Susan L. Welkos

Protection against experimental bubonic and pneumonic plague by a recombinant capsular F1-V antigen fusion protein vaccine.

The current human whole-cell vaccine is ineffective against pneumonic plague caused by typical F1 capsule positive (F1+) strains of Yersinia pestis. The authors found this vaccine to also be ineffective against F1-negative (F1-) Y. pestis strains, which have been isolated from a human case and from rodents. For these reasons, the authors developed a recombinant vaccine composed of a fusion protein of F1 with a second protective immunogen, V antigen. This vaccine protected experimental mice against pneumonic as well as bubonic plague produced by either an F1+ or F1- strain of Y. pestis, gave better protection than F1 or V alone against the F1+ strain, and may provide the basis for an improved human plague vaccine.

The role of antibodies to Bacillus anthracis and anthrax toxin components in inhibiting the early stages of infection by anthrax spores.

Vaccines which are efficacious against anthrax, such as the human vaccine, Anthrax Vaccine Absorbed (AVA), contain the protective antigen (PA) component of the anthrax toxins as the major protective immunogen. Although AVA protects against inhalational anthrax, the immune responses to and role in protection of PA and possibly other antigens have yet to be fully elucidated. Sera from animals immunized with a toxin-producing, unencapsulated live vaccine strain of Bacillus anthracis have been reported to have anti-spore activities associated with the antitoxin humoral response. The authors performed studies to determine whether anti-PA antibody (Ab)-containing preparations stimulated spore uptake by phagocytes and suppressed the germination of spores in vitro. AVA- and PA-immune sera from several species enhanced the phagocytosis by murine peritoneal macrophages of spores of the virulent Ames and the Sterne vaccine strains. Antitoxin Abs appeared to contribute significantly, although not solely, to the enhanced uptake. Rabbit antisera to PA purified from either Sterne or a PA-producing pX01-cured recombinant, affinity-purified anti-PA IgG, and monkey antisera to AVA were used to assess the role of anti-PA ABS: Rabbit anti-PA Abs promoted the uptake of spores of the PA-producing strains Sterne, Ames and RP42, a mutant of Sterne producing only PA, but not of the pX01-Sterne-1 strain, Ames strain, or RP4, a mutant of Sterne with deletions in the loci encoding PA and the oedema factor (EF) toxin component and producing only the lethal factor toxin component. Rabbit anti-PA and monkey anti-AVA Abs also significantly inhibited spore germination in vitro compared to preimmune serum or medium. Spore-associated proteins recognized by anti-PA Abs were detected by electron microscopy and confirmed by immunoblotting of spore coat extracts. Thus, the anti-PA Ab-specific immunity induced by AVA has anti-spore activity and might have a role in impeding the early stages of infection with B. anthracis spores.

Roles of macrophages and neutrophils in the early host response to Bacillus anthracis spores in a mouse model of infection

ABSTRACT The development of new approaches to combat anthrax requires that the pathogenesis and host response to Bacillus anthracis spores be better understood. We investigated the roles that macrophages and neutrophils play in the progression of infection by B. anthracis in a mouse model. Mice were treated with a macrophage depletion agent (liposome-encapsulated clodronate) or with a neutrophil depletion agent (cyclophosphamide or the rat anti-mouse granulocyte monoclonal antibody RB6-8C5), and the animals were then infected intraperitoneally or by aerosol challenge with fully virulent, ungerminated B. anthracis strain Ames spores. The macrophage-depleted mice were significantly more susceptible to the ensuing infection than the saline-pretreated mice, whereas the differences observed between the neutropenic mice and the saline-pretreated controls were generally not significant. We also found that augmenting peritoneal neutrophil populations before spore challenge did not increase resistance of the mice to infection. In addition, the bacterial load in macrophage-depleted mice was significantly greater and appeared significantly sooner than that observed with the saline-pretreated mice. However, the bacterial load in the neutropenic mice was comparable to that of the saline-pretreated mice. These data suggest that, in our model, neutrophils play a relatively minor role in the early host response to spores, whereas macrophages play a more dominant role in early host defenses against infection by B. anthracis spores.

