Peptide-Protein Conjugate Vaccines

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BACILLUS ANTHRACIS, a potential cause of lethal human infection, has 2 essential virulence factors without either of which it is not pathogenic for humans: the anthrax toxin and the capsule. The toxin is composed of 3 peptides: Lethal Factor, Edema Factor, and Protective Antigen (PA). PA is the toxin part that binds to mammalian cells. A 20 KDa peptide must be hydrolyzed off it exposing a site to which LF or EF may bind, rendering toxins that enzymatically modify substrates in the mammalian cell cytosol. The capsule is composed of poly-D-gamma-glutamic acid (PGA).It is non-immunogenic and its protective effect not clear. The licensed vaccine, PA based, is safe and protective but has limitations that justify development of improved vaccines. A recombinant PA was isolated from an uncapsulated strain. Several formulations with formaldehyde treated and alum adsorbed materials were found to be immunogenic in mice. Clinical evaluation of these formulations is underway. The capsule has been isolated from a non toxic strain and it or corresponding synthetic peptides were bound to BSA, rEPA, rPA or tetanus toxoid. Thioether, hydrazone and oxime linkages between the gamma D-PGA and the proteins, with active groups at the C or N termini yielded conjugates immunogenic in mice, with no statistical difference between them. These antibodies were opsonophagocytic. Peptides 10 to 20-mers long, and 10-15 mole gamma D-PGA per mole protein were the most immunogenic. Dose response experiments of an rPA-PGA conjugate, with doses between 0.31 and 20 mcg/mouse showed 1.25 mcg to be the optimal dose for a PGA response, while PA antibody levels increased with higher immunizing doses. The use of alum adjuvant increased PA antibody levels while having little effect upon anti PGA levels. Serum IgG anti PA was measured in 246 sera of recruits injected with the Anthrax Vaccine Adsorbed (AVA ) stored at the Department of Defense Serum Repository. Paired sera were analyzed by ELISA. Serum conversion rates of !Y 4-fold increase in antibody levels were: pre-post 3rd 85.3%, pre 4th-post 4th 67.9% and pre 6th-post 6th 45%. Geometric mean levels of all individuals were 59.9 microgr/mL following the 3rd injection, 157.4 microgr/mL following the 4th and 277 microgram/mL following the 6th. PLASMODIUM FALCIPARUM: Malaria is a leading cause of morbidity and mortality globally, especially in children, estimated to cause over a million childhood deaths annually. P. falciparum causes the most severe form of disease. Experimental vaccines have been described and some tested clinically but no licensed vaccine is available. The most studied is the circumsporozoite protein (CS), expressed extracellularly on the sporozoite, and various forms of its synthesized repeat unit, NANP. These vaccines were safe and immunogenic but poorly protective, even when administered with adjuvants. Based on our studies with peptides of the B. anthracis capsule, peptides of 4 repeat units of NANP were synthesized and bound to carrier proteins and their immunogenicity studied in general purpose mice without adjuvants and by a scheme suitable for humans. High levels of antibodies were induced, with circumsporozoite neutralizing activity roughly correlated to levels measured by ELISA. In another approach to provide a transmission blocking vaccine, Pfs25, a low molecular weight protein, non immunogenic by itself, was bound to itself or to carrier proteins by several methods: amide, hydrazone or thioether linkages and injected into mice to evaluate their antibody responses. All conjugates were immunogenic with booster responses upon reinjection. The best immunogens were created using Adipic acid dihydrazide as the linker.