Elucidating functional epitopes within the N-terminal region of malaria transmission blocking vaccine antigen Pfs230

Pfs230 is a leading malaria transmission blocking vaccine (TBV) candidate. Comprising 3135 amino acids (aa), the large size of Pfs230 necessitates the use of sub-fragments as vaccine immunogens. Therefore, determination of which regions induce functional antibody responses is essential. We previously reported that of 27 sub-fragments spanning the entire molecule, only five induced functional antibodies. A “functional” antibody is defined herein as one that inhibits Plasmodium falciparum parasite development in mosquitoes in a standard membrane-feeding assay (SMFA). These five sub-fragments were found within the aa 443–1274 range, and all contained aa 543–730. Here, we further pinpoint the location of epitopes within Pfs230 that are recognized by functional antibodies using antibody depletion and enrichment techniques. Functional epitopes were not found within the aa 918–1274 region. Within aa 443–917, further analysis showed the existence of functional epitopes not only within the aa 543–730 region but also outside of it. Affinity-purified antibodies using a synthetic peptide matching aa 543–588 showed activity in the SMFA. Immunization with a synthetic peptide comprising this segment, formulated either as a carrier-protein conjugate vaccine or with a liposomal vaccine adjuvant system, induced antibodies in mice that were functional in the SMFA. These findings provide key insights for Pfs230-based vaccine design and establish the feasibility for the use of synthetic peptide antigens for a malaria TBV.

Vector microbiota manipulation by host antibodies: the forgotten strategy to develop transmission-blocking vaccines

Human and animal pathogens that are transmitted by arthropods are a global concern, particularly those vectored by ticks (e.g. Borrelia burgdorferi and tick-borne encephalitis virus) and mosquitoes (e.g. malaria and dengue virus). Breaking the circulation of pathogens in permanent foci by controlling vectors using acaricide-based approaches is threatened by the selection of acaricide resistance in vector populations, poor management practices and relaxing of control measures. Alternative strategies that can reduce vector populations and/or vector-mediated transmission are encouraged worldwide. In recent years, it has become clear that arthropod-associated microbiota are involved in many aspects of host physiology and vector competence, prompting research into vector microbiota manipulation. Here, we review how increased knowledge of microbial ecology and vector-host interactions is driving the emergence of new concepts and tools for vector and pathogen control. We focus on the immune functions of host antibodies taken in the blood meal as they can target pathogens and microbiota bacteria within hematophagous arthropods. Anti-microbiota vaccines are presented as a tool to manipulate the vector microbiota and interfere with the development of pathogens within their vectors. Since the importance of some bacterial taxa for colonization of vector-borne pathogens is well known, the disruption of the vector microbiota by host antibodies opens the possibility to develop novel transmission-blocking vaccines.

Assessment in vitro of the antimalarial and transmission blocking activities of Cipargamin and Ganaplacide in artemisinin resistant Plasmodium falciparum

Artemisinin resistance in Plasmodium falciparum has emerged and spread widely in the Greater Mekong Subregion threatening current first line artemisinin combination treatments. New antimalarial drugs are needed urgently. Cipargamin (KAE609) and ganaplacide (KAF156) are promising novel antimalarial compounds in advanced stages of development. Both compounds have potent asexual blood stage activities, inhibit P. falciparum gametocytogenesis and reduce oocyst development in anopheline mosquitoes. In this study, we compared the asexual and sexual stage activities of cipargamin, ganaplacide and artesunate in artemisinin resistant P. falciparum isolates (N=7, K13 mutation; C580Y, G449A and R539T) from Thailand and Cambodia. Asexual blood stage antimalarial activity was evaluated in a SYBR-green I based 72h in vitro assay, and the effects on male and female mature stage V gametocytes were assessed in the P. falciparum dual gamete formation assay. Ganaplacide had higher activities when compared to cipargamin and artesunate, with a mean (SD) IC50 against asexual stages of 5.5 (1.1) nM, 7.8 (3.9) nM for male gametocytes and 57.9 (59.6) nM for female gametocytes. Cipargamin had a similar potency against male and female gametocytes, with a mean (SD) IC50 of 123.1 (80.2) nM for male gametocytes, 88.5 (52.7) nM for female gametocytes and 2.4 (0.6) nM for asexual stages. Both cipargamin and ganaplacide showed significant transmission-blocking activities against artemisinin resistant P. falciparum in vitro .

Malaria transmission-blocking conjugate vaccine in ALFQ adjuvant induces durable functional immune responses in rhesus macaques

Malaria transmission-blocking vaccines candidates based on Pfs25 and Pfs230 have advanced to clinical studies. Exoprotein A (EPA) conjugate of Pfs25 in Alhydrogel® developed functional immunity in humans, with limited durability. Pfs230 conjugated to EPA (Pfs230D1-EPA) with liposomal adjuvant AS01 is currently in clinical trials in Mali. Studies with these conjugates revealed that non-human primates are better than mice to recapitulate the human immunogenicity and functional activity. Here, we evaluated the effect of ALFQ, a liposomal adjuvant consisting of TLR4 agonist and QS21, on the immunogenicity of Pfs25-EPA and Pfs230D1-EPA in Rhesus macaques. Both conjugates generated strong antibody responses and functional activity after two vaccinations though activity declined rapidly. A third vaccination of Pfs230D1-EPA induced functional activity lasting at least 9 months. Antibody avidity increased with each vaccination and correlated strongly with functional activity. IgG subclass analysis showed induction of Th1 and Th2 subclass antibody levels that correlated with activity.

