protecting humans and cattle against tuberculosis has prompted a search for a more effective vaccination regimen. A prime-boost strategy was investigated in cattle naturally sensitized to environmental mycobacteria by using a combination of three DNA vaccines coding for Hsp 65, Hsp 70, and Apa for priming, followed by a boost with BCG prior to experimental challenge with virulent M. bovis. Controls were vaccinated with DNA or BCG alone or were not vaccinated. The immune responses were monitored throughout the study, and protection was assessed based on reductions in the numbers of lesions and viable mycobacteria in lymph node samples. Vaccination with BCG alone or with a DNA prime-BCG boost regimen induced high levels of antigen-specific gamma interferon (IFN-gamma) in whole-blood cultures. In the prime-boost group there were fewer animals with severe lung lesions, fewer lymph nodes with lesions per animal, a smaller proportion of animals with lesions, lower mean lung and lymph node lesion scores, and less M. bovis isolated from retropharyngeal and thoracic lymph nodes compared to the results obtained for the nonvaccinated animals. The prime-boost regimen induced significant enhancement of protection in six parameters, compared with significant enhancement of protection in only two parameters for BCG alone. In addition, following challenge, in vitro IFN-gamma responses against ESAT-6 and CFP-10, as well as bovine tuberculin-induced skin test and in vitro IFN-gamma responses, were identified as immunological markers that predicted protection. The use of the prime-boost strategy suggested that a combination of vaccines may be better than a single vaccine for protection against tuberculosis.