Anthrax lethal factor as an immune target in humans and transgenic mice and the impact of HLA polymorphism on CD4+ T cell immunity.

Bacillus anthracis produces a binary toxin composed of protective antigen (PA) and one of two subunits, lethal factor (LF) or edema factor (EF). Most studies have concentrated on induction of toxin-specific antibodies as the correlate of protective immunity, in contrast to which understanding of cellular immunity to these toxins and its impact on infection is limited. We characterized CD4+ T cell immunity to LF in a panel of humanized HLA-DR and DQ transgenic mice and in naturally exposed patients. As the variation in antigen presentation governed by HLA polymorphism has a major impact on protective immunity to specific epitopes, we examined relative binding affinities of LF peptides to purified HLA class II molecules, identifying those regions likely to be of broad applicability to human immune studies through their ability to bind multiple alleles. Transgenics differing only in their expression of human HLA class II alleles showed a marked hierarchy of immunity to LF. Immunogenicity in HLA transgenics was primarily restricted to epitopes from domains II and IV of LF and promiscuous, dominant epitopes, common to all HLA types, were identified in domain II. The relevance of this model was further demonstrated by the fact that a number of the immunodominant epitopes identified in mice were recognized by T cells from humans previously infected with cutaneous anthrax and from vaccinated individuals. The ability of the identified epitopes to confer protective immunity was demonstrated by lethal anthrax challenge of HLA transgenic mice immunized with a peptide subunit vaccine comprising the immunodominant epitopes that we identified.

CD4+ T cell epitopes of FliC conserved between strains of Burkholderia: implications for vaccines against melioidosis and cepacia complex in cystic fibrosis.

Burkholderia pseudomallei is the causative agent of melioidosis characterized by pneumonia and fatal septicemia and prevalent in Southeast Asia. Related Burkholderia species are strong risk factors of mortality in cystic fibrosis (CF). The B. pseudomallei flagellar protein FliC is strongly seroreactive and vaccination protects challenged mice. We assessed B. pseudomallei FliC peptide binding affinity to multiple HLA class II alleles and then assessed CD4 T cell immunity in HLA class II transgenic mice and in seropositive individuals in Thailand. T cell hybridomas were generated to investigate cross-reactivity between B. pseudomallei and the related Burkholderia species associated with Cepacia Complex CF. B. pseudomallei FliC contained several peptide sequences with ability to bind multiple HLA class II alleles. Several peptides were shown to encompass strong CD4 T cell epitopes in B. pseudomallei-exposed individuals and in HLA transgenic mice. In particular, the p38 epitope is robustly recognized by CD4 T cells of seropositive donors across diverse HLA haplotypes. T cell hybridomas against an immunogenic B. pseudomallei FliC epitope also cross-reacted with orthologous FliC sequences from Burkholderia multivorans and Burkholderia cenocepacia, important pathogens in CF. Epitopes within FliC were accessible for processing and presentation from live or heat-killed bacteria, demonstrating that flagellin enters the HLA class II Ag presentation pathway during infection of macrophages with B. cenocepacia. Collectively, the data support the possibility of incorporating FliC T cell epitopes into vaccination programs targeting both at-risk individuals in B. pseudomallei endemic regions as well as CF patients.

Repertoire of HLA-DR1-restricted CD4 T-cell responses to capsular Caf1 antigen of Yersinia pestis in human leukocyte antigen transgenic mice.

Yersinia pestis is the causative agent of plague, a rapidly fatal infectious disease that has not been eradicated worldwide. The capsular Caf1 protein of Y. pestis is a protective antigen under development as a recombinant vaccine. However, little is known about the specificity of human T-cell responses for Caf1. We characterized CD4 T-cell epitopes of Caf1 in "humanized" HLA-DR1 transgenic mice lacking endogenous major histocompatibility complex class II molecules. Mice were immunized with Caf1 or each of a complete set of overlapping synthetic peptides, and CD4 T-cell immunity was measured with respect to proliferative and gamma interferon T-cell responses and recognition by a panel of T-cell hybridomas, as well as direct determination of binding affinities of Caf1 peptides to purified HLA-DR molecules. Although a number of DR1-restricted epitopes were identified following Caf1 immunization, the response was biased toward a single immunodominant epitope near the C terminus of Caf1. In addition, potential promiscuous epitopes, including the immunodominant epitope, were identified by their ability to bind multiple common HLA alleles, with implications for the generation of multivalent vaccines against plague for use in humans.

