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Individuals infected with P. falciparum develop antibody responses to intra-erythrocytic gametocyte proteins and exported gametocyte proteins present on the surface of infected erythrocytes. However, there is currently limited knowledge on the immunogenicity of gametocyte antigens and the specificity of gametocyte-induced antibody responses. In this study, we assessed antibody responses in participants of two controlled human malaria infection (CHMI) studies by ELISA, multiplexed bead-based antibody assays and protein microarray. By comparing antibody responses in participants with and without gametocyte exposure, we aimed to disentangle the antibody response induced by asexual and sexual stage parasites. We showed that after a single malaria infection, a significant anti-sexual stage humoral response is induced in malaria-naïve individuals, even after exposure to relatively low gametocyte densities (up to ~1,600 gametocytes/mL). In contrast to antibody responses to well-characterised asexual blood stage antigens that were detectable by day 21 after infection, responses to sexual stage antigens (including transmission blocking vaccine candidates Pfs48/45 and Pfs230) were only apparent at 51 days after infection. We found antigens previously associated with early gametocyte or anti-gamete immunity were highly represented among responses linked with gametocyte exposure. Our data provide detailed insights on the induction and kinetics of antibody responses to gametocytes and identify novel antigens that elicit antibody responses exclusively in individuals with gametocyte exposure. Our findings provide target identification for serological assays for surveillance of the malaria infectious reservoir, and support vaccine development by describing the antibody response to leading vaccine antigens after primary infection.

Transmission of Plasmodium parasites to the mosquito requires the formation and development of gametocytes. Studies in infected humans have shown that only the most mature forms of Plasmodium falciparum gametocytes are present in circulation, whereas immature forms accumulate in the hematopoietic environment of the bone marrow. We used the rodent model Plasmodium berghei to study gametocyte behavior through time under physiological conditions. Intravital microscopy demonstrated preferential homing of early gametocyte forms across the intact vascular barrier of the bone marrow and the spleen early during infection and subsequent development in the extravascular environment. During the acute phase of infection, we observed vascular leakage resulting in further parasite accumulation in this environment. Mature gametocytes showed high deformability and were found entering and exiting the intact vascular barrier. We suggest that extravascular gametocyte localization and mobility are essential for gametocytogenesis and transmission of Plasmodium to the mosquito.

Naturally acquired immune responses target infected red blood cell surface antigens of immature malaria transmission stages. Targeting transmission: Effective malaria vaccines to block transmission of Plasmodium falciparum may require targeting of different antigens than those that are currently being studied. To identify candidate antigens, Dantzler et al. studied immune responses from large cohorts of people that had been infected with P. falciparum. They used multiple complementary assays to study how antibodies recognize different stages of gametocytes, the sexual stage that allows transmission from human blood into mosquitoes. Immune sera recognized immature but not mature gametocytes inside red blood cells. They further studied a selection of candidate antigens that are not present in other stages and are relatively conserved across P. falciparum strains. Strong natural immunity to these antigens indicates that a vaccine may be designed to induce protective immune responses that could potentially interfere with transmission. The recent decline in global malaria burden has stimulated efforts toward Plasmodium falciparum elimination. Understanding the biology of malaria transmission stages may provide opportunities to reduce or prevent onward transmission to mosquitoes. Immature P. falciparum transmission stages, termed stages I to IV gametocytes, sequester in human bone marrow before release into the circulation as mature stage V gametocytes. This process likely involves interactions between host receptors and potentially immunogenic adhesins on the infected red blood cell (iRBC) surface. Here, we developed a flow cytometry assay to examine immune recognition of live gametocytes of different developmental stages by naturally exposed Malawians. We identified strong antibody recognition of the earliest immature gametocyte-iRBCs (giRBCs) but not mature stage V giRBCs. Candidate surface antigens (n = 30), most of them shared between asexual- and gametocyte-iRBCs, were identified by mass spectrometry and mouse immunizations, as well as correlations between responses by protein microarray and flow cytometry. Naturally acquired responses to a subset of candidate antigens were associated with reduced asexual and gametocyte density, and plasma samples from malaria-infected individuals were able to induce immune clearance of giRBCs in vitro. Infected RBC surface expression of select candidate antigens was validated using specific antibodies, and genetic analysis revealed a subset with minimal variation across strains. Our data demonstrate that humoral immune responses to immature giRBCs and shared iRBC antigens are naturally acquired after malaria exposure. These humoral immune responses may have consequences for malaria transmission potential by clearing developing gametocytes, which could be leveraged for malaria intervention. [ABSTRACT FROM AUTHOR]

Individuals infected with P. falciparum develop antibody responses to intra-erythrocytic gametocyte proteins and exported gametocyte proteins present on the surface of infected erythrocytes. However, there is currently limited knowledge on the immunogenicity of gametocyte antigens and the specificity of gametocyte-induced antibody responses. In this study, we assessed antibody responses in participants of two controlled human malaria infection (CHMI) studies by ELISA, multiplexed bead-based antibody assays and protein microarray. By comparing antibody responses in participants with and without gametocyte exposure, we aimed to disentangle the antibody response induced by asexual and sexual stage parasites. We showed that after a single malaria infection, a significant anti-sexual stage humoral response is induced in malaria-naïve individuals, even after exposure to relatively low gametocyte densities (up to ~1,600 gametocytes/mL). In contrast to antibody responses to well-characterised asexual blood stage antigens that were detectable by day 21 after infection, responses to sexual stage antigens (including transmission blocking vaccine candidates Pfs48/45 and Pfs230) were only apparent at 51 days after infection. We found antigens previously associated with early gametocyte or anti-gamete immunity were highly represented among responses linked with gametocyte exposure. Our data provide detailed insights on the induction and kinetics of antibody responses to gametocytes and identify novel antigens that elicit antibody responses exclusively in individuals with gametocyte exposure. Our findings provide target identification for serological assays for surveillance of the malaria infectious reservoir, and support vaccine development by describing the antibody response to leading vaccine antigens after primary infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 de Jong, Alkema, Oulton, Dumont, Teelen, Nakajima, de Assis, Press, Ngotho, Tetteh, Felgner, Marti, Collins, Drakeley, Bousema and Stone.)