Transmission-blocking Vaccine Candidate Research Articles

Immunogenicity and transmission-blocking potential of quiescin sulfhydryl oxidase in Plasmodium vivax.

Transmission-blocking vaccines (TBVs) can effectively prevent the community's spread of malaria by targeting the antigens of mosquito sexual stage parasites. At present, only a few candidate antigens have demonstrated transmission-blocking activity (TBA) potential in P. vivax. Quiescin-sulfhydryl oxidase (QSOX) is a sexual stage protein in the rodent malaria parasite Plasmodium berghei and is associated with a critical role in protein folding by introducing disulfides into unfolded reduced proteins. Here, we reported the immunogenicity and transmission-blocking potency of the PvQSOX in P. vivax. The full-length recombinant PvQSOX protein (rPvQSOX) was expressed in the Escherichia coli expression system. The anti-rPvQSOX antibodies were generated following immunization with the rPvQSOX in rabbits. A parasite integration of the pvqsox gene into the P. berghei pbqsox gene knockout genome was developed to express full-length PvQSOX protein in P. berghei (Pv-Tr-PbQSOX). In western blot, the anti-rPvQSOX antibodies recognized the native PvQSOX protein expressed in transgenic P. berghei gametocyte and ookinete. In indirect immunofluorescence assays, the fluorescence signal was detected in the sexual stages, including gametocyte, gamete, zygote, and ookinete. Anti-rPvQSOX IgGs obviously inhibited the ookinetes and oocysts development both in vivo and in vitro using transgenic parasites. Direct membrane feeding assays of anti-rPvQSOX antibodies were conducted using four field P. vivax isolates (named isolates #1-4) in Thailand. Oocyst density in mosquitoes was significantly reduced by 32.00, 85.96, 43.52, and 66.03% with rabbit anti-rPvQSOX antibodies, respectively. The anti-rPvQSOX antibodies also showed a modest reduction of infection prevalence by 15, 15, 20, and 22.22%, respectively, as compared to the control, while the effect was insignificant. The variation in the DMFA results may be unrelated to the genetic polymorphisms. Compared to the P.vivax Salvador (Sal) I strain sequences, the pvqsox in isolate #1 showed no amino acid substitution, whereas isolates #2, #3, and #4 all had the M361I substitution. Our results suggest that PvQSOX could serve as a potential P. vivax TBVs candidate, which warrants further evaluation and optimization.

Malaria transmission blocking activity of Anopheles stephensi alanyl aminopeptidase N antigen formulated with MPL, CpG, and QS21 adjuvants.

Malaria, a preventive and treatable disease, is still responsible for annual deaths reported in most tropical regions, principally in sub-Saharan Africa. Subunit recombinant transmission-blocking vaccines (TBVs) have been proposed as promising vaccines to succeed in malaria elimination and eradication. Here, a provisional study was designed to assess the immunogenicity and functional activity of alanyl aminopeptidase N (APN1) of Anopheles stephensi, as a TBV candidate, administered with MPL, CpG, and QS21 adjuvants in the murine model. The mouse groups were immunized with recombinant APN1 (rAPN1) alone or formulated with CpG, MPL, QS-21, or a combination of adjuvants (CMQ), and the elicited immune responses were evaluated after the third immunization. The standard membrane feeding assay (SMFA) measured the functional activity of antibodies against bacterial-expressed APN1 protein in adjuvanted vaccine groups on transmission of P. falciparum (NF54) to An. stephensi mosquitoes. Evaluation of mice vaccinated with rAPN1 formulated with distinct adjuvants manifested a significant increase in the high-avidity level of anti-APN1 IgG and IgG subclasses; however, rAPN1 induced the highest level of high-avidity anti-APN1 IgG1, IgG2a, and IgG2b antibodies in the immunized vaccine group 5 (APN1/CMQ). In addition, vaccine group 5 (receiving APN1/CMQ), had still the highest level of anti-APN1 IgG antibodies relative to other immunized groups after six months, on day 180. The SMFA data indicates a trend towards higher transmission-reducing activity in groups 2 and 5, which received the antigen formulated with CpG or a combination of three adjuvants. The results have shown the capability of admixture to stimulate high-affinity and long-lasting antibodies against the target antigen to hinder Plasmodium parasite development in the mid-gut of An. stephensi. The attained results authenticated APN1/CMQ and APN1/CpG as a potent APN1-based TBV formulation which will be helpful in designing a vaccine in the future.

