Malaria Transmission-blocking Vaccine Research Articles

Evaluation of transmission-blocking potential of PvPSOP25 using transgenic murine malaria parasite and clinical isolates.

Malaria transmission-blocking vaccines (TBVs) aim to inhibit malaria parasite development in mosquitoes and prevent further transmission to the human host. The putative-secreted ookinete protein 25 (PSOP25), highly conserved in Plasmodium spp., is a promising TBV target. Here, we investigated PvPSOP25 from P. vivax as a TBV candidate using transgenic murine parasite P. berghei and clinical P. vivax isolates. A transgenic P. berghei line expressing PvPSOP25 (TrPvPSOP25Pb) was generated. Full-length PvPSOP25 was expressed in the yeast Pichia pastoris and used to immunize mice to obtain anti-rPvPSOP25 sera. The transmission-blocking activity of the anti-rPvPSOP25 sera was evaluated through in vitro assays and mosquito-feeding experiments. The antisera generated by immunization with rPvPSOP25 specifically recognized the native PvPSOP25 antigen expressed in TrPvPSOP25Pb ookinetes. In vitro assays showed that the immune sera significantly inhibited exflagellation and ookinete formation of the TrPvPSOP25Pb parasite. Mosquitoes feeding on mice infected with the transgenic parasite and passively transferred with the anti-rPvPSOP25 sera showed a 70.7% reduction in oocyst density compared to the control group. In a direct membrane feeding assay conducted with five clinical P. vivax isolates, the mouse anti-rPvPSOP25 antibodies significantly reduced the oocyst density while showing a negligible influence on mosquito infection prevalence. This study supported the feasibility of transgenic murine malaria parasites expressing P. vivax antigens as a useful tool for evaluating P. vivax TBV candidates. Meanwhile, the moderate transmission-reducing activity of the generated anti-rPvPSOP25 sera necessitates further research to optimize its efficacy.

Plasmodium vivax HAP2 genetic diversity and population structure from worldwide clinical samples. A potential Transmission-Blocking malaria vaccine candidate

BackgroundMalaria infections due to Plasmodium vivax are increasingly becoming a significant public health problem and are directly impacting malaria elimination efforts in many countries. Recent functional studies have shown that a pre-fertilising antigen; gamete membrane fusion protein HAP2/Generative Cell specific 1 (GCS1) domain significantly reduces the formation of P. vivax oocysts in mosquitos, implying that it could be developed as a transmission-blocking vaccine. MethodsIn this study, the genetic diversity, natural selection, haplotype network analysis and population structure of PvHAP2/GCS1 full-length gene using worldwide samples from 16 countries are determined and the implications for its use as a potential vaccine candidate against P. vivax are discussed. ResultsThe study revealed low levels of nucleotide diversity (π ∼ 0.003, SNPs = 95) and purifying selection for the full-length PvHAP2 gene indicating functional constraints. Among the 95 SNPs (65 synonymous and 30 non-synonymous), 15 non-synonymous substitutions were localised to the C-terminal domain of the protein (674a.a − 862a.a) majority of these non-synonymous substitutions were from two countries of Central Africa i.e. Cameroon and Gabon. Phylogeographic haplotype network analysis indicated that a major haplotype (Hap_2, n = 34) is shared among countries from Asian countries and South American countries. Population structure analysis using a robust Bayesian algorithm indicated the existence of two sub-populations of PvHAP2/GCS1 among the worldwide samples. ConclusionThis study suggests that, due to low genetic diversity across 15 countries, and shared haplotype identified, the HAP2 gene can be a good target for vaccine development as a transmission-blocking vaccine with the potential to provide strain-transcending immunity. The C-terminal region from two countries from Central Africa (Cameroon and Gabon) identified high number of non-synonymous substitutions, which may indicate new adaption of parasites from these countries. However, more detailed field based studies with a larger sample size from these countries are needed.

