Bacillus anthracis. B. anthracis, a cause of lethal human infection with potential for bioterrorism, has two essential virulence factors without either of which it is not pathogenic for humans: the anthrax toxin and the capsule. The toxin is composed of three peptides: Lethal Factor, Edema Factor, and Protective Antigen (PA). The capsule is composed of poly-d-gamma-glutamic acid (PGA). By itself, the capsule is non-immunogenic and its protective effect is not clear. Anthrax is transmitted indirectly, via spores, of different structure and composition than the vegetative organism. The PA-based licensed vaccine is safe and protective under normal conditions, but enhancement of its immunogenicity may be needed in the event of bioterrorism. We sought to induce capsular and anti-spore antibodies as a potential means to expand the immunity conferred by the available anthrax vaccines. We isolated a recombinant PA from an uncapsulated strain, several formaldehydetreated and/or alum-adsorbed formulations of which were immunogenic in mice. The formulations were safe in adult volunteers, and local and systemic reactions were rare and minor. Preliminary antibody assays compare favorably with those of the licensed vaccine. We isolated the capsule from a non-toxic strain and bound it or corresponding synthetic peptides to BSA, rEPA, rPA, or tetanus toxoid. Thioether, hydrazone, and oxime linkages between the PGA and the proteins with active groups at the C- or N-termini yielded conjugates immunogenic in mice, with no statistically different responses to the conjugates. The induced antibodies were opsonophagocytic. Peptides 10- to 20-mer long and 10 to 15 mole PGA per mole protein were the most immunogenic. Dose-response experiments of an rPA-PGA conjugate, using between 0.31 and 20 g PGA per mouse, showed 1.25 g to be optimal for a PGA response while PA antibody levels increased with higher immunizing dosages. The use of alum increased PA antibody levels with little effect on antiPGA levels. Chimpanzees were immunized, 10 mcg PGA/animal, sc, one with PA-PGA another with TT-PGA, with the goal of preparing humanized monoclonal antibodies to PGA. Both chimps responded with antibodies to both vaccine components. Higher anti PGA levels were obtained in the TT-PGA injected chimp. PGS specific IgG1 and IgG3 were prepared. The glycosyl part of the glycoprotein BclA (collagen-like protein of B. anthracis spore) is an oligosaccharide composed of 2-O-methyl-4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy-d-glucose (referred to as anthrose) and three rhamnose residues. We found structures similar to anthrose in the sidechain of the CP of Shewanella spp. MR-4: 4-(3-hydroxy-3-methylbutanamido)-4,6-dideoxy-dglucose. Under certain growth conditions, the bacteria produce a variant CP lacking one methyl group on the hydroxybutyrate: 4-(3-hydroxybutanamido)-4,6-dideoxy-d-glucose. In contrast to anthrose, neither of the Shewanella CPs is 2-O-methylated. Both Shewanella CP variants reacted with anti B. anthracis spore sera. We also found structures similar to anthrose in the flagellae of Pseudomonas syringae, which are reportedly glycosylated with a similar terminal saccharide: 4-(3-hydroxybutanamido)-4,6-dideoxy-2-O-methyl-d-glucose. In a fluorescence microscopy assay, sera produced by immunization with Shewanella or P. syringae cells bound to B. anthracis spores but not to B. cereus spores. Protein conjugates of the two variants of Shewanella CP induced antibodies that bound to both Shewanella CP variants, by ELISA, and to B. anthracis spores, detected by fluorescence microscopy. We propose the use of Shewanella CP conjugates as a component of an anthrax vaccine. We also elucidated the structure of the Shewanella MR-4 LPS carbohydrate backbone but found no anthrose-like sugar, confirming the presence of such a sugar in the CP only. Plasmodium falciparum. Malaria, a leading cause of morbidity and mortality globally, especially in children, is estimated to cause over a million childhood deaths annually. P. falciparum causes the most severe form of the disease. Experimental vaccines have been described and some tested clinically, but no licensed vaccine is available. The goal of this study is to provide vaccine candidates. The most studied experimental malaria vaccines are the circumsporozoite protein (CSP), expressed extracellularly on the sporozoite, and various forms of its synthesized repeat unit, NANP. The vaccines have been shown to be safe and immunogenic, but their protection is poor and of limited duration even when administered with adjuvants. We used two approaches to provide experimental malaria vaccines. 1. Directed to the sexual, mosquito parasite stage, to provide a transmission blocking vaccine. Pfs25, a low molecular mass protein, non immunogenic by itself, was bound onto itself or to carrier proteins by amide, hydrazone or thioether linkages. Injected into mice, all conjugates were immunogenic with booster responses upon reinjection. Remarkably, the serum antibody levels 3 and 7 months after immunization were higher than 1 week after the last injection. The best immunogens used adipic acid dihydrazide as the linker. Adsorption of the conjugates onto alum increased further the antibody levels. Transmission blocking activity of immune sera correlated with antibody levels measured by ELISA. 2. Directed to the asexual, human stage of the parasite;CSP was cloned into E. coli, isolated and several formulations, including adsorption onto alum, were evaluated in young outbred mice. Based on our studies with peptides of the B. anthracis capsule, peptides of 4-5 repeat units of NANP were synthesized and bound to carrier proteins at different molar ratios and end groups. Injected into general purpose mice, all tested preparations induced high levels of antibodies that bound to circumsporozoites in IFA. NANP-Pfs25-Pfs25 conjugates induced long lasting antibodies to both vaccine components;3 months serum levels higher than 1 week after the last injection. Alum adsorbed CSP induced higher antibody levels than the non-adsorbed protein. The terminal amino acid of the CSP-derived peptides was shown to be an important determinant, with NPNA-protein being the best immunogen.
|Chen, Zhaochun; Schneerson, Rachel; Lovchik, Julie et al. (2011) Pre- and postexposure protection against virulent anthrax infection in mice by humanized monoclonal antibodies to Bacillus anthracis capsule. Proc Natl Acad Sci U S A 108:739-44|
|Kubler-Kielb, Joanna; Majadly, Fathy; Biesova, Zuzana et al. (2010) A bicomponent Plasmodium falciparum investigational vaccine composed of protein-peptide conjugates. Proc Natl Acad Sci U S A 107:1172-7|
|Vinogradov, Evgeny; Kubler-Kielb, Joanna; Korenevsky, Anton (2008) The structure of the carbohydrate backbone of the LPS from Shewanella spp. MR-4. Carbohydr Res 343:2701-5|