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.

A Pfs48/45-based vaccine to block Plasmodium falciparum transmission: phase 1, open-label, clinical trial

BackgroundThe stalling global progress in malaria control highlights the need for novel tools for malaria elimination, including transmission-blocking vaccines. Transmission-blocking vaccines aim to induce human antibodies that block parasite development in the mosquito and mosquitoes becoming infectious. The Pfs48/45 protein is a leading Plasmodium falciparum transmission-blocking vaccine candidate. The R0.6C fusion protein, consisting of Pfs48/45 domain 3 (6C) and the N-terminal region of P. falciparum glutamate-rich protein (R0), has previously been produced in Lactococcus lactis and elicited functional antibodies in rodents. Here, we assess the safety and transmission-reducing efficacy of R0.6C adsorbed to aluminium hydroxide with and without Matrix-M™ adjuvant in humans.MethodsIn this first-in-human, open-label clinical trial, malaria-naïve adults, aged 18–55 years, were recruited at the Radboudumc in Nijmegen, the Netherlands. Participants received four intramuscular vaccinations on days 0, 28, 56 and 168 with either 30 µg or 100 µg of R0.6C and were randomised for the allocation of one of the two different adjuvant combinations: aluminium hydroxide alone, or aluminium hydroxide combined with Matrix-M1™ adjuvant. Adverse events were recorded from inclusion until 84 days after the fourth vaccination. Anti-R0.6C and anti-6C IgG titres were measured by enzyme-linked immunosorbent assay. Transmission-reducing activity of participants’ serum and purified vaccine-specific immunoglobulin G was assessed by standard membrane feeding assays using laboratory-reared Anopheles stephensi mosquitoes and cultured P. falciparum gametocytes.ResultsThirty-one participants completed four vaccinations and were included in the analysis. Administration of all doses was safe and well-tolerated, with one related grade 3 adverse event (transient fever) and no serious adverse events occurring. Anti-R0.6C and anti-6C IgG titres were similar between the 30 and 100 µg R0.6C arms, but higher in Matrix-M1™ arms. Neat participant sera did not induce significant transmission-reducing activity in mosquito feeding experiments, but concentrated vaccine-specific IgGs purified from sera collected two weeks after the fourth vaccination achieved up to 99% transmission-reducing activity.ConclusionsR0.6C/aluminium hydroxide with or without Matrix-M1™ is safe, immunogenic and induces functional Pfs48/45-specific transmission-blocking antibodies, albeit at insufficient serum concentrations to result in transmission reduction by neat serum. Future work should focus on identifying alternative vaccine formulations or regimens that enhance functional antibody responses.Trial registrationThe trial is registered with ClinicalTrials.gov under identifier NCT04862416.

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.

A Novel Ex Vivo Assay to Evaluate Functional Effectiveness of Plasmodium vivax Transmission-Blocking Vaccine Using Pvs25 Transgenic Plasmodium berghei.

