Evaluation of the precision of the Plasmodium knowlesi growth inhibition assay for Plasmodium vivax Duffy-binding protein-based malaria vaccine development.

Recent data indicate increasing disease burden and importance of Plasmodium vivax (Pv) malaria. A robust assay will be essential for blood-stage Pv vaccine development. Results of the in vitro growth inhibition assay (GIA) with transgenic P. knowlesi (Pk) parasites expressing the Pv Duffy-binding protein region II (PvDBPII) correlate with in vivo protection in the first PvDBPII controlled human malaria infection (CHMI) trials, making the PkGIA an ideal selection tool once the precision of the assay is defined. To determine the precision in percentage of inhibition in GIA (%GIA) and in GIA50 (antibody concentration that gave 50 %GIA), ten GIAs with transgenic Pk parasites were conducted with four different anti-PvDBPII human monoclonal antibodies (mAbs) at concentrations of 0.016 to 2 mg/mL, and three GIAs with eighty anti-PvDBPII human polyclonal antibodies (pAbs) at 10 mg/mL. A significant assay-to-assay variation was observed, and the analysis revealed a standard deviation (SD) of 13.1 in the mAb and 5.94 in the pAb dataset for %GIA, with a LogGIA50 SD of 0.299 (for mAbs). Moreover, the ninety-five percent confidence interval (95 %CI) for %GIA or GIA50 in repeat assays was calculated in this investigation. The error range determined in this study will help researchers to compare PkGIA results from different assays and studies appropriately, thus supporting the development of future blood-stage malaria vaccine candidates, specifically second-generation PvDBPII-based formulations.

Vaccines and monoclonal antibodies: new tools for malaria control.

SUMMARYMalaria remains one of the biggest health problems in the world. While significant reductions in malaria morbidity and mortality had been achieved from 2000 to 2015, the favorable trend has stalled, rather significant increases in malaria cases are seen in multiple areas. In 2022, there were 249 million estimated cases, and 608,000 malaria-related deaths, mostly in infants and children aged under 5 years, globally. Therefore, in addition to the expansion of existing anti-malarial control measures, it is critical to develop new tools, such as vaccines and monoclonal antibodies (mAbs), to fight malaria. In the last 2 years, the first and second malaria vaccines, both targeting Plasmodium falciparum circumsporozoite proteins (PfCSP), have been recommended by the World Health Organization to prevent P. falciparum malaria in children living in moderate to high transmission areas. While the approval of the two malaria vaccines is a considerable milestone in vaccine development, they have much room for improvement in efficacy and durability. In addition to the two approved vaccines, recent clinical trials with mAbs against PfCSP, blood-stage vaccines against P. falciparum or P. vivax, and transmission-blocking vaccine or mAb against P. falciparum have shown promising results. This review summarizes the development of the anti-PfCSP vaccines and mAbs, and recent topics in the blood- and transmission-blocking-stage vaccine candidates and mAbs. We further discuss issues of the current vaccines and the directions for the development of next-generation vaccines.

Cross-reactivity of rPvs48/45, a recombinant Plasmodium vivax protein, with sera from Plasmodium falciparum endemic areas of Africa.

