Search for correlates of protective immunity conferred by anthrax vaccine

Vaccination by anthrax protective antigen (PA)-based vaccines requires multiple immunization, underlying the need to develop more efficacious vaccines or alternative vaccination regimens. In spite of the vast use of PA-based vaccines, the definition of a marker for protective immunity is still lacking. Here we describe studies designed to help define such markers. To this end we have immunized guinea pigs by different methods and monitored the immune response and the corresponding extent of protection against a lethal challenge with anthrax spores. Active immunization was performed by a single injection using one of two methods: (i) vaccination with decreasing amounts of PA and (ii) vaccination with constant amounts of PA that had been thermally inactivated for increasing periods. In both studies a direct correlation between survival and neutralizing-antibody titer was found (r(2) = 0.92 and 0.95, respectively). Most significantly, in the two protocols a similar neutralizing-antibody titer range provided 50% protection. Furthermore, in a complementary study involving passive transfer of PA hyperimmune sera to naive animals, a similar correlation between neutralizing-antibody titers and protection was found. In all three immunization studies, neutralization titers of at least 300 were sufficient to confer protection against a dose of 40 50% lethal doses (LD(50)) of virulent anthrax spores of the Vollum strain. Such consistency in the correlation of protective immunity with anti-PA antibody titers was not observed for antibody titers determined by an enzyme-linked immunosorbent assay. Taken together, these results clearly demonstrate that neutralizing antibodies to PA constitute a major component of the protective immunity against anthrax and suggest that this parameter could be used as a surrogate marker for protection.     

The role of T cells in pathogenesis and protective immunity to murine malaria.

T-cell-mediated immunity to a virulent strain of Plasmodium berghei NK65 (Pb NK65) and to an attenuated derivative (Pb XAT) of the strain were examined in CBA mice by the administration of monoclonal antibodies against T-cell subsets or interferon-gamma (IFN-gamma). The injection of anti-CD8+ or anti-IFN-gamma delayed the mortality of mice infected with Pb NK65, although it did not affect the parasitaemia. In the late stage of PB NK65 infection, T cells, especially CD8+ T cells, were increased in number in the liver at the expense of splenic CD8+ T cells. These CD8+ T cells released IFN-gamma in culture without antigen stimulation and were thought to induce tumour necrosis factor-alpha (TNF-alpha) production by the cells in the liver. In mice infected with Pb XAT, or mice primed with Pb XAT and then challenged with Pb NK65, CD4+ T cells had a crucial role in preventing parasite growth and in protective immunity. IFN-gamma was again the key molecule in protective immunity. These results suggest that T cells stimulated with malaria antigen play important roles both in protective immunity and pathogenesis depending upon their subsets; CD8+ T cells in pathogenesis, and CD4+ T cells in protective immunity. These apparently contradictory responses may be mediated by the same cytokine, IFN-gamma.     

Immunological characterization of a protective antigen of Erysipelothrix rhusiopathiae: identification of the region responsible for protective immunity

The gene encoding a protective protein antigen of the gram-positive bacterium Erysipelothrix rhusiopathiae, an important veterinary pathogen responsible for erysipelas in swine and a variety of diseases in animals, was cloned and sequenced. The gene encodes a polypeptide of 597 amino acids plus a putative signal sequence of 29 amino acids, resulting in a mature protein with a molecular mass of 69,017 Da. Sequence analysis of the gene product revealed a C-terminal region composed of nine tandem repeats of 20 amino acids and a total sequence that is nearly identical to that of the 64-kDa cell surface protein (SpaA) of the bacterium. Because of this similarity, the protein was designated SpaA.1. In this study, we examined whether the SpaA.1 protein could induce protective antibodies and whether we could identify the region involved in protective immunity. Both the mature SpaA.1 protein and its C-terminal repeat region, but not the N-terminal segment, were expressed in Escherichia coli and purified as a histidine-tagged fusion recombinant protein. Rabbit antiserum raised against the mature SpaA.1 protein passively protected mice from lethal challenge with a virulent homologous strain, Fujisawa-SmR, suggesting that protection is mediated by humoral antibodies. To determine which domain of the SpaA.1 protein is responsible for the observed protection, mice were actively immunized with either the mature SpaA. 1 protein or the C-terminal repeat region and then challenged with Fujisawa-SmR. The result showed that mice immunized with the mature SpaA.1 protein, but not the C-terminal repeat region, were protected, suggesting that the protection-eliciting epitope(s) is located within the N-terminal two-thirds of the SpaA.1 molecule. This was confirmed by passive immunization experiments in which the protective activity of rabbit antiserum, raised against mature SpaA. 1 protein, was not abolished by absorption with the purified recombinant C-terminal repeat region. In addition, antibodies specific for the C-terminal repeat region were unable to protect mice from lethal challenge. These results show that the N-terminal two-thirds of the SpaA.1 molecule may constitute a good vaccine candidate against erysipelas.     

