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.

Several highly attenuated spore-forming nontoxinogenic and nonencapsulated Bacillus anthracis vaccines differing in levels of expression of recombinant protective antigen (rPA) were constructed. Biochemical analyses (including electrospray mass spectroscopy and N terminus amino acid sequencing) as well as biological and immunological tests demonstrated that the rPA retains the characteristics of native PA. A single immunization of guinea pigs with 5 ؋ 10 7 spores of one of these recombinant strains, MASC-10, expressing high levels of rPA (>100 g/ml) from a constitutive heterologous promoter induced high titers of neutralizing anti-PA antibodies. This immune response was long lasting (at least 12 months) and provided protection against a lethal challenge of virulent (Vollum) anthrax spores. The recombinant B. anthracis spore vaccine appears to be more efficacious than the vegetative cell vaccine. Furthermore, while results clearly suggest a direct correlation between the level of expression of PA and the potency of the vaccine, they also suggest that some B. anthracis spore-associated antigen(s) may contribute in a significant manner to protective immunity.The etiological agent of anthrax disease in animals and humans is the spore-forming bacterium Bacillus anthracis. The major factors of virulence of B. anthracis are located on two plasmids, pXO1 and pXO2. pXO2 encodes a poly-D-glutamic acid capsule (19, 41), while pXO1 encodes two binary exotoxins, the lethal toxin (LT) and the edema toxin (ET) (43,46,61). These two toxins are composed of three different proteins: protective antigen (PA), edema factor (EF), and lethal factor (LF) (for a review, see reference 36). PA is the common receptor binding domain of the toxins and can interact with the two different effector domains, EF and LF, to mediate their entry into target cells (14). EF is a calmodulin-dependent adenylate cyclase (37) responsible for the edema seen at the site of infection in experimental animals (17). The LF is a metalloprotease (34) recently shown to cleave the amino termini of the mitogen-activated protein kinase kinases 1 and 2, which results in their inactivation (13). It remains to be determined whether these are the main physiological substrates for the LT activity in vivo (5,22).Two types of anthrax vaccines are licensed for use in humans: the spores of the toxigenic, nonencapsulated B. anthracis STI-1 strain (55) and the cell-free PA-based vaccines consisting of aluminum hydroxide-adsorbed supernatant material from cultures of the toxigenic, nonencapsulated B. anthracis strain V770-NPI-R (49) or alum-precipitated culture filtrate from the Sterne strain (6). The use of the live attenuated STI-1 occasionally results in general and local adverse responses, observed both after primary application and revaccination, and the frequency of responses increases with the number of vaccinations (58). Furthermore, it was reported that the STI-1 vaccine has a relatively low immunogenicity (reviewed by Stepanov et al. in reference 58). To increase the i...

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The most aggressive form of anthrax results from inhalation of airborne spores of Bacillus anthracis and usually progresses unnoticed in the early stages because of unspecific symptoms. The only reliable marker of anthrax is development of bacteremia, which increases with disease progress. Rapid diagnosis of anthrax is imperative for efficient treatment and cure. Herein we demonstrate that the presence and level of a bacterial antigen, the protective antigen (PA), a component of B. anthracis toxins, in host sera can serve as a reliable marker of infection. This was tested in two animal models of inhalation anthrax, rabbits and guinea pigs infected by intranasal instillation of Vollum spores. In both models, we demonstrated qualitative and quantitative correlations between levels of bacteremia and PA concentrations in the sera of sick animals. The average time to death in infected animals was about 16 h after the appearance of bacteremia, leaving a small therapeutic window. As the time required for immunodetection of PA can be very short, the use of this marker will be beneficial for faster diagnosis and treatment of inhalation anthrax.

The neuropathogenicity of West Nile virus (WNV) and two derived attenuated strains WN25 and WN25A, was studied in young adult ICR mice and in severe combined immunodeficient (SCID) mice. Similarity in serology and RNA fingerprints were found between WNV and WN25. The viral envelope proteins of the attenuates differed from WNV in their slower mobility in SDS-PAGE due probably to the presence of N-linked glycan. The three strains were lethal to ICR mice by intracerebral (IC) inoculation, but when inoculated intraperitoneally (IP), WNV caused viremia, invaded the CNS and was lethal, whereas the attenuates showed no viremia or invasion of the CNS. The attenuates elicited antibodies to comparable levels as WNV in IP-infected mice, conferring upon them immunity to IC challenge with the wild type. In IP-inoculated SCID mice the three strains exhibited similar high viremiae that lasted until death of the animals. All strains invaded the CNS and proliferated in the mouse brain to similar high titers, but differed largely in the time of invasion: WNV invaded the CNS of SCID mice (and two other mouse strains) much earlier than the attenuates, which showed large intervals in their time of invasion into individual mouse brains within the group. The data presented for SCID mice indicate that WN25 and WN25A have truly lost the neuroinvasive property, and that this property materialized by a prescribed, active process specific for WNV.