Plasmodium falciparum and Plasmodium vivax account for >90% global malaria burden. Transmission intervention strategies encompassing transmission-blocking vaccines (TBV) and drugs represent ideal public health tools to eliminate malaria at the population level. The availability of mature P. falciparum gametocytes through in vitro culture has facilitated development of a standard membrane feeding assay to assess efficacy of transmission interventions against P. falciparum. The lack of in vitro culture for P. vivax has significantly hampered similar progress on P. vivax and limited studies have been possible using blood from infected patients in endemic areas. The ethical and logistical limitations of on-time access to blood from patients have impeded the development of P. vivax TBVs. Transgenic murine malaria parasites (Plasmodium berghei) expressing TBV candidates offer a promising alternative for evaluation of P. vivax TBVs through in vivo studies in mice, and ex vivo membrane feeding assay (MFA). We describe the development of transmission-competent transgenic TgPbvs25 parasites and optimization of parameters to establish an ex vivo MFA to evaluate P. vivax TBV based on Pvs25 antigen. The MFA is expected to expedite Pvs25-based TBV development without dependence on blood from P. vivax-infected patients in endemic areas for evaluation.

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.

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.

Evaluation of a new fusion antigen, cd loop and HAP2-GCS1 domain (cd-HAP) of Plasmodium falciparum Generative Cell Specific 1 antigen formulated with various adjuvants, as a transmission blocking vaccine

BackgroundMalaria is a major global health challenge, and for the elimination and eradication of this disease, transmission-blocking vaccines (TBVs) are a priority. Plasmodium falciparum Generative Cell Specific 1 (PfGCS1), a promising TBV candidate, is essential for gamete fertilization. The HAP2-GCS1 domain of this antigen as well as its cd loop could induce antibodies that partially inhibit transmission of P. falciparum.MethodsIn the current study, a new synthetic fusion antigen containing cd loop and HAP2-GCS1 domain (cd-HAP) of PfGCS1 was evaluated as a transmission blocking vaccine candidate. Initially, the profile of naturally acquired IgG antibodies to the cd-HAP antigen was analysed in Iranian individuals infected with P. falciparum, to confirm that this new fusion protein has the appropriate structure containing common epitopes with the native form of PfGCS1. Then, the immunogenicity of cd-HAP was evaluated in BALB/c mice, using different adjuvant systems such as CpG, MPL, QS-21, and a combination of them (CMQ). Furthermore, the blocking efficacy of polyclonal antibodies induced against these formulations was also assessed by oocyst intensity and infection prevalence in the Standard Membrane Feeding Assay (SMFA).ResultsThe naturally acquired antibodies (dominantly IgG1 and IgG3 subclasses) induced in P. falciparum-infected individuals could recognize the cd-HAP antigen which implies that the new fusion protein has a proper conformation that mimics the native structure of PfGCS1. Concerning the immunogenicity of cd-HAP antigen, the highest IgG levels and titers, by a Th1-type immune profile, and elevated antibody avidity were induced in mice immunized with the cd-HAP antigen formulated with a combination of adjuvants (P < 0.0001). Additionally, cytokine profiling of the immunized mice displayed that a high level of IFN-γ response, a Th1-type immune response, was produced by splenocytes from immunized mice that received cd-HAP antigen in combination with CMQ adjuvants (P < 0.0001). This formulation of cd-HAP antigen with CMQ adjuvants could reduce oocyst intensity and infection prevalence by 82%, evidenced by the SMFA and hold significant implications for future malaria vaccine development.ConclusionAltogether, the results showed that cd-HAP antigen formulated with a combination of the adjuvants (CMQ), could be a promising formulation to develop a PfGCS1-based transmission-blocking vaccine.

Vaccination of cattle with the Babesia bovis sexual-stage protein HAP2 abrogates parasite transmission by Rhipicephalus microplus ticks