Background: Ps48/45, a Plasmodium gametocyte surface protein, is a promising candidate for malaria transmission-blocking (TB) vaccine. Due to its relevance for a multispecies vaccine, we explored the cross-reactivity and TB activity of a recombinant P. vivax Ps48/45 protein (rPvs48/45) with sera from P. falciparum-exposed African donors. Methods: rPvs48/45 was produced in Chinese hamster ovary cell lines and tested by ELISA for its cross-reactivity with sera from Burkina Faso, Tanzania, Mali, and Nigeria - In addition, BALB/c mice were immunized with the rPvs48/45 protein formulated in Montanide ISA-51 and inoculated with a crude extract of P. falciparum NF-54 gametocytes to evaluate the parasite-boosting effect on rPvs48/45 antibody titers. Specific anti-rPvs48/45 IgG purified from African sera was used to evaluate the ex vivo TB activity on P. falciparum, using standard mosquito membrane feeding assays (SMFA). Results: rPvs48/45 protein showed cross-reactivity with sera of individuals from all four African countries, in proportions ranging from 94% (Tanzania) to 40% (Nigeria). Also, the level of cross-reactive antibodies varied significantly between countries (p<0.0001), with a higher antibody level in Mali and the lowest in Nigeria. In addition, antibody levels were higher in adults (≥ 17 years) than young children (≤ 5 years) in both Mali and Tanzania, with a higher proportion of responders in adults (90%) than in children (61%) (p<0.0001) in Mali, where male (75%) and female (80%) displayed similar antibody responses. Furthermore, immunization of mice with P. falciparum gametocytes boosted anti-Pvs48/45 antibody responses, recognizing P. falciparum gametocytes in indirect immunofluorescence antibody test. Notably, rPvs48/45 affinity-purified African IgG exhibited a TB activity of 61% against P. falciparum in SMFA. Conclusion: African sera (exposed only to P. falciparum) cross-recognized the rPvs48/45 protein. This, together with the functional activity of IgG, warrants further studies for the potential development of a P. vivax and P. falciparum cross-protective TB vaccine.

A randomized first-in-human phase I trial of differentially adjuvanted Pfs48/45 malaria vaccines in Burkinabé adults.

BACKGROUNDMalaria transmission-blocking vaccines aim to interrupt the transmission of malaria from one person to another.METHODSThe candidates R0.6C and ProC6C share the 6C domain of the Plasmodium falciparum sexual-stage antigen Pfs48/45. R0.6C utilizes the glutamate-rich protein (GLURP) as a carrier, and ProC6C includes a second domain (Pfs230-Pro) and a short 36-amino acid circumsporozoite protein (CSP) sequence. Healthy adults (n = 125) from a malaria-endemic area of Burkina Faso were immunized with 3 intramuscular injections, 4 weeks apart, of 30 µg or 100 µg R0.6C or ProC6C each adsorbed to Alhydrogel (AlOH) adjuvant alone or in combination with Matrix-M (15 µg or 50 µg, respectively). The allocation was random and double-blind for this phase I trial.RESULTSThe vaccines were safe and well tolerated with no vaccine-related serious adverse events. A total of 7 adverse events, mild to moderate in intensity and considered possibly related to the study vaccines, were recorded. Vaccine-specific antibodies were highest in volunteers immunized with 100 µg ProC6C-AlOH with Matrix-M, and 13 of 20 (65%) individuals in the group showed greater than 80% transmission-reducing activity (TRA) when evaluated in the standard membrane feeding assay at 15 mg/mL IgG. In contrast, R0.6C induced sporadic TRA.CONCLUSIONAll formulations were safe and well tolerated in a malaria-endemic area of Africa in healthy adults. The ProC6C-AlOH/Matrix-M vaccine elicited the highest levels of functional antibodies, meriting further investigation.TRIAL REGISTRATIONPan-African Clinical Trials Registry (https://pactr.samrc.ac.za) PACTR202201848463189.FUNDINGThe study was funded by the European and Developing Countries Clinical Trials Partnership (grant RIA2018SV-2311).

Naturally Acquired Transmission-Blocking Immunity Against Different Strains of Plasmodium vivax in a Malaria-Endemic Area in Thailand.