CD4+ T Cells Targeting Dominant and Cryptic Epitopes from Bacillus anthracis Lethal Factor.

Anthrax is an endemic infection in many countries, particularly in the developing world. The causative agent, Bacillus anthracis, mediates disease through the secretion of binary exotoxins. Until recently, research into adaptive immunity targeting this bacterial pathogen has largely focused on the humoral response to these toxins. There is, however, growing recognition that cellular immune responses involving IFNγ producing CD4+ T cells also contribute significantly to a protective memory response. An established concept in adaptive immunity to infection is that during infection of host cells, new microbial epitopes may be revealed, leading to immune recognition of so called 'cryptic' or 'subdominant' epitopes. We analyzed the response to both cryptic and immunodominant T cell epitopes derived from the toxin component lethal factor and presented by a range of HLA-DR alleles. Using IFNγ-ELISpot assays we characterized epitopes that elicited a response following immunization with synthetic peptide and the whole protein and tested their capacities to bind purified HLA-DR molecules in vitro. We found that DR1 transgenics demonstrated T cell responses to a greater number of domain III cryptic epitopes than other HLA-DR transgenics, and that this pattern was repeated with the immunodominant epitopes, as a greater proportion of these epitopes induced a T cell response when presented within the context of the whole protein. Immunodominant epitopes LF457-476 and LF467-487 were found to induce a T cell response to the peptide, as well as to the whole native LF protein in DR1 and DR15, but not in DR4 transgenics. The analysis of Domain I revealed the presence of several unique cryptic epitopes all of which showed a strong to moderate relative binding affinity to HLA-DR4 molecules. However, none of the cryptic epitopes from either domain III or I displayed notably high binding affinities across all HLA-DR alleles assayed. These responses were influenced by the specific HLA alleles presenting the peptide, and imply that construction of future epitope string vaccines which are immunogenic across a wide range of HLA alleles could benefit from a combination of both cryptic and immunodominant anthrax epitopes.

Sequential proteolytic processing of the capsular Caf1 antigen of Yersinia pestis for major histocompatibility complex class II-restricted presentation to T lymphocytes.

We studied the mechanisms of antigen presentation of CD4 T cell epitopes of the capsular Caf1 antigen of Yersinia pestis using murine bone marrow macrophages as antigen presenting cells and T cell hybridomas specific for major histocompatibility complex (MHC) class II-restricted epitopes distributed throughout the Caf1 sequence. The data revealed diversity in the pathways used and the degrees of antigen processing required depending on the structural context of epitopes within the Caf1 molecule. Two epitopes in the carboxyl-terminal globular domain were presented by newly synthesized MHC class II after low pH-dependent lysosomal processing, whereas an epitope located in a flexible amino-terminal strand was presented by mature MHC class II independent of low pH and with no detectable requirement for proteolytic processing. A fourth epitope located between the two regions of Caf1 showed intermediate behavior. The data are consistent with progressive unfolding and cleavage of rCaf1 from the amino terminus as it traverses the endosomal pathway, the availability of epitopes determining which pool of MHC class II is preferentially loaded. The Caf1 capsular protein is a component of second generation plague vaccines and an understanding of the mechanisms and pathways of MHC class II-restricted presentation of multiple epitopes from this candidate vaccine antigen should inform the choice of delivery systems and adjuvants that target vaccines successfully to appropriate intracellular locations to induce protective immune responses against as wide a T cell repertoire as possible.

Mechanisms of major histocompatibility complex class II-restricted processing and presentation of the V antigen of Yersinia pestis.

We mapped mouse CD4 T-cell epitopes located in three structurally distinct regions of the V antigen of Yersinia pestis. T-cell hybridomas specific for epitopes from each region were generated to study the mechanisms of processing and presentation of V antigen by bone-marrow-derived macrophages. All three epitopes required uptake and/or processing from V antigen as well as presentation to T cells by newly synthesized major histocompatibility complex (MHC) class II molecules over a time period of 3-4 hr. Sensitivity to inhibitors showed a dependence on low pH and cysteine, serine and metalloproteinase, but not aspartic proteinase, activity. The data indicate that immunodominant epitopes from all three structural regions of V antigen were presented preferentially by the classical MHC class II-restricted presentation pathway. The requirement for processing by the co-ordinated activity of several enzyme families is consistent with the buried location of the epitopes in each region of V antigen. Understanding the structure-function relationship of multiple immunodominant epitopes of candidate subunit vaccines is necessary to inform choice of adjuvants for vaccine delivery. In the case of V antigen, adjuvants designed to target it to lysosomes are likely to induce optimal responses to multiple protective T-cell epitopes.