The apicomplexan parasite Babesia bovis is responsible for bovine babesiosis, a poorly controlled tick-borne disease of global impact. The widely conserved gametocyte protein HAPLESS2/GCS1 (HAP2) is uniquely expressed on the surface of B. bovis sexual stage parasites and is a candidate for transmission-blocking vaccines (TBV). Here, we tested whether vaccination of calves with recombinant HAP2 (rHAP2) interferes with the transmission of B. bovis by competent ticks. Calves vaccinated with rHAP2 (n = 3), but not control animals (n = 3) developed antibodies specific to the vaccine antigen. Vaccinated and control animals were infested with Rhipicephalus microplus larvae and subsequently infected with virulent blood stage B. bovis parasites by needle inoculation, with all animals developing clinical signs of acute babesiosis. Engorged female ticks fed on the infected calves were collected for oviposition, hatching, and obtention of larvae. Transmission feeding was then conducted using pools of larvae derived from ticks fed on rHAP2-vaccinated or control calves. Recipient calves (n = 3) exposed to larvae derived from control animals, but none of the recipient calves (n = 3) challenged with larvae from ticks fed on rHAP2-vaccinated animals, developed signs of acute babesiosis within 11 days after tick infestation. Antibodies against B. bovis antigens and parasite DNA were found in all control recipient animals, but not in any of the calves exposed to larvae derived from HAP2-vaccinated animals, consistent with the absence of B. bovis infection via tick transmission. Overall, our results are consistent with the abrogation of parasite tick transmission in rHAP2-vaccinated calves, confirming this antigen as a prime TBV candidate against B. bovis.

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.

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.

Genome-wide genetic variation and molecular surveillance of drug resistance in Plasmodium falciparum isolates from asymptomatic individuals in Ouélessébougou, Mali

Sequence analysis of Plasmodium falciparum parasites is informative in ensuring sustained success of malaria control programmes. Whole-genome sequencing technologies provide insights into the epidemiology and genome-wide variation of P. falciparum populations and can characterise geographical as well as temporal changes. This is particularly important to monitor the emergence and spread of drug resistant P. falciparum parasites which is threatening malaria control programmes world-wide. Here, we provide a detailed characterisation of genome-wide genetic variation and drug resistance profiles in asymptomatic individuals in South-Western Mali, where malaria transmission is intense and seasonal, and case numbers have recently increased. Samples collected from Ouélessébougou, Mali (2019–2020; n = 87) were sequenced and placed in the context of older Malian (2007–2017; n = 876) and African-wide (n = 711) P. falciparum isolates. Our analysis revealed high multiclonality and low relatedness between isolates, in addition to increased frequencies of molecular markers for sulfadoxine-pyrimethamine and lumefantrine resistance, compared to older Malian isolates. Furthermore, 21 genes under selective pressure were identified, including a transmission-blocking vaccine candidate (pfCelTOS) and an erythrocyte invasion locus (pfdblmsp2). Overall, our work provides the most recent assessment of P. falciparum genetic diversity in Mali, a country with the second highest burden of malaria in West Africa, thereby informing malaria control activities.

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.

Design of a stabilized non-glycosylated Pfs48/45 antigen enables a potent malaria transmission-blocking nanoparticle vaccine

A malaria vaccine that blocks parasite transmission from human to mosquito would be a powerful method of disrupting the parasite lifecycle and reducing the incidence of disease in humans. Pfs48/45 is a promising antigen in development as a transmission blocking vaccine (TBV) against the deadliest malaria parasite Plasmodium falciparum. The third domain of Pfs48/45 (D3) is an established TBV candidate, but production challenges have hampered development. For example, to date, a non-native N-glycan is required to stabilize the domain when produced in eukaryotic systems. Here, we implement a SPEEDesign computational design and in vitro screening pipeline that retains the potent transmission blocking epitope in Pfs48/45 while creating a stabilized non-glycosylated Pfs48/45 D3 antigen with improved characteristics for vaccine manufacture. This antigen can be genetically fused to a self-assembling single-component nanoparticle, resulting in a vaccine that elicits potent transmission-reducing activity in rodents at low doses. The enhanced Pfs48/45 antigen enables many new and powerful approaches to TBV development, and this antigen design method can be broadly applied towards the design of other vaccine antigens and therapeutics without interfering glycans.

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.