BACKGROUND: Human immunity triggered by natural malaria infections impedes parasite transmission from humans to mosquitoes, leading to interest in transmission-blocking vaccines. However, immunity characteristics, especially strain specificity, remain largely unexplored. We investigated naturally acquired transmission-blocking immunity (TBI) against Plasmodium vivax, a major malaria parasite. METHODS: Using the direct membrane-feeding assay, we assessed TBI in plasma samples and examined the role of antibodies by removing immunoglobulins through protein G/L adsorption before mosquito feeding. Strain specificity was evaluated by conducting a direct membrane-feeding assay with plasma exchange. RESULTS: Blood samples from 47 patients with P vivax were evaluated, with 37 plasma samples successfully infecting mosquitoes. Among these, 26 showed inhibition before immunoglobulin depletion. Despite substantial immunoglobulin removal, 4 samples still exhibited notable inhibition, while 22 had reduced blocking activity. Testing against heterologous strains revealed some plasma samples with broad TBI and others with strain-specific TBI. CONCLUSIONS: Our findings indicate that naturally acquired TBI is mainly mediated by antibodies, with possible contributions from other serum factors. The transmission-blocking activity of plasma samples varied by the tested parasite strain, suggesting single polymorphic or multiple targets for naturally acquired TBI. These observations improve understanding of immunity against P vivax and hold implications for transmission-blocking vaccine development.

One-year antibody durability induced by EuCorVac-19, a liposome-displayed COVID-19 receptor binding domain subunit vaccine, in healthy Korean subjects.

OBJECTIVE: EuCorVac-19 (ECV-19), an adjuvanted liposome-displayed receptor binding domain (RBD) COVID-19 vaccine, previously reported interim Phase 2 trial results showing induction of neutralizing antibodies 3 weeks after prime-boost immunization. The objective of this study was to determine the longer-term antibody response of the vaccine. METHODS: To assess immunogenicity 6 and 12 months after vaccination, participants in the Phase 2 trial (NCT04783311) were excluded if they: 1) withdrew, 2) reported COVID-19 infection or additional vaccination, or 3) exhibited increasing Spike (S) antibodies (representing possible non-reported infection). Following exclusions, of the 197 initial subjects, anti-S IgG antibodies and neutralizing antibodies were further assessed in 124 subjects at the 6-month timepoint, and 36 subjects at the 12-month timepoint. RESULTS: Median anti-S antibody half-life was 52 days (interquartile range [IQR]:42-70), in the "early" period from 3 weeks to 6 months, and 130 days (IQR:97-169) in the "late" period from 6 to 12 months. There was a negative correlation between initial antibody titer and half-life. Anti-S and neutralizing antibody responses were correlated. Neutralizing antibody responses showed longer half-lives; the early period had a median half-life of 120 days (IQR:81-207), and the late period had a median half-life of 214 days (IQR:140-550). CONCLUSION: These data establish antibody durability of ECV-19, using a framework to analyze COVID-19 vaccine-induced antibodies during periods of high infection.

Mesenchymal stem cells of the bone marrow raise infectivity of Plasmodium falciparum gametocytes.

Plasmodium falciparum is a parasite that causes the deadly human disease, malaria, and exhibits a complex life cycle in human and mosquito hosts. In the sexual stages of the parasite, gametocytes mature in the human body and propagate malaria when they are picked up by mosquitoes to infect new hosts. Previous research has shown that gametocytes home to the bone marrow of the host, where they complete their maturation and alter the behavior of resident mesenchymal stem cells (MSCs). In this study, we investigated the alternate side of this host-pathogen interaction, whether MSCs could alter the behavior of gametocytes. Gametocytes were co-cultured with MSCs until maturity and subsequently fed to mosquitoes to measure the oocysts produced. Here, we report, for the first time, that MSCs co-culture significantly elevated oocyst numbers in the infected mosquito compared to conventional culture medium. This enhancement appeared to be most effective during the early stages of gametocyte development and was not replicated by other cell types. MSC co-culture also increased the infectivity of field isolated P. falciparum parasites. This effect was partially mediated by soluble factor(s) as conditioned medium harvested from MSCs could also partially raise infectivity of gametocytes to nearly half compared to MSC co-culture. Together, this study reveals novel host-pathogen interactions, where the human MSCs are elevating the infectivity of malaria gametocytes. IMPORTANCE While prior research has established that Plasmodium gametocytes sequester in the bone marrow and can influence resident stem cells, the question of why they would choose this compartment and these cells remained a mystery. This study, for the first time, shows that being in the presence of mesenchymal stem cells (MSCs) alters the biology of the P. falciparum parasite and makes it more infectious to mosquitoes, hinting at novel mechanisms in its life cycle. This method also facilitates mosquito infections with field isolated parasites, affording research teams new infection models with parasites, which are challenging to infect into mosquitos using conventional culture methods. Finally, our findings that MSC-conditioned medium can also raise infectivity open avenues of investigation into mechanisms involved but can also serve as a practical tool for researchers hoping to increase oocyst yields.