Immunogenicity of a Yersinia pestis vaccine antigen monomerized by circular permutation.

Caf1, a chaperone-usher protein from Yersinia pestis, is a major protective antigen in the development of subunit vaccines against plague. However, recombinant Caf1 forms polymers of indeterminate size. We report the conversion of Caf1 from a polymer to a monomer by circular permutation of the gene. Biophysical evaluation confirmed that the engineered Caf1 was a folded monomer. We compared the immunogenicity of the engineered monomer with polymeric Caf1 in antigen presentation assays to CD4 T-cell hybridomas in vitro, as well as in the induction of antibody responses and protection against subcutaneous challenge with Y. pestis in vivo. In C57BL/6 mice, for which the major H-2(b)-restricted immunodominant CD4 T-cell epitopes were intact in the engineered monomer, immunogenicity and protective efficacy were preserved, although antibody titers were decreased 10-fold. Disruption of an H-2(d)-restricted immunodominant CD4 T-cell epitope during circular permutation resulted in a compromised T-cell response, a low postvaccination antibody titer, and a lack of protection of BALB/c mice. The use of circular permutation in vaccine design has not been reported previously.

Major histocompatibility class II molecules prevent destructive processing of exogenous peptides at the cell surface of macrophages for presentation to CD4 T cells.

We studied factors affecting major histocompatibility complex class II (MHC-II)-restricted presentation of exogenous peptides at the surface of macrophages. We have previously shown that peptide presentation is modulated by surface-associated proteolytic enzymes, and in this report the role of the binding of MHC-II molecules in preventing proteolysis of exogenous synthetic peptides was addressed. Two peptides containing CD4 T-cell epitopes were incubated with fixed macrophages expressing binding and non-binding MHC-II, and supernatants were analysed by high-performance liquid chromatography and mass spectrometry to monitor peptide degradation. The proportion of full-length peptides that were degraded and the number of peptide fragments increased when non-binding macrophages were used, leading to reduction in peptide presentation. When MHC-II molecules expressed on the surface of fixed macrophages were blocked with monoclonal antibody and incubated with peptides and the supernatants were transferred to fixed macrophages, a significant reduction in peptide presentation was observed. Peptide presentation was up-regulated at pH 5.5 compared to neutral pH, and the latter was found to be the pH optimum of the proteolytic activity of the surface enzymes involved in the degradation of exogenous peptides and proteins. The data suggest that MHC-II alleles that bind peptides protect them from degradation at the antigen-presenting cell surface for presentation to CD4 T cells and we argue that this mechanism could be particularly pronounced at sites of inflammation.

Differential processing of CD4 T-cell epitopes from the protective antigen of Bacillus anthracis.

We have mapped CD4+ T-cell epitopes located in three domains of the recombinant protective antigen of Bacillus anthracis. Mouse T-cell hybridomas specific for these epitopes were generated to study the mechanisms of proteolytic processing of recombinant protective antigen for antigen presentation by bone marrow-derived macrophages. Overall, epitopes differed considerably in their processing requirements. In particular, the kinetics of presentation, ranging from 15 (fast) to 120 min (slow), suggested sequential liberation of epitopes during proteolytic processing of the intact PA molecule. Pretreatment of macrophages with ammonium chloride or inhibitors of the major enzyme families showed that T-cell responses to an epitope presented with fast kinetics were unaffected by raising endosomal pH or inhibiting cysteine or aspartic proteinases, suggesting presentation independent of lysosomal processing. In contrast, responses to epitopes presented with slower kinetics were dependent on low pH and the activity of cysteine or aspartic proteinases indicating a requirement for lysosomal processing. In addition, responses to all epitopes, whether their presentation was dependent on low pH or not, were prevented by treatment of macrophages with broad spectrum serine proteinase inhibitors. Thus, our data are consistent with a model of sequential antigen processing within the endosomal system, beginning with a pre-processing step mediated by serine or metalloproteinases prior to further processing by lysosomal enzymes. Rapidly presented epitopes seemed to require only limited proteolysis at earlier stages of endocytosis, whereas the majority of epitopes required more extensive processing by neutral proteinases followed by lysosomal enzymes.

