Summary For malaria to be transmitted, the Plasmodium falciparum parasite must invade an erythrocyte and undergo gametocytogenesis. When mature intraerythrocytic gametocytes are taken up in a blood meal by a mosquito they emerge as gametes and, once fertilized, continue to differentiate into infectious sporozoites. One of the major proteins associated with the surface of the parasite during gamete differentiation is Pfs230, a 360 kDa member of a family of P. falciparum proteins that contains a repeated cysteine motif domain. To characterize the role of different regions of Pfs230, the gene was disrupted by targeted integration and clones isolated that expressed distinct sections of Pfs230. Independent clones D1.356 a and b express the first 452 amino acids (aa) of Pfs230 and do not contain a cysteine motif domain, whereas clones D2.850 a and b express the first 950 aa, including the first cysteine motif domain. Although both sets of clones undergo gametogenesis and produce morphologically normal gametes, neither truncated Pfs230 is located on the surface of the gamete. In clones D1.356 a and b, the 452 aa Pfs230 is secreted into the parasitophorous vacuole and released as a soluble protein when the parasite emerges from the erythrocyte as a gamete. In marked contrast, the 950 aa form of Pfs230 expressed by clones D2.850 a and b is sequestered in a novel tubular compartment in the erythrocyte cytoplasm. This sexual‐stage tubular intraerythrocytic compartment (STIC) is not recognized by antibodies specific for proteins associated with the parasitophorous vacuole membrane (Pfs16 or Exp‐1) or Maurer’s clefts (Pfsbp 1 or mAb LWL1) or intraerythrocytic asexual parasite proteins (PfEMP2 or HRP II).

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Proteolytically processed 310 kDa form of Plasmodium falciparum gamete surface antigen, Pfs230, is the target of malaria transmission-blocking monoclonal antibodies. To design a recombinant malaria transmission-blocking subunit vaccine, the amino terminus of the 310 kDa surface-exposed form of Pfs230 was mapped to amino acids (aa) 522 and 584 using a series of peptides and recombinant proteins encoding distinct regions of Pfs230. Antiserum generated against an Escherichia coli-produced recombinant protein, spanning the Pfs230 processing site and extending into the cysteine domains, r230/MBP.C (aa 443-1132), reduced parasite infectivity by 71.2-89.8%. To determine if the region spanning the cleavage site blocked malaria transmission when produced as a secreted protein by Saccharomyces cerevisiae, y230.CA14 (aa 467-584) was generated, purified, emulsified in adjuvant and used to vaccinate mice. In contrast to E. coli-produced r230/MBP.C, the immune response generated against y230. CA14 was very weak. To enhance the response, y230.CA14 was mixed with tetanus toxoid, chemically crosslinked, repurifed, and its immunogenicty compared with unconjugated y230.CA14. Conjugated-y230. CA14/TT required fewer booster injections to induce an immune response against Pfs230 and the antibodies generated reacted with the surface of intact gametes and immunoprecipitated radiolabelled Pfs230 extracted from 125I surface-labelled gametes to a greater extent. After seven injections, all y230.CA14 vaccinated mice developed anti-Pfs230 antibodies and the isotype profile was the same. In addition to enhancing the initial immune response generated against y230.CA14, conjugation focuses the immune response toward epitopes within the region of Pfs230 present on the surface of the gamete.

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