Antibody gene features associated with binding and functional activity in vaccine-derived human mAbs targeting malaria parasites.

Adjuvants have been essential to malaria vaccine development, but their impact on the vaccine-induced antibody repertoire is poorly understood. Here, we used cDNA sequences from antigen-specific single memory B cells to express 132 recombinant human anti-Pfs230 monoclonal antibodies (mAbs). Alhydrogel®-induced mAbs demonstrated higher binding to Pfs230D1, although functional activity was similar between adjuvants. All Alhydrogel® mAbs using IGHV1-69 gene bound to recombinant Pfs230D1, but none blocked parasite transmission to mosquitoes; similarly, no AS01 mAb using IGHV1-69 blocked transmission. Functional mAbs from both Alhydrogel® and AS01 vaccines used IGHV3-21 and IGHV3-30 genes. Antibodies with the longest CDR3 sequences were associated with binding but not functional activity. This study assesses adjuvant effects on antibody clonotype diversity during malaria vaccination.

Structure guided mimicry of an essential P. falciparum receptor-ligand complex enhances cross neutralizing antibodies.

Invasion of human erythrocytes by Plasmodium falciparum (Pf) merozoites relies on the interaction between two parasite proteins: apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2). While antibodies to AMA1 provide limited protection against Pf in non-human primate malaria models, clinical trials using recombinant AMA1 alone (apoAMA1) yielded no protection due to insufficient functional antibodies. Immunization with AMA1 bound to RON2L, a 49-amino acid peptide from its ligand RON2, has shown superior protection by increasing the proportion of neutralizing antibodies. However, this approach relies on the formation of a complex in solution between the two vaccine components. To advance vaccine development, here we engineered chimeric antigens by replacing the AMA1 DII loop, displaced upon ligand binding, with RON2L. Structural analysis confirmed that the fusion chimera (Fusion-FD12) closely mimics the binary AMA1-RON2L complex. Immunization studies in female rats demonstrated that Fusion-FD12 immune sera, but not purified IgG, neutralized vaccine-type parasites more efficiently compared to apoAMA1, despite lower overall anti-AMA1 titers. Interestingly, Fusion-FD12 immunization enhanced antibodies targeting conserved epitopes on AMA1, leading to increased neutralization of non-vaccine type parasites. Identifying these cross-neutralizing antibody epitopes holds promise for developing an effective, strain-transcending malaria vaccine.

Structure-based design of a strain transcending AMA1-RON2L malaria vaccine.

Apical membrane antigen 1 (AMA1) is a key malaria vaccine candidate and target of neutralizing antibodies. AMA1 binds to a loop in rhoptry neck protein 2 (RON2L) to form the moving junction during parasite invasion of host cells, and this complex is conserved among apicomplexan parasites. AMA1-RON2L complex immunization achieves higher growth inhibitory activity than AMA1 alone and protects mice against Plasmodium yoelii challenge. Here, three single-component AMA1-RON2L immunogens were designed that retain the structure of the two-component AMA1-RON2L complex: one structure-based design (SBD1) and two insertion fusions. All immunogens elicited high antibody titers with potent growth inhibitory activity, yet these antibodies did not block RON2L binding to AMA1. The SBD1 immunogen induced significantly more potent strain-transcending neutralizing antibody responses against diverse strains of Plasmodium falciparum than AMA1 or AMA1-RON2L complex vaccination. This indicates that SBD1 directs neutralizing antibody responses to strain-transcending epitopes in AMA1 that are independent of RON2L binding. This work underscores the importance of neutralization mechanisms that are distinct from RON2 blockade. The stable single-component SBD1 immunogen elicits potent strain-transcending protection that may drive the development of next-generation vaccines for improved malaria and apicomplexan parasite control.

