Peptide-Protein Conjugate Vaccines

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Bacillus anthracis: A recombinant PA was isolated from an uncapsulated strain. Several formaldehyde treated and or alum adsorbed formulations were immunogenic in mice. Clinical evaluation of these formulations is underway. 150 18-45 year old volunteers have been injected up to 4 times with no serious adverse effects. Local and systemic reactions were rare and minor. 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 ≥ 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 mcg/mL following the 3rd injection, 157.4 mcg/mL following the 4th and 277microgram/mL following the 6th. 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. Additional conjugating methods using 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 statistical difference between them. The induced antibodies were opsonophagocytic. Peptides 10 to 20-mer long, and 10-15 mole PGA per mole protein were the most immunogenic. Dose response experiments of an rPA-PGA conjugate, using between 0.31 to 20 mcg PGA/mouse showed 1.25 mcg to be optimal for a PGA response, while PA antibody levels increased with higher immunizing dosages. The use of alum increased PA antibody levels while having little effect upon anti PGA 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. Plasmodium falciparum: The most studied experimental malaria vaccine candidate is the circumsporozoite protein (CSP), 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 by a scheme suitable for humans. High levels of antibodies were induced, with circumsporozoite neutralizing activity correlated roughly 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. Remarkably, the serum antibody levels 3 and 7 months after immunization were higher than 1 week after the last injection. The best immunogens were created using 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. The Pfs25 conjugate was also bound to a synthetic (NANP)5 of the repeat unit of the CSP. This conjugate induced IgG antibodies to both its components and adsorption onto alum increased further both levels: from 0.8 to 87g/mL for Pfs25 and from 3.3 to 53 EU/mL for CSP after 2 injections. Three months after the 2nd injection IgG anti Pfs25 increased to 524 mcg/mL and anti CSP to 120 EU/mL.