Evaluation of different types of adjuvants in a malaria transmission-blocking vaccine

Adjuvants are critical components for vaccines, which enhance the strength and longevity of the antibody response and influence the types of immune response. Limited research has been conducted on the immunogenicity and protective efficacy of various adjuvants in malaria transmission-blocking vaccines (TBVs). In this study, we formulated a promising TBV candidate antigen, the P. berghei ookinete surface antigen PSOP25, with different types of adjuvants, including the TLR4 agonist monophosphoryl lipid A (MPLA), the TLR9 agonist cytosine phosphoguanosine oligodeoxynucleotides (CpG ODN 1826) (CpG), a saponin adjuvant QS-21, aluminum hydroxide (Alum), and two combination adjuvants MPLA + QS-21 and QS-21 + CpG. We demonstrated that adjuvanted vaccines results in elevated elicited antibody levels, increased proliferation of plasma cells, and efficient formation of germinal centers (GCs), leading to enhanced long-term protective immune responses. Furthermore, CpG group exhibited the most potent inhibition of ookinete formation and transmission-blocking activity. We found that the rPSOP25 with CpG adjuvant was more effective than MPLA, QS-21, MPLA + QS-21, QS-21 + CpG adjuvants in dendritic cells (DCs) activation and differentiation. Additionally, the CpG adjuvant elicited more rubust immune memory response than Alum adjuvant. CpG and QS-21 adjuvants could activate the Th1 response and promote the secretion of IFN-γ and TNF-α. PSOP25 induced a higher number of Tfh cells in splenocytes when combined with MPLA, CpG, and QS-21 + CpG; and there was no increase in these cell populations when PSOP25 was administered with Alum. In conclusion, CpG may confer enhanced efficacy for the rPSOP25 vaccine, as evidenced by the ability of the elicited antisera to induce protective immune responses and improved transmission-blocking activity.

Nano-differential scanning fluorimetry as a tool for the assessment of refolded antibody fragments: A case study for anti-Pfs25 single-chain antibodies

Differential Scanning Fluorimetry (DSF) is a valuable and versatile tool in the field of protein and antibody stability studies, providing valuable insights into their thermal stability, and aiding in the optimization of experimental conditions for various applications. Antibody fragments provide armamentarium for researchers and practitioners across various fields, enabling advancements in medicine, diagnostics, research, and industrial applications. In the present study, single-chain antibodies derived from mAb 4B7 and mAb 1245 were expressed in E. coli, refolded, and checked for their binding with the antigen - Pfs25 (transmission-blocking malaria vaccine candidate). We used nano-DSF as a valuable tool to assess thermal stability, consequently aiding in predicting the correct folding of single-chain antibodies.. Employing nano-DSF as a checkpoint enables the determination of whether to proceed with functional and binding studies on the refolded single-chain antibodies.

Transmission-reducing and -enhancing monoclonal antibodies against Plasmodium vivax gamete surface protein Pvs48/45.

Gamete surface protein P48/45 has been shown to be important for male gamete fertility and a strong candidate for the development of a malaria transmission-blocking vaccine (TBV). However, TBV development for Plasmodium vivax homolog Pvs48/45 has been slow because of a number of challenges: availability of conformationally suitable recombinant protein; the lack of an in vivo challenge model; and the inability to produce P. vivax gametocytes in culture to test transmission-blocking activity of antibodies. To support ongoing efforts to develop Pvs48/45 as a potential vaccine candidate, we initiated efforts to develop much needed reagents to move the field forward. We generated monoclonal antibodies (mAbs) directed against Pvs48/45 and characterized putative functional domains in Pvs48/45 using recombinant fragments corresponding to domains D1-D3 and their biological functionality through ex vivo direct membrane feeding assays (DMFAs) using P. vivax parasites from patients in a field setting in Brazil. While some mAbs partially blocked oocyst development in the DMFA, one mAb caused a significant enhancement of the infectivity of gametocytes in the mosquitoes. Individual mAbs exhibiting blocking and enhancing activities recognized non-overlapping epitopes in Pvs48/45. Further characterization of precise epitopes recognized by transmission-reducing and -enhancing antibodies will be crucial to design an effective immunogen with optimum transmission-reducing potential.

Optimized Refolding Buffers Oriented Humoral Immune Responses Versus PfGCS1 Self-Assembled Peptide Nanoparticle.