A human antibody epitope map of the malaria vaccine antigen Pfs25.

Pfs25 is a leading antigen for a malaria transmission-blocking vaccine and shows moderate transmission-blocking activity and induction of rapidly decreasing antibody titers in clinical trials. A comprehensive definition of all transmission-reducing epitopes of Pfs25 will inform structure-guided design to enhance Pfs25-based vaccines, leading to potent transmission-blocking activity. Here, we compiled a detailed human antibody epitope map comprising epitope binning data and structures of multiple human monoclonal antibodies, including three new crystal structures of Pfs25 in complex with transmission-reducing antibodies from Malian volunteers immunized with Pfs25 conjugated to EPA and adjuvanted with AS01. These structures revealed additional epitopes in Pfs25 capable of reducing transmission and expanded this characterization to malaria-exposed humans. This work informs immunogen design to focus the antibody response to transmission-reducing epitopes of Pfs25, enabling development of more potent transmission-blocking vaccines for malaria.

A potent and durable malaria transmission-blocking vaccine designed from a single-component 60-copy Pfs230D1 nanoparticle.

Malaria transmission-blocking vaccines (TBVs) reduce disease transmission by breaking the continuous cycle of infection between the human host and the mosquito vector. Domain 1 (D1) of Pfs230 is a leading TBV candidate and comprises the majority of transmission-reducing activity (TRA) elicited by Pfs230. Here we show that the fusion of Pfs230D1 to a 60-copy multimer of the catalytic domain of dihydrolipoyl acetyltransferase protein (E2p) results in a single-component nanoparticle composed of 60 copies of the fusion protein with high stability, homogeneity, and production yields. The nanoparticle presents a potent human transmission-blocking epitope within Pfs230D1, indicating the antigen is correctly oriented on the surface of the nanoparticle. Two vaccinations of New Zealand White rabbits with the Pfs230D1 nanoparticle elicited a potent and durable antibody response with high TRA when formulated in two distinct adjuvants suitable for translation to human use. This single-component nanoparticle vaccine may play a key role in malaria control and has the potential to improve production pipelines and the cost of manufacturing of a potent and durable TBV.

Superior antibody immunogenicity of a viral-vectored RH5 blood-stage malaria vaccine in Tanzanian infants as compared to adults.

BACKGROUND: RH5 is a leading blood-stage candidate antigen for a Plasmodium falciparum vaccine; however, its safety and immunogenicity in malaria-endemic populations are unknown. METHODS: A phase 1b, single-center, dose-escalation, age-de-escalation, double-blind, randomized, controlled trial was conducted in Bagamoyo, Tanzania (NCT03435874). Between 12th April and 25th October 2018, 63 healthy adults (18-35 years), young children (1-6 years), and infants (6-11 months) received a priming dose of viral-vectored ChAd63 RH5 or rabies control vaccine. Sixty participants were boosted with modified vaccinia virus Ankara (MVA) RH5 or rabies control vaccine 8 weeks later and completed 6 months of follow-up post priming. Primary outcomes were the number of solicited and unsolicited adverse events post vaccination and the number of serious adverse events over the study period. Secondary outcomes included measures of the anti-RH5 immune response. FINDINGS: Vaccinations were well tolerated, with profiles comparable across groups. No serious adverse events were reported. Vaccination induced RH5-specific cellular and humoral responses. Higher anti-RH5 serum immunoglobulin G (IgG) responses were observed post boost in young children and infants compared to adults. Vaccine-induced antibodies showed growth inhibition activity (GIA) in vitro against P. falciparum blood-stage parasites; their highest levels were observed in infants. CONCLUSIONS: The ChAd63-MVA RH5 vaccine shows acceptable safety and reactogenicity and encouraging immunogenicity in children and infants residing in a malaria-endemic area. The levels of functional GIA observed in RH5-vaccinated infants are the highest reported to date following human vaccination. These data support onward clinical development of RH5-based blood-stage vaccines to protect against clinical malaria in young African infants. FUNDING: Medical Research Council, London, UK.