Comparative safety and efficacy against Bacillus anthracis of protective antigen and live vaccines in mice

The efficacy and mechanisms of protection of two live vaccines and of a protective antigen (PA) vaccine against Bacillus anthracis were studied in inbred mice. Mice that differed in their natural resistance to killing by Sterne, a non-encapsulated, toxigenic vaccine strain of B. anthracis, were used. Vaccination with live Sterne spores protected Sterne-resistant mice against challenge with the virulent Vollum 1B (V1B) strain of B. anthracis, but only at doses of Sterne greater than or equal to 0.1 50% lethal dose. The live B. subtilis recombinant strain PA2, which produces the PA component of anthrax toxin, fully protected (CBA/J) or partially protected (BALB/cJ) Sterne-resistant mice against V1B. Neither immunization with the cell-free PA vaccine nor passive administration of anti-PA antiserum protected Sterne-resistant mice against V1B. Sterne-susceptible A/J mice were not protected against V1B by either live vaccine or by the PA vaccine. However, immunization with strain PA2 induced anti-PA antibody and protected A/J mice against Sterne. A/J mice passively treated with antitoxin antibodies also survived Sterne, and survivors were then partially protected against V1B. Thus, immunity to Sterne correlated with an effective anti-PA response. Immunity to fully virulent V1B also required PA but may involve mechanisms in addition to humoral immunity.

Cutting Edge: Resistance to Bacillus anthracis Infection Mediated by a Lethal Toxin Sensitive Allele of Nalp1b/Nlrp1b

Pathogenesis of Bacillus anthracis is associated with the production of lethal toxin (LT), which activates the murine Nalp1b/Nlrp1b inflammasome and induces caspase-1–dependent pyroptotic death in macrophages and dendritic cells. In this study, we investigated the effect of allelic variation of Nlrp1b on the outcome of LT challenge and infection by B. anthracis spores. Nlrp1b allelic variation did not alter the kinetics or pathology of end-stage disease induced by purified LT, suggesting that, in contrast to previous reports, macrophage lysis does not contribute directly to LT-mediated pathology. However, animals expressing a LT-sensitive allele of Nlrp1b showed an early inflammatory response to LT and increased resistance to infection by B. anthracis. Data presented here support a model whereby LT-mediated activation of Nlrp1b and subsequent lysis of macrophages is not a mechanism used by B. anthracis to promote virulence, but rather a protective host-mediated innate immune response.

RECENT ADVANCES IN THE DEVELOPMENT OF AN IMPROVED HUMAN ANTHRAX VACCINE

Human anthrax vaccines currently licensed in the United States and Western Europe consist of alum-precipitated or aluminum hydroxide-adsorbed supernatant material from fermentor cultures of toxigenic, nonencapsulated strains of Bacillus anthracis. These vaccines have several drawbacks, including the need for frequent boosters, the apparent inability to protect adequately against certain strains of B. anthracis, and occasional local reactogenicity.Studies are being undertaken to develop an improved human anthrax vaccine which is safe and efficacious, and which provides long-lasting immunity. Aspects being studied include the identification of antigens and epitopes responsible for eliciting protective immunity, the mechanisms of resistance to anthrax infection, the role of specific antibody in resistance, the differences in immunity elicited by living and chemical vaccines, the potential of new adjuvants to augment immunity, and the feasibility of developing safe vaccine strains having mutationally altered toxin genes. Both living and non-living (chemical) prototype vaccines are being developed and tested.

Morphogenesis of the Bacillus anthracis Spore

Bacillus spp. and Clostridium spp. form a specialized cell type, called a spore, during a multistep differentiation process that is initiated in response to starvation. Spores are protected by a morphologically complex protein coat. The Bacillus anthracis coat is of particular interest because the spore is the infective particle of anthrax. We determined the roles of several B. anthracis orthologues of Bacillus subtilis coat protein genes in spore assembly and virulence. One of these, cotE, has a striking function in B. anthracis: it guides the assembly of the exosporium, an outer structure encasing B. anthracis but not B. subtilis spores. However, CotE has only a modest role in coat protein assembly, in contrast to the B. subtilis orthologue. cotE mutant spores are fully virulent in animal models, indicating that the exosporium is dispensable for infection, at least in the context of a cotE mutation. This has implications for both the pathophysiology of the disease and next-generation therapeutics. CotH, which directs the assembly of an important subset of coat proteins in B. subtilis, also directs coat protein deposition in B. anthracis. Additionally, however, in B. anthracis, CotH effects germination; in its absence, more spores germinate than in the wild type. We also found that SpoIVA has a critical role in directing the assembly of the coat and exosporium to an area around the forespore. This function is very similar to that of the B. subtilis orthologue, which directs the assembly of the coat to the forespore. These results show that while B. anthracis and B. subtilis rely on a core of conserved morphogenetic proteins to guide coat formation, these proteins may also be important for species-specific differences in coat morphology. We further hypothesize that variations in conserved morphogenetic coat proteins may play roles in taxonomic variation among species.