Generation of protective immunity by inactivated recombinant staphylococcal enterotoxin B vaccine in nonhuman primates and identification of correlates of immunity

At this time there are no vaccines or therapeutics to protect against staphylococcal enterotoxin B (SEB) exposure. Here, we report vaccine efficacy of an attenuated SEB in a nonhuman primate model following lethal aerosol challenge and identify several biomarkers of protective immunity. Initial in vitro results indicated that the mutation of key amino acid residues in the major histocompatibility complex (MHC) class II binding sites of SEB produced a nontoxic form of SEB, which had little to no detectable binding to MHC class II molecules, and lacked T-cell stimulatory activities. When examined in a mouse model, we found that the attenuated SEB retained antigenic structures and elicited protective immune responses against wild-type SEB challenge. Subsequently, a vaccine regimen against SEB in a nonhuman primate model was partially optimized, and investigations of immune biomarkers as indicators of protection were performed. SEB-naïve rhesus monkeys were vaccinated two or three times with 5 or 20 μg of the attenuated SEB and challenged by aerosol with wild-type SEB toxin. Unlike exposure to the native toxin, the vaccine did not trigger the release of inflammatory cytokines (TNFα, IL6, or IFNγ). All rhesus monkeys that developed anti-SEB serum titers ≥10⁴ and elicited high levels of neutralizing antibody survived the aerosol challenge. These findings suggest that the attenuated SEB is fully protective against aerosolized toxin when administered to unprimed subjects. Moreover, experiments presented in this study identified various biomarkers that showed substantial promise as correlates of immunity and surrogate endpoints for assessing in vivo biological responses in primates, and possibly in humans, to vaccines against SEs.     

Genetic dissection of immunity in leprosy

Leprosy is a chronic infectious disease caused by Mycobacterium leprae that affects an estimated 700,000 new individuals each year. A strong contribution of host genetics to susceptibility to leprosy has long been suggested to account for the considerable variability observed between individuals exposed to M. leprae. As there is no relevant animal model for human leprosy, forward genetics is the main strategy used to identify the genes and, consequently, the immunological pathways involved in protective immunity to M. leprae. With respect to genome-wide screens, a major breakthrough has been reported this year; variants in the regulatory region shared by PARK2 and PACRG have been identified as being common risk factors for leprosy.     

Cellular immunity in lepromatous and tuberculoid leprosy

The depression of cellular immunity in lepromatous patients is not understood. While the blood monocytes of leprosy patients appear to be activated normally by lymphokines, T cell proliferation and production of lymphokines in response to Mycobacterium leprae are impaired in lepromatous patients. Attempts to restore responsiveness in cells from these patients have been unsuccessful in our hands. The addition of exogenous IL-2 to leukocyte cultures does not appear to restore responsiveness to M. leprae in cells from nonresponder patients. Rather, some enhancement, often not antigen specific, is observed in cells from patients with a preexisting response. Similarly, depletion of monocytes does not restore responsiveness to M. leprae in non-responder patients, but a nonspecific enhancement of proliferation is observed in monocyte-free cultures from patients that do respond to M. leprae. Thus, the defect in lepromatous non-responder patients does not result from a simple lack of IL-2 production or suppression by monocytes and/or their products. Possibly, there is a low level or lack of M. leprae responsive T cells in the circulation of these patients.     

Immunological aspects of fungal pathogenesis

The incidence of infection with the pathogenic fungi continues to escalate, especially in the era of the acquired immune deficiency syndrome. To the clinician, this heterogeneous group of organisms poses both a diagnostic and a therapeutic challenge. Consequently, growing numbers of investigators are seeking to elucidate the pathogenetic mechanisms involved in disease caused by medically important fungi. In this review, many of the recent scientific advances that have been made in the immunological aspects of the pathogenesis of fungal infections are presented. The topics covered include 1) the receptors for fungi on the surface of professional phagocytes; 2) the mechanisms for killing and growth inhibition of fungi by phagocytes; 3) the means by which fungi evade host defenses; 4) the role of humoral immunity in fungal infection; 5) immunoregulation in fungal infections; and 6) the influence of cytokines on host defenses against pathogenic fungi.     