Developing a novel class of vaccine is pivotal for eliminating and eradicating malaria. Preceding investigations demonstrated partial blocking activity in malaria transmission against recombinant vaccine PfHAP2-GCS1 and conserved region of the cd loop. The effectiveness of immune response varies with the size and shape of the self-assembly of peptide nanoparticles (SAPNs) displaying antigen, affected by different components in refolding buffers. Plasmodium falciparum Generative Cell Specific 1 (PfGCS1), a promising malaria transmission-blocking vaccine (TBV) candidate, was expressed, purified, and followed by a four-step refolding process to form nanoparticles (PfGCS1-SAPNs). The influence of buffer components on the size and shape of SAPNs was investigated by DLS and FESEM. Furthermore, the immunogenicity of nanostructures was assessed in different mouse groups. The results showed that PfGCS1-SAPN was immunogenic and its administration with Poly (I:C), stimulated humoral and cellular responses in the mouse model. In the immunized mice groups, the level of IgG antibodies against PfGCS1-SAPN was significantly increased in different time points (second and third boost) and heterogeneous boosters. The various IgG-subclasses profile shifted to Th1, Th2, or Th1/Th2 mix responses in mice immunized with PfGCS1-SAPN refolded in different buffers, indicating a prerequisite for further investigations to optimize vaccine formulation to enhance and modulate Th1/cellular responses. Such studies pave the way to improve biophysical features related to the nanoparticles' size, shape, and conformational epitopes of candidate antigens and T- and B-cells presented on the superficial structure to elicit robust immune responses.

MRNA vaccines expressing malaria transmission-blocking antigens Pfs25 and Pfs230D1 induce a functional immune response

Malaria transmission-blocking vaccines (TBV) are designed to inhibit the sexual stage development of the parasite in the mosquito host and can play a significant role in achieving the goal of malaria elimination. Preclinical and clinical studies using protein–protein conjugates of leading TBV antigens Pfs25 and Pfs230 domain 1 (Pfs230D1) have demonstrated the feasibility of TBV. Nevertheless, other promising vaccine platforms for TBV remain underexplored. The recent success of mRNA vaccines revealed the potential of this technology for infectious diseases. We explored the mRNA platform for TBV development. mRNA constructs of Pfs25 and Pfs230D1 variously incorporating signal peptides (SP), GPI anchor, and Trans Membrane (TM) domain were assessed in vitro for antigen expression, and selected constructs were evaluated in mice. Only mRNA constructs with GPI anchor or TM domain that resulted in high cell surface expression of the antigens yielded strong immune responses in mice. These mRNA constructs generated higher transmission-reducing functional activity versus the corresponding alum-adjuvanted protein-protein conjugates used as comparators. Pfs25 mRNA with GPI anchor or TM maintained >99% transmission reducing activity through 126 days, the duration of the study, demonstrating the potential of mRNA platform for TBV.

Pfs230 Domain 7 is targeted by a potent malaria transmission-blocking monoclonal antibody

Malaria transmission-blocking vaccines (TBVs) aim to induce antibodies that block Plasmodium parasite development in the mosquito midgut, thus preventing mosquitoes from becoming infectious. While the Pro-domain and first of fourteen 6-Cysteine domains (Pro-D1) of the Plasmodium gamete surface protein Pfs230 are known targets of transmission-blocking antibodies, no studies to date have discovered other Pfs230 domains that are functional targets. Here, we show that a murine monoclonal antibody (mAb), 18F25.1, targets Pfs230 Domain 7. We generated a subclass-switched complement-fixing variant, mAb 18F25.2a, using a CRISPR/Cas9-based hybridoma engineering method. This subclass-switched mAb 18F25.2a induced lysis of female gametes in vitro. Importantly, mAb 18F25.2a potently reduced P. falciparum infection of Anopheles stephensi mosquitoes in a complement-dependent manner, as assessed by standard membrane feeding assays. Together, our data identify Pfs230 Domain 7 as target for transmission-blocking antibodies and provide a strong incentive to study domains outside Pfs230Pro-D1 as TBV candidates.