Vaccination with Plasmodium vivax Duffy-binding protein inhibits parasite growth during controlled human malaria infection.

There are no licensed vaccines against Plasmodium vivax. We conducted two phase 1/2a clinical trials to assess two vaccines targeting P. vivax Duffy-binding protein region II (PvDBPII). Recombinant viral vaccines using chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) vectors as well as a protein and adjuvant formulation (PvDBPII/Matrix-M) were tested in both a standard and a delayed dosing regimen. Volunteers underwent controlled human malaria infection (CHMI) after their last vaccination, alongside unvaccinated controls. Efficacy was assessed by comparisons of parasite multiplication rates in the blood. PvDBPII/Matrix-M, given in a delayed dosing regimen, elicited the highest antibody responses and reduced the mean parasite multiplication rate after CHMI by 51% (n = 6) compared with unvaccinated controls (n = 13), whereas no other vaccine or regimen affected parasite growth. Both viral-vectored and protein vaccines were well tolerated and elicited expected, short-lived adverse events. Together, these results support further clinical evaluation of the PvDBPII/Matrix-M P. vivax vaccine.

Structure guided mimicry of an essential P. falciparum receptor-ligand complex enhances cross neutralizing antibodies.

Invasion of human red blood cells (RBCs) by Plasmodium falciparum (Pf) merozoites relies on the interaction between two parasite proteins, apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2) 1,2 . Antibodies to AMA1 confer limited protection against P. falciparum in non-human primate malaria models 3,4 . However, clinical trials with recombinant AMA1 alone (apoAMA1) saw no protection, likely due to inadequate levels of functional antibodies 5-8 . Notably, immunization with AMA1 in its ligand bound conformation using RON2L, a 49 amino acid peptide from RON2, confers superior protection against P. falciparum malaria by enhancing the proportion of neutralizing antibodies 9,10 . A limitation of this approach, however, is that it requires the two vaccine components to form a complex in solution. To facilitate vaccine development, we engineered chimeric antigens by strategically replacing the AMA1 DII loop that is displaced upon ligand binding with RON2L. Structural characterization of the fusion chimera, Fusion-F D12 to 1.55 Å resolution showed that it closely mimics the binary receptor-ligand complex. Immunization studies showed that Fusion-F D12 immune sera neutralized parasites more efficiently than apoAMA1 immune sera despite having an overall lower anti-AMA1 titer, suggesting improvement in antibody quality. Furthermore, immunization with Fusion-F D12 enhanced antibodies targeting conserved epitopes on AMA1 resulting in greater neutralization of non-vaccine type parasites. Identifying epitopes of such cross-neutralizing antibodies will help in the development of an effective, strain-transcending malaria vaccine. Our fusion protein design is a robust vaccine platform that can be enhanced by incorporating polymorphisms in AMA1 to effectively neutralize all P. falciparum parasites.

Assessment of precision in growth inhibition assay (GIA) using human anti-PfRH5 antibodies.

