A bicomponent Plasmodium falciparum investigational vaccine composed of protein-peptide conjugates.

There is yet no licensed vaccine against malaria, a serious human disease affecting mostly children, with an annual death rate of about one million. Plasmodia, the malaria-causing parasites, have two obligatory hosts: mammals or birds, in which they multiply asexually, and mosquitoes with sexual multiplication. The most common and serious type of malaria is caused by Plasmodium falciparum. The circumsporozoite protein (CSP), a major surface antigen of sporozoites, is a protective antigen. A unique feature of P. falciparum CSP is its large central domain composed of over 30 tetrapeptide repeats of Asn-Ala-Asn-Pro (NANP). Several NANP peptide-protein conjugates were tested clinically but elicited a low level of CSP antibodies for a short duration. To provide a CSP-based candidate vaccine, we investigated recombinant CSP and NANP conjugates of various peptide lengths, with different N-terminal amino acids, bound at different ratios to various carrier proteins. Injected into mice, CSP alone and CSP or NANP conjugates induced antibodies with booster responses and were positive by the sporozoite immunofluorescent assay. The use of the mosquito stage P. falciparum ookinete surface protein, Pfs25, cross-linked onto itself as a carrier for NANP, induced in mice high levels of uniquely long-lasting antibodies to both vaccine components with secondary biological activities, that will provide immunity to liver infection by sporozoites and block transmission by mosquitoes.

Additional conjugation methods and immunogenicity of Bacillus anthracis poly-gamma-D-glutamic acid-protein conjugates.

The capsule of Bacillus anthracis, composed of poly-gamma-d-glutamic acid (gammaDPGA), is an essential virulence factor of B. anthracis. The capsule inhibits innate host defense through its antiphagocytic action. gammaDPGA is a poor immunogen, but when covalently bound to a carrier protein, it elicits serum antibodies. To identify the optimal construct for clinical use, synthetic gammaDPGAs of different lengths were bound to carrier proteins at different densities. The advantages of the synthetic over the natural polypeptide are the homogeneous chain length and end groups, allowing conjugates to be accurately characterized and standardized and their chemical compositions to be related to their immunogenicities. In the present study, we evaluated, in addition to methods reported by us, hydrazone, oxime, and thioether linkages between gammaDPGA and several proteins, including bovine serum albumin, recombinant Pseudomonas aeruginosa exotoxin A, recombinant B. anthracis protective antigen (rPA), and tetanus toxoid (TT). The effects of the dosage and formulation on the immunogenicities of the conjugates were evaluated in mice. All conjugates were immunogenic. The optimal gammaDPGA chain length of 10 to 15 amino acids and the density, an average of 15 mol gammaDPGA per mol of protein, were confirmed. The thioether bond was the optimal linkage type, and TT and rPA were the best carriers. The optimal dosage was 1.2 to 2.5 microg of gammaDPGA per mouse, and adsorption of the conjugates onto aluminum hydroxide significantly increased the antibody response to the protein with a lesser effect on anti-gammaDPGA levels.

Poly(gamma-D-glutamic acid) protein conjugates induce IgG antibodies in mice to the capsule of Bacillus anthracis: a potential addition to the anthrax vaccine.

Both the protective antigen (PA) and the poly(gamma-d-glutamic acid) capsule (gamma dPGA) are essential for the virulence of Bacillus anthracis. A critical level of vaccine-induced IgG anti-PA confers immunity to anthrax, but there is no information about the protective action of IgG anti-gamma dPGA. Because the number of spores presented by bioterrorists might be greater than encountered in nature, we sought to induce capsular antibodies to expand the immunity conferred by available anthrax vaccines. The nonimmunogenic gamma dPGA or corresponding synthetic peptides were bound to BSA, recombinant B. anthracis PA (rPA), or recombinant Pseudomonas aeruginosa exotoxin A (rEPA). To identify the optimal construct, conjugates of B. anthracis gamma dPGA, Bacillus pumilus gamma dLPGA, and peptides of varying lengths (5-, 10-, or 20-mers), of the d or l configuration with active groups at the N or C termini, were bound at 5-32 mol per protein. The conjugates were characterized by physico-chemical and immunological assays, including GLC-MS and matrix-assisted laser desorption ionization time-of-flight spectrometry, and immunogenicity in 5- to 6-week-old mice. IgG anti-gamma dPGA and antiprotein were measured by ELISA. The highest levels of IgG anti-gamma dPGA were elicited by decamers of gamma dPGA at 10 -20 mol per protein bound to the N- or C-terminal end. High IgG anti-gamma dPGA levels were elicited by two injections of 2.5 microg of gamma dPGA per mouse, whereas three injections were needed to achieve high levels of protein antibodies. rPA was the most effective carrier. Anti-gamma dPGA induced opsonophagocytic killing of B. anthracis tox-, cap+. gamma dPGA conjugates may enhance the protection conferred by PA alone. gamma dPGA-rPA conjugates induced both anti-PA and anti-gamma dPGA.

Study of capsular polysaccharide from Vibrio parahaemolyticus.

The leading cause of food poisoning in both Taiwan and Japan is Vibrio parahaemolyticus infection, whose mechanism of enteropathogenesis is still unclear. To evaluate whether surface components are responsible for the intestinal adhesion of V. parahaemolyticus, we have developed a novel method for isolating the capsular polysaccharide (CPS) from V. parahaemolyticus (serotype O4:K8). We found that culturing of V. parahaemolyticus in broth for 1 week or more changed the colony form of the bacteria on an agar plate from opaque to translucent. The translucent colonies of V. parahaemolyticus contained little CPS and exhibited a much lower level of adherence to epithelial cells (Int-407) than the opaque colonies of the bacteria. Incubation of V. parahaemolyticus in medium supplemented with bile increased the levels of CPS and adherence. Treatment of V. parahaemolyticus with anti-CPS but not anti-LPS serum decreased the level of bacterial adherence. In addition, purified CPS bound to epithelial cells in a dose-dependent manner. Intranasal administration of CPS to mice in the presence of adjuvants such as immunostimulatory sequence oligodeoxynucleotides or cholera toxin elicited CPS-specific mucosal and systemic immune responses. These results indicate that CPS plays an important role in the adherence of V. parahaemolyticus to its target cells and may be considered a potential target for the development of a vaccine against this pathogen.