Functional characterization of a conserved membrane protein, Pbs54, involved in gamete fertilization in Plasmodium berghei

The successful completion of gamete fertilization is essential for malaria parasite transmission, and this process can be targeted by intervention strategies. In this study, we identified a conserved gene (PBANKA_0813300) in the rodent malaria parasite Plasmodium berghei, which encodes a protein of 54 kDa (designated as Pbs54). Localization studies indicated that Pbs54 is associated with the plasma membranes of gametes and ookinetes. Functional studies by gene disruption showed that the Δpbs54 parasites had no defect in asexual proliferation, gametocyte development, or gametogenesis. However, the interactions between male and female gametes were significantly decreased compared with wild-type parasites. The Δpbs54 lines did not show a further reduction in zygote and ookinete numbers during in vitro culture, indicating that the defects were probably restricted to gamete fertilization. Consistent with this finding, mosquitoes fed on Δpbs54-infected mice showed a 30.1% reduction in infection prevalence and a 74.7% reduction in oocyst intensity. Cross-fertilization assay indicated that both male and female gametes were impaired in the Δpbs54 parasites. To evaluate its transmission-blocking potential, we obtained polyclonal antibodies from mice immunized with the recombinant Pbs54 (rPbs54) protein. In vitro assays showed that anti-rPbs54 sera inhibited ookinete formation by 42.7%. Our experiments identified Pbs54 as a fertility factor required for mosquito transmission and a novel candidate for a malaria transmission-blocking vaccine.

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.

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.

Malaria transmission-blocking vaccines Pfs230D1-EPA and Pfs25-EPA in Alhydrogel in healthy Malian adults; a phase 1, randomised, controlled trial

Malaria transmission-blocking vaccines target mosquito-stage parasites and will support elimination programmes. Gamete vaccine Pfs230D1-EPA/Alhydrogel induced superior activity to zygote vaccine Pfs25-EPA/Alhydrogel in malaria-naive US adults. Here, we compared these vaccines in malaria-experienced Malians. We did a pilot safety study then double-blind, block-randomised, comparator-controlled main-phase trial in malaria-intense Bancoumana, Mali. 18-50-year-old healthy non-pregnant, non-breastfeeding consenting adult residents were randomly assigned (1:1:1:1) to receive four doses at months 0, 1, 4·5, and 16·5 of either 47 μg Pfs25, 40 μg Pfs230D1 or comparator (Twinrix or Menactra)-all co-administered with normal saline for blinding-or 47 μg Pfs25 plus 40 μg Pfs230D1 co-administered. We documented safety and tolerability (primary endpoint in the as-treated populations) and immunogenicity (secondary endpoint in the as-treated populations: ELISA, standard-membrane-feeding assay, and mosquito direct skin feed assay). This trial is registered at ClinicalTrials.gov, NCT02334462. Between March 19, and June 2, 2015, we screened 471 individuals. Of 225 enrolled for the pilot and main cohorts, we randomly assigned 25 participants to pilot safety cohort groups of five (20%) to receive a two-dose series of Pfs25-EPA/Alhydrogel (16 μg), Pfs230D1-EPA/Alhydrogel (15 μg) or comparator, followed by Pfs25-EPA/Alhydrogel (16 μg) plus Pfs230D1-EPA/Alhydrogel (15 μg) or comparator plus saline. For the main cohort, we enrolled 200 participants between May 11 and June 2, 2015, to receive a four-dose series of 47 μg Pfs25-EPA/Alhydrogel plus saline (n=50 [25%]; Pfs25), 40 μg Pfs230D1-EPA/Alhydrogel plus saline (n=49 [25%]; Pfs230D1), 47 μg Pfs25-EPA/Alhydrogel plus 40 μg Pfs230D1-EPA/Alhydrogel (n=50 [25%]; Pfs25 plus Pfs230D1), or comparator (Twinrix or Menactra) plus saline (n=51 [25%]). Vaccinations were well tolerated in the pilot safety and main phases. Most vaccinees became seropositive after two Pfs230D1 or three Pfs25 doses; peak titres increased with each dose thereafter (Pfs230D1 geometric mean: 77·8 [95% CI 56·9-106·3], 146·4 [108·3-198·0], and 410·2 [301·6-558·0]; Pfs25 geometric mean 177·7 [130·3-242·4] and 315·7 [209·9-474·6]). Functional activity (mean peak transmission-reducing activity) appeared for Pfs230D1 (74·5% [66·6-82·5]) and Pfs25 plus Pfs230D1 (68·6% [57·3-79·8]), after the third dose and after the fourth dose (88·9% [81·7-96·2] for Pfs230D1 and 85·0% [78·4-91·5] Pfs25 plus Pfs230D1) but not for Pfs25 (58·2% [49·1-67·3] after the third dose and 58·2% [48·5-67·9] after the fourth dose). Pfs230D1 transmission-reducing activity (73·7% [64·1-83·3]) persisted 10 weeks after the fourth dose. Transmission-reducing activity of 80% was estimated at 1659 ELISA units for Pfs25, 218 for Pfs230D1, and 223 for Pfs230D1 plus Pfs25. After 3850 direct skin feed assays, 35 participants (12 Pfs25, eight Pfs230D1, five Pfs25 plus Pfs230D1, and ten comparator) had transmitted parasites at least once. The proportion of positive assays in vaccine groups (Pfs25 33 [3%] of 982 [-0·013 to 0·014], Pfs230D1 22 [2%] of 954 [-0·005 to 0·027], and combination 11 [1%] of 940 [-0·024 to 0·002]) did not differ from that of the comparator (22 [2%] of 974), nor did Pfs230D1 and combination groups differ (-0·024 to 0·001). Pfs230D1 but not Pfs25 vaccine induces durable serum functional activity in Malian adults. Direct skin feed assays detect parasite transmission to mosquitoes but increased event rates are needed to assess vaccine effectiveness. Intramural Research Program of the National Institute of Allergy and Infectious Diseases and US National Institutes of Health.