Regulation of peptide presentation by major histocompatibility complex class II molecules at the surface of macrophages.

We studied major histocompatibility complex class II-dependent presentation of two T cell epitopes delivered as synthetic peptides by fixed macrophages. Treatment of bone marrow macrophages with inhibitors of proteinases of the metallo-, aspartic and serine proteinase families enhanced presentation of peptides, indicating that several enzyme families participate in destructive antigen processing of exogenous peptides. High performance liquid chromatography and mass spectrometry analysis demonstrated the presence of peptide fragments in macrophage supernatants, and permitted identification of the cleavage sites which confirmed the enzyme families involved. Peptide fragments were shown to be competitive inhibitors of presentation of the full-length peptide to CD4 T cells by fixed and live macrophages. The results indicate that several classes of proteinases can modulate antigen presentation by at least two mechanisms: (1) degradation of extracellular oligopeptides and (2) generation of natural peptide ligands that block antigen presentation to CD4 T cells. The generation of inhibitory natural peptide ligands is a new mechanism of immunoregulation which could operate during the induction of T cell responses in a variety of situations.

Processing of viable Salmonella typhimurium for presentation of a CD4 T cell epitope from the Salmonella invasion protein C (SipC).

We have identified Salmonella invasion protein C (SipC) as a target antigen for CD4 T cell recognition in mice infected with Salmonella typhimurium. SipC is a product of the type III secretion system encoded by S. typhimurium pathogenicity island 1. A SipC-specific T cell response was induced by infection with either the C5 wild type or attenuated SL3261 vaccine strain of S. typhimurium. We localized the response of T cell lines from infected mice to an epitope near the carboxyl terminus of SipC (SipC(381-394)) and studied the way it was processed from viable S. typhimurium. We demonstrated that CD4 T cell recognition of this epitope required actin-dependent uptake of S. typhimurium. Presentation also occurred when transport of newly synthesized MHC class II from the endoplasmic reticulum was disrupted and when the pH of intracellular compartments was raised, suggesting presentation by mature MHC class II recycled from the macrophage surface into neutral intracellular compartments. Salmonellae are known to colonize macrophages by localizing to compartments that do not make contact with the bactericidal environment of late endosomes or lysosomes, and thus might avoid lysosomal antigen processing. However, we demonstrate that a CD4 T cell response to S. typhimurium-secreted proteins may be induced by an alternative pathway capable of antigen presentation in conditions similar to those in the compartments where Salmonella localize.

Attenuated Salmonella typhimurium htrA mutants cause fatal infections in mice deficient in NADPH oxidase and destroy NADPH oxidase-deficient macrophage monolayers.

Salmonella live vaccine strains harbouring mutations in htrA, a stress protein gene, display increased susceptibility to oxidative stress in vitro. This is believed to be connected to their reduced virulence, perhaps due to impaired survival inside phagocytes, although this has never been formally proven. We report that the in vitro phenotype of increased susceptibility to oxidative stress of Salmonella typhimurium htrA mutants newly prepared by transduction is rapidly lost on subculture, with the mutants becoming as resistant as the parent for reasons that remain unclear. However, despite this change, htrA mutants are still attenuated in normal mice. In contrast, they were found to be lethal for gene targeted gp91phox-/- mice deficient in NADPH oxidase, as was a S. typhimurium SPI-2 mutant known to be virulent in gp9lphox-/- mice. Infection with htrA mutants caused little damage to primary bone marrow macrophage cultures from normal mice; conversely, they caused extensive damage to macrophages from gp9lphox-/- mice, with more than 60% reduction in cell numbers 2.5h after being infected. The parental wild type strain similarly caused extensive damage to macrophages from both normal and gp9lphox-/- mice, whereas an aroA live vaccine strain had no effect on either normal or gp9lphox-/- macrophages. Taken collectively, the present results suggest that htrA is somehow involved in resistance to oxidative stress in vivo, with the avirulence of htrA mutants in mice being due to mechanisms which involve NADPH oxidase and suppression of bacterial growth within macrophages.