BACKGROUND: For blood-stage malaria vaccine development, the in vitro growth inhibition assay (GIA) has been widely used to evaluate functionality of vaccine-induced antibodies (Ab), and Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) is a leading blood-stage antigen. However, precision, also called "error of assay (EoA)", in GIA readouts and the source of EoA has not been evaluated systematically. METHODS: In the Main GIA experiment, 4 different cultures of P. falciparum 3D7 parasites were prepared with red blood cells (RBC) collected from 4 different donors. For each culture, 7 different anti-RH5 Ab (either monoclonal or polyclonal Ab) were tested by GIA at two concentrations on three different days (168 data points). To evaluate sources of EoA in % inhibition in GIA (%GIA), a linear model fit was conducted including donor (source of RBC) and day of GIA as independent variables. In addition, 180 human anti-RH5 polyclonal Ab were tested in a Clinical GIA experiment, where each Ab was tested at multiple concentrations in at least 3 independent GIAs using different RBCs (5,093 data points). The standard deviation (sd) in %GIA and in GIA50 (Ab concentration that gave 50%GIA) readouts, and impact of repeat assays on 95% confidence interval (95%CI) of these readouts was estimated. RESULTS: The Main GIA experiment revealed that the RBC donor effect was much larger than the day effect, and an obvious donor effect was also observed in the Clinical GIA experiment. Both %GIA and log-transformed GIA50 data reasonably fit a constant sd model, and sd of %GIA and log-transformed GIA50 measurements were calculated as 7.54 and 0.206, respectively. Taking the average of three repeat assays (using three different RBCs) reduces the 95%CI width in %GIA or in GIA50 measurements by ~ half compared to a single assay. CONCLUSIONS: The RBC donor effect (donor-to-donor variance on the same day) in GIA was much bigger than the day effect (day-to-day variance using the same donor's RBC) at least for the RH5 Ab evaluated in this study; thus, future GIA studies should consider the donor effect. In addition, the 95%CI for %GIA and GIA50 shown here help when comparing GIA results from different samples/groups/studies; therefore, this study supports future malaria blood-stage vaccine development.

Extending the range of Plasmodium falciparum transmission blocking antibodies.

Recent work demonstrating that asymptomatic carriers of P. falciparum parasites make up a large part of the infectious reservoir highlights the need for an effective malaria vaccine. Given the historical challenges of vaccine development, multiple parasite stages have been targeted, including the sexual stages required for transmission. Using flow cytometry to efficiently screen for P. falciparum gamete/zygote surface reactivity, we identified 82 antibodies that bound live P. falciparum gametes/zygotes. Ten antibodies had significant transmission-reducing activity (TRA) in a standard membrane feeding assay and were subcloned along with 9 nonTRA antibodies as comparators. After subcloning, only eight of the monoclonals obtained have significant TRA. These eight TRA mAbs do not recognize epitopes present in any of the current recombinant transmission-blocking vaccine candidates, Pfs230D1M, Pfs48/45.6C, Pf47 D2 and rPfs25. One TRA mAb immunoprecipitates two surface antigens, Pfs47 and Pfs230, that are expressed by both gametocytes and gametes/zygotes. These two proteins have not previously been reported to associate and the recognition of both by a single TRA mAb suggests the Pfs47/Pfs230 complex is a new vaccine target. In total, Pfs230 was the dominant target antigen, with five of the eight TRA mAbs and 8 of 11 nonTRA gamete/zygote surface reactive mAbs interacting with Pfs230. Of the three remaining TRA mAbs, two recognized non-reduced, parasite-produced Pfs25 and one bound non-reduced, parasite-produced Pfs48/45. None of the TRA mAbs bound protein on an immunoblot of reduced gamete/zygote extract and two TRA mAbs were immunoblot negative, indicating none of the new TRA epitopes are linear. The identification of eight new TRA mAbs that bind epitopes not included in any of the constructs currently under advancement as transmission-blocking vaccine candidates may provide new targets worthy of further study.

Novel Transmission-Blocking Antimalarials Identified by High-Throughput Screening of Plasmodium berghei Ookluc.