Plasmid-associated virulence factors of non-toxigenic (pX01−) Bacillus anthracis

The anthrax toxins and capsule, encoded by plasmids pX01 and pX02, respectively, are the only known virulence factors of Bacillus anthracis and are considered essential for full virulence. Some B. anthracis strains cured of pX01, such as delta Ames-1, remained virulent for mice. The virulence was partially mediated by pX02, as determined by phage transduction. pX02 plasmids from the delta Ames-1 and Pasteur strains were mutagenized with transposon Tn917 to identify loci associated with virulence. The capsule phenotype, virulence and pX02 restriction pattern of the insertion mutants were characterized. Two mutants that produced no detectable capsule were avirulent. One had a deletion of more than 20 kb, which included the structural genes required for capsule synthesis (cap); the second had an insertion outside of cap. Two mutants with reduced encapsulation had insertions at different sites outside cap and were less virulent, whereas one that was normally encapsulated, but had a high rate of pX02 curing, was unaltered in virulence. Mutants that produced greater amounts of capsule than the parental strain were more virulent, and a few that produced wild-type levels of capsule were less virulent.

Resistance to the Sterne strain of B. anthracis: phagocytic cell responses of resistant and susceptible mice

Inflammatory responses were compared in vivo, and host phagocytic cell functions compared in vitro, of mice resistant (CBA/J) and susceptible (A/J) to lethal infection with the Sterne strain of Bacillus anthracis. Polymorphonuclear leukocyte (PMN) and macrophage responses at the initial site of infection were slower in A/J mice than in CBA/J mice. Whereas in A/J mice, the number of PMN ultimately responding to infection was equal to, or greater than, that in CBA/J mice, fewer macrophages accumulated. A/J mice failed to clear relatively low doses of the organisms and died. In vitro, chemotactic responses to both serum- and bacteria-derived attractants were similar for macrophages from A/J and CBA/J mice but were reduced for PMN from A/J mice. PMN and macrophages from the two mouse strains phagocytosed and killed spores in vitro to a similar extent, although killing by A/J PMN could be blocked by prior uptake of large numbers of killed spores. Thus susceptibility to lethal infection with Sterne strain correlated with the delayed influx (PMN) and reduced accumulation (macrophages) of phagocytes at the initial site of infection, but not with defective in vitro uptake or killing of spores.

Bacillus anthracis Spores of the bclA Mutant Exhibit Increased Adherence to Epithelial Cells, Fibroblasts, and Endothelial Cells but Not to Macrophages

ABSTRACT Bacillus anthracis is the causative agent of anthrax, and the spore form of the bacterium represents the infectious particle introduced into a host. The spore is surrounded by an exosporium, a loose-fitting membrane composed of proteins and carbohydrates from which hair-like projections extend. These projections are composed mainly of BclA (Bacillus-collagen-like protein of B. anthracis). To date, exact roles of the exosporium structure and BclA protein remain undetermined. We examined differences in spore binding of wild-type Ames and a bclA mutant of B. anthracis to bronchial epithelial cells as well as to the following other epithelial cells: A549, CHO, and Caco-2 cells; the IMR-90 fibroblast line; and human umbilical vein vascular endothelium cells. The binding of wild-type Ames spores to bronchial epithelial cells appeared to be a dose-dependent, receptor-ligand-mediated event. There were similar findings for the bclA mutant, with an additional nonspecific binding component likely leading to significantly more adherence to all nonprofessional phagocytic cell types. In contrast, we detected no difference in adherence and uptake of spores by macrophages for either the wild-type Ames or the bclA mutant strain. These results suggest that one potential role of the BclA fibers may be to inhibit nonspecific interactions between B. anthracis spores with nonprofessional phagocytic cells and thus direct the spores towards uptake by macrophages during initiation of infection in mammals.