Long-lasting and transmission-blocking activity of antibodies toPlasmodium falciparumelicited in mice by protein conjugates of Pfs25

Abstract

Malaria is a leading cause of morbidity and mortality, estimated to cause >1 million childhood deaths annually. Plasmodium falciparum causes the most severe form of the disease. There is as yet no licensed vaccine for this disease, despite over a half century of research. In this study, we investigated a transmission-blocking vaccine candidate, the ookinete surface protein Pfs25. Antibodies against Pfs25, drawn in during a bite, can block parasite development in the mosquito midgut, preventing transmission to other individuals. Pfs25 is a low-molecular-weight protein, by itself not immunogenic. To increase its immunogenicity, we investigated several methods of conjugating Pfs25 to itself and to other proteins: recombinant Pseudomonas aeruginosa exotoxin A, and ovalbumin, using amide, hydrazone, or thioether linkages. All conjugates were immunogenic and induced booster responses in mice. The scheme to form amide bonds between proteins by using adipic acid dihydrizide as a linker produced the most immunogenic conjugates. Adsorption of the conjugates onto aluminum hydroxide further increased the antibody response. Remarkably, the antibody levels 3 or 7 months after the last injection were significantly higher than those 1 wk after that injection. The observed transmission-blocking activity of immune sera correlated with antibody levels measured by ELISA. malaria ͉ vaccine O ne approach for a vaccine against malaria is to block transmission of the parasite from mosquitoes to humans. When ingested by a mosquito with the blood meal, antibodies against the sexual and the mosquito stage-specific surface antigens can block parasite development inside the vector (1). Four proteins have been identified as potential inducers of transmission-blocking antibodies (2-5). Two of these are expressed on the surface of gametes and in intracellular gameotocytes. The other two are the Plasmodium falciparum surface proteins with apparent molecular masses of 25 kDa (Pfs25) and 28 kDa (Pfs28), expressed exclusively on the zygote and ookinete surfaces during the mosquito stage of the infection. No antibody response to these two proteins has been shown in people infected with malaria and living in endemic countries (6). Pfs25 from different parts of the world has shown minimal variation in its amino acid sequence (7). This relative homogeneity, likely a result from not being subjected to immune pressure in the human host, makes Pfs25 an attractive candidate for a malaria transmission-blocking vaccine (8). Pfs25 is poorly immunogenic in mice and in humans, even if administered with adjuvant (9, 10). In this article, we show that Pfs25 bound onto itself or onto another protein induced high levels of transmission-blocking antibodies in mice.

Addition of CpG ODN to recombinant Pseudomonas aeruginosa ExoProtein A conjugates of AMA1 and Pfs25 greatly increases the number of responders

“…The antibodies elicited by Pfs25-rEPA F1/Alhydrogel + CPG 7909 displayed almost the same activity in TBA as the antibodies elicited by Pfs25-rEPA F1/Alhydrogel. The TBA activities at 2600 units of antibody were also comparable to those previously published [6,7]. These results demonstrated that the immune sera induced by the formulation with CPG 7909 were as functional as the immune sera induced by the formulation without CPG 7909 at the same ELISA units.…”

“…Conjugation of Pfs25 to outer-membrane protein complex of Neisseria meningitidis (OMPC) greatly increased and sustained the specific antibody levels in animals [5]. Similar results were observed with the conjugation of Pfs25 to itself or to Pseudomonas aeruginosa ExoProtein A (rEPA) [6]. Our study showed that conjugation of AMA1 and Pfs25 to rEPA significantly enhanced the immune response against both malarial antigens, with a 3-fold increase for AMA1-rEPA/Alhydrogel and over a 1000-fold increase for Pfs25-rEPA/ Alhydrogel compared to unconjugated antigens [7].…”

“…Enhancing the immunogenicities of these antigens to obtain a protective and sustained response in humans remains a formidable task that confronts malarial vaccine researchers today [3,4]. One strategy that we have used to increase immunogenicity was to chemically conjugate malarial antigens to a carrier protein [5][6][7][8][9]. Conjugation of Pfs25 to outer-membrane protein complex of Neisseria meningitidis (OMPC) greatly increased and sustained the specific antibody levels in animals [5].…”

A novel plant-produced Pfs25 fusion subunit vaccine induces long-lasting transmission blocking antibody responses

“…32 Furthermore, chemical conjugation of PpPfs25H-A to either ExoProtein A (EPA) of Pseudomonas aeruginosa or an outer membrane protein complex of Neisseria meningitides was shown to have a dramatically enhancing effect on both anti-Pfs25 and TB antibody responses. [13][14][15][16] Chemically conjugated Pfs25-EPA nanoparticles were subsequently manufactured under current Good Manufacturing Practices conditions and evaluated in mice in the presence of Alhydrogel, demonstrating superior immunogenicity compared with unconjugated Pfs25 plus Alhydrogel. 33 A Phase 1 clinical trial (NCT01434381) in which 30 healthy malaria-naïve adult subjects received up to three doses of the Pfs25-EPA TBV candidate (8 or 16 µg of Pfs25 at 0 and 2 mo or 47 µg of Pfs25 at 0, 2, and 4 mo) has recently been completed.…”

“…Despite these challenges, recent success has been achieved with recombinant versions of Pfs25 produced in yeast. [10][11][12][13][14][15][16][17] During the last two decades, several groups have demonstrated the potential of plants as an effective and highly scalable platform for production of recombinant vaccine antigens and therapeutic proteins (for a review see refs. 18 and 19).…”