Preclinical evaluations of Pfs25-EPA and Pfs230D1-EPA in AS01 for a vaccine to reduce malaria transmission

Malaria transmission-blocking vaccine candidates Pfs25-EPA and Pfs230D1-EPA target sexual stage development of Plasmodium falciparum parasites in the mosquito host, thereby reducing mosquito infectivity. When formulated on Alhydrogel, Pfs25-EPA has demonstrated safety and immunogenicity in a phase 1 field trial, while Pfs230D1-EPA has shown superior activity to Pfs25-EPA in a phase 1US trial and has entered phase 2 field trials. Development continues to enhance immunogenicity of these candidates toward producing a vaccine to reduce malaria transmission (VRMT) with both pre-erythrocytic (i.e., anti-infection) and transmission-blocking components. GSK Adjuvant Systems have demonstrated successful potency in pre-erythrocytic vaccine trials and might offer a common platform for VRMT development. Here, we describe preclinical evaluations of Pfs25-EPA and Pfs230D1-EPA nanoparticles with GSK platforms. Formulations were stable after a series of assessments and induced superior antibody titers and functional activity in CD-1 mice, compared to Alhydrogel formulations of the same antigens.

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, yetall 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.

Highly potent, naturally acquired human monoclonal antibodies against Pfs48/45 block Plasmodium falciparum transmission to mosquitoes

Malaria transmission-blocking vaccines (TBVs) aim to induce antibodies that interrupt malaria parasite development in the mosquito, thereby blocking onward transmission, and provide a much-needed tool for malaria control and elimination. The parasite surface protein Pfs48/45 is a leading TBV candidate. Here, we isolated and characterized a panel of 81 human Pfs48/45-specific monoclonal antibodies (mAbs) from donors naturally exposed to Plasmodium parasites. Genetically diverse mAbs against each of the three domains (D1-D3) of Pfs48/45 were identified. The most potent mAbs targeted D1 and D3 and achieved >80% transmission-reducing activity in standard membrane-feeding assays, at 10 and 2μg/mL, respectively. Co-crystal structures of D3 in complex with four different mAbs delineated two conserved protective epitopes. Altogether, these Pfs48/45-specific human mAbs provide important insight into protective and non-protective epitopes that can further our understanding of transmission and inform the design of refined malaria transmission-blocking vaccine candidates.

