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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.

SummaryAnthrax is a toxin-mediated disease, the lethal effects of which are initiated by the binding of protective antigen (PA) with one of three reported cell surface toxin receptors (ANTXR). Receptor binding has been shown to influence host susceptibility to the toxins. Despite this crucial role for ANTXR in the outcome of disease, and the reported immunomodulatory consequence of the anthrax toxins during infection, little is known about ANTXR expression on human leucocytes. We characterized the expression levels of ANTXR1 (TEM8) on human leucocytes using flow cytometry. In order to assess the effect of prior toxin exposure on ANTXR1 expression levels, leucocytes from individuals with no known exposure, those exposed to toxin through vaccination and convalescent individuals were analysed. Donors could be defined as either 'low' or 'high' expressers based on the percentage of ANTXR1-positive monocytes detected. Previous exposure to toxins appears to modulate ANTXR1 expression, exposure through active infection being associated with lower receptor expression. A significant correlation between low receptor expression and high anthrax toxin-specific interferon (IFN)-g responses was observed in previously infected individuals. We propose that there is an attenuation of ANTXR1 expression post-infection which may be a protective mechanism that has evolved to prevent reinfection.

A temperature-sensitive mutant of Bacillus subtilis W23, dna-20 (Ts), has been isolated and shown to be defective in initiation of rounds of chromosome replication at the nonpermissive temperature. Upon transfer of dna-20(Ts) from 30 to 45 C, deoxyribonucleic acid synthesis, as measured by [3H]thymine incorporation, gradually ceases. The distribution of genetic markers among unreplicated and replicated deoxyribonucleic acid, isolated from dna-20(Ts) after a period at 43 C in a medium containing 5-bromouracil, and fractionated in a CsCl gradient, shows that the cessation of initiation at the higher temperature is immediate. On the other hand, ribonucleic acid and protein synthesis continues at elevated or unaltered rates for some time after the shift to 45 C. Marker frequency analysis shows that all rounds of replication in progress at the time of the temperature shift terminate rapidly (within 40 min), even when chromosomes are replicating dichotomously in rich media. dna-20(Ts) remains 100% viable for at least 2 h at 45 C. Over a 5-h period at 45 C the nuclear bodies remain compact; a small number (less than 5%) of deoxyribonucleic acid-less cells are produced, but there is no morphological distortion of the cells. When the cells are returned to 30 C after 2 h at 45 C, chromosome replication is initiated rapidly at the normal origin and then proceeds in the normal established sequence. However, a second round of replication is initiated soon after the first. dna-20(Ts) has been shown to map as a B-group mutation, the major class of initiation mutants identified in B. subtillus 168.