T cell and cytokine patterns in leprosy skin lesions

Conclusion The skin lesions of leprosy provide a window for directly studying immune responses to a pathogen. Analysis of the local immune responses in these lesions revealed distinct T cell and cytokine patterns which correlate with resistance versus susceptibility to infection. Similar information was not often obtainable from the study of blood, underscoring the importance of investigation of the immune response at the site of disease activity. The techniques employed in these investigations are generally useful for the study of other inflammatory diseases of skin.     

Suppressors of cytokine signaling and immunity

The suppressors of cytokine signaling (SOCS) and cytokine-inducible SH2 protein are key physiological regulators of the immune system. Principally, SOCS1 and SOCS3 regulate T cells as well as antigen-presenting cells, including macrophages and dendritic cells. Here we review the function of SOCS1 and SOCS3 in innate and adaptive immunity, with particular emphasis on the relationship between immune regulation and SOCS.     

Balancing protective immunity and immunopathology

The immune system is fighting a constant war against pathogens in its own territory. This requires a potent arsenal for efficient control of pathogens but also requires tight regulatory mechanisms in order to avoid excessive collateral damage. Maintaining equilibrium is the daily challenge of the immune system. Interactions between pathogens, antigen-presenting cells (APCs) and lymphocytes are critical in this balancing act. Of particular importance for the generation of protective immune responses is the induction of activation programs in APCs directly by pathogens or by T cell derived factors, such as CD40L, RANKL or cytokines. In order to counterbalance overshooting immune responses, T cells and APCs secrete anti-inflammatory cytokines that are key for maintaining a healthy balance between protection and immunopathology.     

Risk factors in the pathogenesis of invasive group A streptococcal infections: role of protective humoral immunity

An impressive change in the epidemiology and severity of invasive group A streptococcal infections occurred in the 1980s, and the incidence of streptococcal toxic shock syndrome cases continues to rise. The reason for the resurgence of severe invasive cases remains a mystery-has there been a change in the pathogen or in host protective immunity? To address these questions, we have studied 33 patients with invasive infection caused by genotypically indistinguishable M1T1 strains of Streptococcus pyogenes who had different disease outcomes. Patients were classified as having severe (n = 21) and nonsevere (n = 12) invasive infections based on the presence or absence of shock and organ failure. Levels of anti-M1 bactericidal antibodies and of anti-streptococcal superantigen neutralizing antibodies in plasma were significantly lower in both groups than in age- and geographically matched healthy controls (P < 0.01). Importantly, the levels of these protective antibodies in plasma samples from severe and nonsevere invasive cases were not different. Together the data suggest that low levels of protective antibodies may contribute to host susceptibility to invasive streptococcal infection but do not modulate disease outcome. Other immunogenetic factors that regulate superantigen responses may influence the severity of systemic manifestations associated with invasive streptococcal infection.     

Transcutaneous immunization using colonization factor and heat-labile enterotoxin induces correlates of protective immunity for enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) diarrheal disease is a worldwide problem that may be addressed by transcutaneous delivery of a vaccine. In several human settings, protective immunity has been associated with immune responses to E. coli colonization factors and to the heat-labile toxin that induces the diarrhea. In this set of animal studies, transcutaneous immunization (TCI) using recombinant colonization factor CS6 and cholera toxin (CT) or heat-labile enterotoxin (LT) as the adjuvant induced immunoglobulin G (IgG) and IgA anti-CS6 responses in sera and stools and antibody responses that recognized CS6 antigen in its native configuration. The antitoxin immunity induced by TCI was also shown to protect against enteric toxin challenge. Although immunization with LT via the skin induced mucosal secretory IgA responses to LT, protection could also be achieved by intravenous injection of the immune sera. Finally, a malaria vaccine antigen, merzoite surface protein 1(42) administered with CT as the adjuvant, induced both merzoite surface protein antibodies and T-cell responses while conferring protective antitoxin immunity, suggesting that both antiparasitic activity and antidiarrheal activity can be obtained with a single vaccine formulation. Overall, our results demonstrate that relevant colonization factor and antitoxin immunity can be induced by TCI and suggest that an ETEC traveler's diarrhea vaccine could be delivered by using a patch.     