Engineering of a plant-produced virus-like particle to improve the display of the Plasmodium falciparum Pfs25 antigen and transmission-blocking activity of the vaccine candidate.

Malaria kills around 409,000 people a year, mostly children under the age of five. Malaria transmission-blocking vaccines work to reduce malaria prevalence in a community and have the potential to be part of a multifaceted approach required to eliminate the parasites causing the disease. Pfs25 is a leading malaria transmission-blocking antigen and has been successfully produced in a plant expression system as both a subunit vaccine and as a virus-like particle. This study demonstrates an improved version of the virus-like particle antigen display molecule by eliminating known protease sites from the prior A85 variant. This re-engineered molecule, termed B29, displays three times the number of Pfs25 antigens per virus-like particle compared to the original Pfs25 virus-like particle. An improved purification scheme was also developed, resulting in a substantially higher yield and improved purity. The molecule was evaluated in a mouse model and found to induce improved transmission-blocking activity at lower doses and longer durations than the original molecule.

366. Optimization of a Surrogate Assay for Efficacy of a Malaria Transmission Blocking Vaccine

Abstract Background An estimated 241 million malaria cases and 627,000 malaria deaths occurred worldwide in 2020. Efforts to develop a higher efficacy malaria vaccine are a global priority, but parasites pose a complex biological puzzle for vaccine development. Innovative vaccine strategies include transmission blocking vaccines (TBV) that target the mosquito stages of parasite development to interrupt parasite transmission. The current leading TBV called Pfs230D1-EPA targets Pfs230, a Plasmodium falciparum gamete surface protein that mediates binding of microgametes to red blood cells. Antibodies against Pfs230 bind and lyse gametes in a complement-dependent manner to block transmission. Methods Here, we are exploiting a panel of human monoclonal antibodies (hmAb) generated from human volunteers who received Pfs230D1-EPA formulated in Alhydrogel or AS01 adjuvants, to develop a competitive ELISA platform. This assay will quantify epitope-specific serum antibodies along with their features and effector functions, expanding the conventionally used functional assay of standard membrane feeding (SMFA). Results The competitive ELISA is being developed with single-chain variable fragments (ScFv) to define epitope-specific binding of antibodies in sera from vaccinated individuals. Sera of human volunteers from trials in Mali, Africa will be assayed to assess epitope-specific serum antibody binding that is competed off in the presence of saturating levels of ScFv. Preliminary optimization of the competitive assay has shown concentration-dependent epitope-specific competition between ScFv and hmAbs and confirmation of ScFv binding to Pfs230D1 antigen. Conclusion Further optimization of this assay will characterize the complement-binding and isotype/subclass-specific features of epitope-specific antibodies. These efforts will lay the foundations for standardized assays that can assess correlates of vaccine efficacy and durability of this leading TBV. Disclosures All Authors: No reported disclosures.

Vectorial capacity and TEP1 genotypes of Anopheles gambiae sensu lato mosquitoes on the Kenyan coast