X marks the shot against malaria

The development of a transmission-blocking vaccine (TBV) against malaria is hampered by poor understanding of functional antibody responses. In this issue of Immunity, Fabra-Garcia etal., Ivanochko etal., and Tang etal. isolate human monoclonal antibodies against the two most promising TBV candidates, Pfs48/45 and Pfs230, and map the epitopes responsible for potent transmission-reducing activity.

Evaluation of transmission-blocking potential of Pv22 using clinical Plasmodium vivax infections and transgenic Plasmodium berghei.

Antigens expressed during the sexual development of malaria parasites are transmission-blocking vaccine (TBV) targets. Pb22, a protein expressed and localized to the plasma membrane of gametes and ookinetes in Plasmodium berghei, is an excellent TBV candidate. Here, we evaluated the TB potential of the Plasmodium vivax ortholog Pv22 using a transgenic P. berghei parasite line and P. vivax clinical isolates. The full-length recombinant Pv22 (rPv22) protein was produced and used to immunize mice and rabbits to obtain antibodies. We generated a transgenic P. berghei line (TrPv22Pb) by inserting the pv22 gene into the pb22 locus and showed that Pv22 expression completely rescued the defects in male gametogenesis of the pb22 deletion parasite. Since Pv22 in the transgenic parasite showed similar expression and localization patterns to Pb22, we used the TrPv22Pb parasite as a surrogate to evaluate the TB potential of Pv22. In mosquito feeding assays, mosquitoes feeding on rPv22-immunized mice infected with TrPv22Pb parasites showed a 49.3-53.3% reduction in the oocyst density compared to the control group. In vitro assays showed that the rPv22 immune sera significantly inhibited exflagellation and ookinete formation of the TrPv22Pb parasites. In a direct membrane feeding assay using three clinical P. vivax isolates, the rabbit anti-rPv22 antibodies also significantly decreased the oocyst density by 53.7, 30.2, and 26.2%, respectively. This study demonstrated the feasibility of using transgenic P. berghei parasites expressing P. vivax antigens as a potential tool to evaluate TBV candidates. However, the much weaker TB activity of Pv22 obtained from two complementary assays suggest that Pv22 may not be a promising TBV candidate for P. vivax.

Genetic diversity of transmission-blocking vaccine candidate antigens Pvs25 and Pvs28 in Plasmodium vivax isolates from China

BackgroundTransmission-blocking vaccines (TBVs) target the sexual stages of malaria parasites to reduce or interrupt the transmission cycle in human and mosquito populations. The genetic diversity of TBVs candidate antigens, Pvs25 and Pvs28, in Plasmodium vivax could provide evidence for the development of TBVs.MethodsDry blood spots from P. vivax patients were collected from Dandong, Suining, Hainan, Nyingchi, Tengchong, and Yingjiang in China. The pvs25 and pvs28 genes were amplified and sequenced. The genetic diversity of pvs25 and pvs28 were analyzed using DNASTAR, MEGA6, and DnaSP 5.0 programs.ResultsA total of 377 samples were collected, among which 324 and 272 samples were successfully amplified in the pvs25 and pvs28 genes, respectively. Eight haplotypes were identified in Pvs25, for which the predominant mutation was I130T with 100% prevalence. A variety of 22 haplotypes in Pvs28 were identified. The number of GSGGE/D repeats of Pvs28 was a range of 4–8, among which, high (7–8) and low (4–5) copy numbers of tandem repeats were found in haplotypes H2 and H17, respectively. The nucleotide diversity of pvs28 (π = 0.00305 ± 0.00061) was slightly higher than that of pvs25 (π = 0.00146 ± 0.00007), thus they were not significantly different (P > 0.05). The Tajima's D value of pvs25 was positive whereas pvs28 was negative, which indicated that both genes were affected by natural selection.ConclusionThe genetic diversity of pvs25 and pvs28 genes in China was relatively limited, which provided valuable information for TBVs design and optimization.