Plasmodium berghei HAP2 induces strong malaria transmission-blocking immunity in vivo and in vitro

Abstract

Fertilization in Plasmodium is a complex process that occurs in the gut of the female Anopheles mosquito upon uptake of a bloodmeal. It requires the emergence of the gametocyte from the RBC and release of eight flagellate male gametes from each male cell, and subsequent fertilization of a similarly emerged immotile extracellular female macrogamete. Previous studies have demonstrated that antibodies against male gamete surface proteins ingested from the blood of an infected and immunized host inhibit parasite transmission. Gene disruption studies in Plasmodium berghei and complimentary studies on the green alga Chlamydomonas have shown that a conserved male gamete sterility gene, HAP2, is essential for fusion of male and female gametes. Genetic disruption of the HAP2 locus revealed that parasite fertilization is prevented, yet hap2 KO male gametes still retained the ability to form tight pre-fusion membrane attachments with females.We demonstrate that heterologous expression of the P. berghei HAP2 protein in Escherichia coli, and subsequent immunization of rabbits, has produced anti-sera that react specifically with recombinant HAP2, and with the native protein on the male gamete. Additionally, anti-HAP2 sera reduces in vitro formation of ookinetes by up to 81%, and, using standard membrane feeding assays, reduces oocyst burden within the mosquito host by up to 81.1%, and prevalence of in vivo infection by up to 34%. Inhibition is dose dependent. These results indicate that HAP2 should be considered as a potential target for any future anti-malarial transmission-blocking vaccine.

Introduction

Malaria is a serious, acute and relapsing disease caused by protozoan parasites of the genus Plasmodium. The parasites of man/mammals are transmitted exclusively by mosquitoes of the genus Anopheles. There are an estimated 247 million malaria cases annually, causing approximately 1 million deaths per year, the majority of whom are African children under the age of five [1]. Successful transmission of Plasmodium from humans to mosquitoes depends on the presence of male (micro) and female (macro) gametocytes in the peripheral blood, which rapidly differentiate into gametes upon uptake by the Anopheles vector. Following fertilization, zygotes develop into motile ookinetes, which traverse the peritrophic matrix and midgut epithelium, lodge under the basal lamina of the midgut, and develop into oocysts. Sporozoites develop in the oocyst, and migrate through the haemocoel to the salivary glands from where they are injected into the next vertebrate host.

Transmission of Plasmodium through Anopheles is potentially regulated by a variety of factors derived from the parasite, the vertebrate host and the mosquito vector [2]. Amongst these are transmission-blocking (TB) antibodies. Both fertilization and ookinete/oocyst development within the mosquito midgut can be inhibited by both naturally occurring or vaccine induced antibodies ingested within the mosquito bloodmeal, therefore preventing the completion of the parasite lifecycle. Fertilization can be blocked through agglutination or complement-mediated lysis of gametes, or by steric inhibition of fertilization [3], [4]. Post-fertilization, ookinete differentiation can be inhibited by complement-mediated lysis, melanisation, antibody dependent phagocytosis or by blocking recognition of the midgut epithelial cells [5], [6]. It has previously been demonstrated in various studies that immunization with various surface proteins of the malarial sexual stages can completely inhibit transmission (see below).

A number of different antigenic targets that potentially induce TB activity in malaria have been investigated over the last 20 years. At present, transmission-blocking vaccine (TBV) immunogens for which evidence is most compelling are; P48/45 and P230 (target proteins on the male and female gametes), and P25 and P28 (expressed on the surface of zygotes and ookinetes). P48/45 is expressed by gametocytes [7], [8], and is present on the surface of both micro- and macrogametocytes. Pfs48/45 plays an essential role in parasite fertilization, and targeted disruption of the gene affects the male gamete's capacity to bind to female gametes [9]. Correctly folded Pfs48/45 protein elicits TB immunity in mice by antibodies that, on current evidence, exclusively target conformation dependent epitopes [10]. Anti-P48/45 antibodies can be found in human sera from endemic areas, and correlate with TB activity [11], [12]. Pfs230, another gametocyte surface antigen, is present on the gamete surface following emergence. Antibodies against Pfs230 prevent oocyst development [13], and lyse gametes in a complement-dependent manner [41]. Targeted disruption of the Pfs230 locus can result in the production of truncated proteins that are not retained on the surface of the gametocyte or gamete [13]. Additionally, these mutant Δ pfs230 male gametes cannot interact with erythrocytes, or form exflagellation centres [14].

P25 and P28 are lead targets for the development of TBVs [15], and are secreted to the surface of the macrogamete and ookinete. The proteins appear to have overlapping redundant functions [16]. Mouse antiserum against native [33], [39], or heterologously expressed P25 or P28 completely inhibits parasite development in mosquitoes [15], [17]. Additionally, phase I human clinical trials using recombinant Pfs25 and Pvs25 have resulted in the production of antibodies that significantly inhibit transmission of the parasites, further confirming the potential of these immunogens [18], [19], [42]. Recombinant histidine₆-tagged Pvs25 expressed in yeast (Pvs25H) induces antibodies that block transmission by up to 80% in terms of mean oocyst intensity (number per midgut), and by 20–30% in reduction of prevalence of infection (number of infected mosquitoes) [18].

In seeking to expand the potential repertoire of available TB vaccines, to permit potential combination of immunogens, there is a real need for the identification of novel target candidates. A male-specific sterility gene (HAP2) was originally identified in Arabidopsis thaliana[20]. A homologous family member was identified during a screen for lily genes whose transcripts were upregulated in sperm [21]. Subsequently, HAP2 homologues have been identified in higher plants, non-pathogenic and pathogenic protists, including Plasmodium spp. Studies carried out on the green alga Chlamydomonas and in the rodent malaria model Plasmodium berghei[22] identified that in Plasmodium, HAP2 is expressed exclusively in gametocytes and localised on the male gametocyte and microgamete. Gene disruption revealed that HAP2 is not necessary for adhesion of male and female gametes (which presumably utilize species specific molecules such as P48/45), but is essential for the fusion of gamete surface membranes, and is therefore required for successful fertilization of the sexual stages of the parasite. Given its expression pattern, localisation on the surface of the male gametocytes/gametes, and essential role in fertilization, HAP2 is an obvious molecule to consider as a TBV candidate. Here, we raise polyclonal rabbit anti-serum against residues 355–609 of the P. berghei HAP2 gene expressed in Escherichia coli. We test its ability to recognise both the recombinant immunogen, and native P. berghei HAP2. We then show that purified anti-P. berghei HAP2 sera inhibits ookinete formation in vitro, and oocyst formation in vivo. Our data suggests HAP2 in Plasmodium spp. could be a promising new TBV target candidate.