Malaria is one of the most serious infectious diseases impacting global public health and economic development. According to the World Malaria Report 2019, there were 228 million malaria cases and 405,000 malaria deaths globally in 2018 . Malaria control measures such as insecticide-treated bed nets, indoor residual sprays of insecticides, and artemisinin combination therapies altogether have contributed to a significant decrease in the morbidity and mortality of malaria. However, the emergence of drug-resistant parasites and insecticide-resistant mosquitoes poses great challenges to malaria control and elimination . Vaccines, in general, have been the most successful intervention against many viral diseases, but an effective vaccine against malaria infection or transmission is not yet available . Among the vaccine designs against the malaria parasites, transmission-blocking vaccines (TBVs), which target the sexual and/or sporogonic development of the parasite, are intended to reduce the transmission of malaria parasites from humans to mosquitoes .
Plasmodium parasites have a complex lifecycle, including developmental stages in both the human host and the mosquito vector. The transmission of malaria begins with the formation of the sexual precursor stage, gametocytes, in humans. Once ingested by a mosquito, male and female gametocytes, experiencing environmental changes such as a lower temperature, higher PH, and the presence of xanthurenic acid, are activated to form gametes, which fertilize to form a diploid zygote inside the midgut. Within 24 h, the zygote transforms into a motile ookinete, which penetrates the midgut epithelium to develop into an oocyst under the basal lamina . In the next two weeks, each oocyst produces thousands of sporozoites, which migrate to the salivary glands and become ready to be transmitted during subsequent bites of the mosquito [5, 6]. During the sexual development of the malaria parasites, antigens expressed in gametocytes and gametes are called pre-fertilization antigens, while those expressed in zygotes and ookinetes are considered post-fertilization antigens .
The fundamental principle of TBVs is to immunize humans with sexual-stage surface antigens of the parasites to produce antibodies that arrest subsequent development of the parasites in mosquitoes. Though TBVs do not directly protect the vaccinated people from the morbidity of malaria, they play a key role in controlling the spread of the parasites in a community . Several promising candidates have been investigated for TBV development, including the pre-fertilization antigens P230, P48/45, and HAP2, and the post-fertilization antigens P25 and P28. P48/45 and P230 are essential for the adhesion of male gametes to female gametes. Antibodies against pre-fertilization antigens such as P48/45 are found in human sera from endemic areas and correlate with transmission-blocking activity (TBA) [9, 10]. Immunization against the first cysteine-motif domain of Pfs230 and the conserved HAP2 cd loop peptides can elicit antibodies with strong TBA [11, 12]. The post-fertilization antigens P25 and P28 have received much attention, and immunization against recombinant P25 and P28 can completely inhibit parasite development in mosquitoes . To date, Pfs25 and Pvs25 have been studied in several clinical trials [14, 15]. However, most of the TBV candidates could only induce incomplete blocking of malaria transmission . Thus, efforts have been undertaken to discover additional antigens and develop immunization methods to enhance antibody production .
Since subunit vaccines based on a single malaria antigen may fail to produce 100% efficacy, a multi-antigen and multi-stage vaccine, by which immune responses are elicited against more than one antigen and antigens from different stages of the parasite life cycle, might be a more effective vaccination strategy. Several studies have investigated whether a multiple antigen combination would be able to enhance the immune efficacy of single antigens and whether the inclusion of multiple antigens could cause immune interference . It has been shown that the combination of two blood-stage antigens MSP1 and AMA1 caused immune interference by the immunodominant antigen , whereas the two ookinete antigens Pfs25 and Pfs28 did not show immune interference [20, 21, 22]. Further, two studies showed that dual-antigen vaccines based on Pfs25 and Pfs230C did not elicit better TRA compared to the mono-antigen vaccines [18, 23]. However, the Pfs230 and Pfs48/45 fusion proteins were found to elicit functional antibodies in mice with higher TBA than the single proteins alone . These studies suggest that the strength of functional TBA from vaccination with multiple antigens may depend on the antigens used in the combination and how they are combined.
We have recently identified several new TBV candidates, including a gametocyte plasma membrane protein Pbg37 and an ookinete surface protein PSOP25 [25, 26]. The recombinant Pbg37 protein targeting the N-terminal 63 amino acids (aa) was able to elicit strong antibody responses with TBA. Consistent with it being a pre-fertilization antigen, the major inhibitory effects of the Pbg37 antisera were on the exflagellation and fertilization processes . Similarly, antisera against the aa 45–245 fragment of PSOP25 also showed significant in vitro and in vivo TBA . Here, we aim to evaluate whether the combination of these two new antigens targeting different stages of sexual development could improve TBA.