Immunological and microbiological factors in the pathogenesis of atherosclerosis

Among the several pathological events that lead to the formation of an atheromatous lesion, endothelial cell damage, smooth muscle cell proliferation, and foam cell formation, are considered as particularly significant. In this review we summarize data suggesting that immunological and microbial factors may cause, directly or indirectly, these pathological events. Binding of immunocomplexes to endothelial cells, phagocytic cells, platelets, or erythrocytes could be the starting point for a variety of circuits leading to endothelial cell cytotoxicity and to the release of a variety of mediators, including cell proliferative factors. Endothelial cell toxicity could also be induced, directly or indirectly, by endotoxin; however, the possibility that endotoxin and other microbial factors may induce abnormalities in lipid metabolism at the monocyte/macrophage level which eventually result in intracellular accumulation of cholesterol (particularly if cholesterol levels are elevated) is specially attractive as a potential pathogenic mechanism. The various pathologic pathways discussed in this review appear plausible on the basis of our current knowledge and point to the need to investigate the potential role of infectious processes, autoimmune reactions, and administration of antigenic compounds as possible risk factors for the development of atherosclerosis.     

Immunological correlates for protection against intranasal challenge of Bacillus anthracis spores conferred by a protective antigen-based vaccine in rabbits

Correlates between immunological parameters and protection against Bacillus anthracis infection in animals vaccinated with protective antigen (PA)-based vaccines could provide surrogate markers to evaluate the putative protective efficiency of immunization in humans. In previous studies we demonstrated that neutralizing antibody levels serve as correlates for protection in guinea pigs (S. Reuveny et al., Infect. Immun. 69:2888-2893, 2001; H. Marcus et al., Infect. Immun. 72:3471-3477, 2004). In this study we evaluated similar correlates for protection by active and passive immunization of New Zealand White rabbits. Full immunization and partial immunization were achieved by single and multiple injections of standard and diluted doses of a PA-based vaccine. Passive immunization was carried out by injection of immune sera from rabbits vaccinated with PA-based vaccine prior to challenge with B. anthracis spores. Immunized rabbits were challenged by intranasal spore instillation with one of two virulent strains (strains Vollum and ATCC 6605). The immune competence was estimated by measuring the level of total anti-PA antibodies, the neutralizing antibody titers, and the conferred protective immunity. The results indicate that total anti-PA antibody titers greater than 1 x 10(5) conferred protection, whereas lower titers (between 10(4) and 10(5)) provided partial protection but failed to predict protection. Neutralizing antibody titers between 500 and 800 provided partial protection, while titers higher than 1,000 conferred protection. In conclusion, this study emphasizes that regardless of the immunization regimen or the time of challenge, neutralizing antibody titers are better predictors of protection than total anti-PA titers.     

Cell-mediated immunity in leprosy and transfer of delayed hypersensitivity reactions

Eighty-nine patients with leprosy, 65 classified as lepromatous and 24 as tuberculoid, were examined in this study. Skin test responses to protein antigens and dinitrochlorobenzene (DNCB) were depressed in lepromatous patients compared to controls. Tuberculoid patients did not exhibit a significant depression to microbial antigens, but they showed a definite depression in the ability to be sensitized with DNCB. The transfer of delayed hypersensitivity reactions to tuberculin, trichophytin, and lepromin (Fernandez and Mitsuda reactions) was accomplished in lepromatous and indeterminate leprosy patients using viable lymphocytes from donors presenting positive reactions to these antigens. The lepromin reaction was also transferred to patients with South American blastomycosis and cutaneous leishmaniasis. The positive reactions of adoptive immunity were confirmed by histologic examination of skin biopsies.     

Immunological enhancement in the pathogenesis of pyelonephritis.

The role of the immune response in pyelonephritis was investigated by manipulation of the host's immune capacity using the immunosuppressive drugs 6-mercaptopurine, cyclophosphamide and thiamphenicol. Treatment with 6-mercaptopurine depressed the humoral immune response but did not have an adverse effect on the course of renal infection. Thiamphenicol administration prevented the development of pathological lesions but this was due to the anti-bacterial activity of thiamphenicol and not to its immunosuppressive activity. Pyelonephritic animals treated with cyclophosphamide did not produce anti-bacterial antibody. Despite this, cyclophosphamide-treated animals were able to eliminate organisms more readily from the infected kidney than untreated animals with a normal humoral immune response. We believe that blocking of the phenomenon of immunological enhancement explains these unexpected results and that the immune response to renal infection may have an immunoenhancing role protecting the bacterial cell from otherwise effective host defence mechanisms.