Safe and effective malaria transmission-blocking chemotherapeutics would allow a community-level approach to malaria control and eradication efforts by targeting the mosquito sexual stage of the parasite life cycle. However, only a single drug, primaquine, is currently approved for use in reducing transmission, and drug toxicity limits its widespread implementation. To address this limitation in antimalarial chemotherapeutics, we used a recently developed transgenic Plasmodium berghei line, Ookluc, to perform a series of high-throughput in vitro screens for compounds that inhibit parasite fertilization, the initial step of parasite development within the mosquito. Screens of antimalarial compounds, approved drug collections, and drug-like molecule libraries identified 185 compounds that inhibit parasite maturation to the zygote form. Seven compounds were further characterized to block gametocyte activation or to be cytotoxic to formed zygotes. These were further validated in mosquito membrane-feeding assays using Plasmodium falciparum and P. vivax. This work demonstrates that high-throughput screens using the Ookluc line can identify compounds that are active against the two most relevant human Plasmodium species and provides a list of compounds that can be explored for the development of new antimalarials to block transmission.

A human antibody epitope map of Pfs230D1 derived from analysis of individuals vaccinated with a malaria transmission-blocking vaccine.

Pfs230 domain 1 (Pfs230D1) is an advanced malaria transmission-blocking vaccine antigen demonstrating high functional activity in clinical trials. However, the structural and functional correlates of transmission-blocking activity are not defined. Here, we characterized a panel of human monoclonal antibodies (hmAbs) elicited in vaccinees immunized with Pfs230D1. These hmAbs exhibited diverse transmission-reducing activity, yet all bound to Pfs230D1 with nanomolar affinity. We compiled epitope-binning data for seventeen hmAbs and structures of nine hmAbs complexes to construct a high-resolution epitope map and revealed that potent transmission-reducing hmAbs bound to one face of Pfs230D1, while non-potent hmAbs bound to the opposing side. The structure of Pfs230D1D2 revealed that non-potent transmission-reducing epitopes were occluded by the second domain. The hmAb epitope map delineated binary hmAb combinations that synergized for extremely high-potency, transmission-reducing activity. This work provides a high-resolution guide for structure-based design of enhanced immunogens and informs diagnostics that measure the transmission-reducing response.

Delayed boosting improves human antigen-specific Ig and B cell responses to the RH5.1/AS01B malaria vaccine.

Modifications to vaccine delivery that increase serum antibody longevity are of great interest for maximizing efficacy. We have previously shown that a delayed fractional (DFx) dosing schedule (0-1-6 month) - using AS01B-adjuvanted RH5.1 malaria antigen - substantially improves serum IgG durability as compared with monthly dosing (0-1-2 month; NCT02927145). However, the underlying mechanism and whether there are wider immunological changes with DFx dosing were unclear. Here, PfRH5-specific Ig and B cell responses were analyzed in depth through standardized ELISAs, flow cytometry, systems serology, and single-cell RNA-Seq (scRNA-Seq). Data indicate that DFx dosing increases the magnitude and durability of circulating PfRH5-specific B cells and serum IgG1. At the peak antibody magnitude, DFx dosing was distinguished by a systems serology feature set comprising increased FcRn binding, IgG avidity, and proportion of G2B and G2S2F IgG Fc glycans, alongside decreased IgG3, antibody-dependent complement deposition, and proportion of G1S1F IgG Fc glycan. Concomitantly, scRNA-Seq data show a higher CDR3 percentage of mutation from germline and decreased plasma cell gene expression in circulating PfRH5-specific B cells. Our data, therefore, reveal a profound impact of DFx dosing on the humoral response and suggest plausible mechanisms that could enhance antibody longevity, including improved FcRn binding by serum Ig and a potential shift in the underlying cellular response from circulating short-lived plasma cells to nonperipheral long-lived plasma cells.