BackgroundMalaria remains one of the most important infectious diseases in sub-Saharan Africa, responsible for approximately 228 million cases and 602,000 deaths in 2020. In this region, malaria transmission is driven mainly by mosquitoes of the Anopheles gambiae and, more recently, Anopheles funestus complex. The gains made in malaria control are threatened by insecticide resistance and behavioural plasticity among these vectors. This, therefore, calls for the development of alternative approaches such as malaria transmission-blocking vaccines or gene drive systems. The thioester-containing protein 1 (TEP1) gene, which mediates the killing of Plasmodium falciparum in the mosquito midgut, has recently been identified as a promising target for gene drive systems. Here we investigated the frequency and distribution of TEP1 alleles in wild-caught malaria vectors on the Kenyan coast.MethodsMosquitoes were collected using CDC light traps both indoors and outdoors from 20 houses in Garithe village, along the Kenyan coast. The mosquitoes were dissected, and the different parts were used to determine their species, blood meal source, and sporozoite status. The data were analysed and visualised using the R (v 4.0.1) and STATA (v 17.0).ResultsA total of 18,802 mosquitoes were collected, consisting of 77.8% (n = 14,631) Culex spp., 21.4% (n = 4026) An. gambiae sensu lato, 0.4% (n = 67) An. funestus, and 0.4% (n = 78) other Anopheles (An. coustani, An. pharoensis, and An. pretoriensis). Mosquitoes collected were predominantly exophilic, with the outdoor catches being higher across all the species: Culex spp. 93% (IRR = 11.6, 95% Cl [5.9–22.9] P < 0.001), An. gambiae s.l. 92% (IRR = 7.2, 95% Cl [3.6–14.5]; P < 0.001), An. funestus 91% (IRR = 10.3, 95% Cl [3.3–32.3]; P < 0.001). A subset of randomly selected An. gambiae s.l. (n = 518) was identified by polymerase chain reaction (PCR), among which 77.2% were An. merus, 22% were An. arabiensis, and the rest were not identified. We were also keen on identifying and describing the TEP1 genotypes of these mosquitoes, especially the *R3/R3 allele that was identified recently in the study area. We identified the following genotypes among An. merus: *R2/R2, *R3/R3, *R3/S2, *S1/S1, and *S2/S2. Among An. arabiensis, we identified *R2/R2, *S1/S1, and *S2/S2. Tests on haplotype diversity showed that the most diverse allele was TEP1*S1, followed by TEP1*R2. Tajima’s D values were positive for TEP1*S1, indicating that there is a balancing selection, negative for TEP1*R2, indicating there is a recent selective sweep, and as for TEP1*R3, there was no evidence of selection. Phylogenetic analysis showed two distinct clades: refractory and susceptible alleles.ConclusionsWe find that the malaria vectors An. gambiae s.l. and An. funestus are predominantly exophilic. TEP1 genotyping for An. merus revealed five allelic combinations, namely *R2/R2, *R3/R3, *R3/S2, *S1/S1 and *S2/S2, while in An. arabiensis we only identified three allelic combinations: *R2/R2, *S1/S1, and *S2/S2. The TEP1*R3 allele was restricted to only An. merus among these sympatric mosquito species, and we find that there is no evidence of recombination or selection in this allele.Graphical

Structural analysis of Plasmodium falciparum ookinete surface antigen Pfs28 relevant for malaria vaccine design

Pfs28 is a Plasmodium falciparum malaria transmission-blocking vaccine candidate that is anchored to the parasite surface through a C-terminal glycosylphosphatidylinositol (GPI) moiety, and plays a role in parasite survival in the mosquito midgut. Pfs28 contains epidermal growth factor (EGF)-like domains and is part of a family of sexual stage malaria proteins that includes the related vaccine antigen Pfs25. The lack of structural definition of Pfs28 and the immune response to this candidate has limited further malaria vaccine development for this antigen. Here, we present the crystal structure of Pfs28, examine its conservation with P. vivax Pvs28, and evaluate the cross-reactivity of Pfs28 to antibodies that recognize Pfs25. Pfs28 is comprised of four EGF-like domains stabilized by ten disulfide bridges with an overall architecture that highly resembles Pfs25. Despite the high sequence and structural similarity between these antigens, no cross reactivity of Pfs28 to anti-Pfs25 monoclonal antibodies could be demonstrated.

Structure of the malaria vaccine candidate Pfs48/45 and its recognition by transmission blocking antibodies

An effective malaria vaccine remains a global health priority and vaccine immunogens which prevent transmission of the parasite will have important roles in multi-component vaccines. One of the most promising candidates for inclusion in a transmission-blocking malaria vaccine is the gamete surface protein Pfs48/45, which is essential for development of the parasite in the mosquito midgut. Indeed, antibodies which bind Pfs48/45 can prevent transmission if ingested with the parasite as part of the mosquito bloodmeal. Here we present the structure of full-length Pfs48/45, showing its three domains to form a dynamic, planar, triangular arrangement. We reveal where transmission-blocking and non-blocking antibodies bind on Pfs48/45. Finally, we demonstrate that antibodies which bind across this molecule can be transmission-blocking. These studies will guide the development of future Pfs48/45-based vaccine immunogens.