Identification of Novel Malaria Transmission-Blocking Vaccine Candidates

Control measures have significantly reduced malaria morbidity and mortality in the last two decades; however, the downward trends have stalled and have become complicated by the emergence of COVID-19. Significant efforts have been made to develop malaria vaccines, but currently only the RTS,S/AS01 vaccine against Plasmodium falciparum has been recommended by the WHO, for widespread use among children in sub-Saharan Africa. The efficacy of RTS,S/AS01 is modest, and therefore the development of more efficacious vaccines is still needed. In addition, the development of transmission-blocking vaccines (TBVs) to reduce the parasite transmission from humans to mosquitoes is required toward the goal of malaria elimination. Few TBVs have reached clinical development, and challenges include low immunogenicity or high reactogenicity in humans. Therefore, novel approaches to accelerate TBV research and development are urgently needed, especially novel TBV candidate discovery. In this mini review we summarize the progress in TBV research and development, novel TBV candidate discovery, and discuss how to accelerate novel TBV candidate discovery.

Molecular interactions between parasite and mosquito during midgut invasion as targets to block malaria transmission

Despite considerable effort, malaria remains a major public health burden. Malaria is caused by five Plasmodium species and is transmitted to humans via the female Anopheles mosquito. The development of malaria vaccines against the liver and blood stages has been challenging. Therefore, malaria elimination strategies advocate integrated measures, including transmission-blocking approaches. Designing an effective transmission-blocking strategy relies on a sophisticated understanding of the molecular mechanisms governing the interactions between the mosquito midgut molecules and the malaria parasite. Here we review recent advances in the biology of malaria transmission, focusing on molecular interactions between Plasmodium and Anopheles mosquito midgut proteins. We provide an overview of parasite and mosquito proteins that are either targets for drugs currently in clinical trials or candidates of promising transmission-blocking vaccines.

Profiling the Quality and Quantity of Naturally Induced Antibody Responses Against Pfs230 and Pfs48/45 Among Non-Febrile Children Living in Southern Ghana: A Longitudinal Study

A clear understanding of the properties of naturally induced antibody responses against transmission-blocking vaccine candidates can accelerate the understanding of the development of transmission-blocking immunity. This study characterized the naturally induced IgG responses against two leading transmission-blocking vaccine antigens, Pfs230 and Pfs48/45, in non-febrile children living in Simiw, Ghana. Consecutive sampling was used to recruit 84 non-febrile children aged from 6 to 12 years old into the 6-month (November 2017 until May 2018) longitudinal study. Venous blood (1 ml) was collected once every 2 months and used to determine hemoglobin levels, P. falciparum prevalence using microscopy and polymerase chain reaction, and the levels and relative avidity of IgG responses against Pfs230 and Pfs48/45 using indirect ELISA. IgG levels against Pfs230 and Pfs48/45 decreased from the start (November) to the middle (January) and end (March) of the dry season respectively, then they began to increase. Participants, especially older children (10–12 years old) with active infections generally had lower antibody levels against both antigens. The relative avidities of IgG against both antigens followed the trend of IgG levels until the middle of the dry season, after which the relative avidities of both antigens correlated inversely with the antibody levels. In conclusion, although IgG antibody levels against both Pfs48/45 and Pfs230 began to increase by the early rainy season, they were inversely correlated to their respective relative avidities.

Characterization of Naturally Acquired Immunity to a Panel of Antigens Expressed in Mature P. falciparum Gametocytes

IntroductionNaturally acquired immune responses against antigens expressed on the surface of mature gametocytes develop in individuals living in malaria-endemic areas. Evidence suggests that such anti-gametocyte immunity can block the development of the parasite in the mosquito, thus playing a role in interrupting transmission. A better comprehension of naturally acquired immunity to these gametocyte antigens can aid the development of transmission-blocking vaccines and improve our understanding of the human infectious reservoir.MethodsAntigens expressed on the surface of mature gametocytes that had not previously been widely studied for evidence of naturally acquired immunity were identified for protein expression alongside Pfs230-C using either the mammalian HEK293E or the wheat germ cell-free expression systems. Where there was sequence variation in the candidate antigens (3D7 vs a clinical isolate PfKE04), both variants were expressed. ELISA was used to assess antibody responses against these antigens, as well as against crude stage V gametocyte extract (GE) and AMA1 using archived plasma samples from individuals recruited to participate in malaria cohort studies. We analyzed antibody levels (estimated from optical density units using a standardized ELISA) and seroprevalence (defined as antibody levels greater than three standard deviations above the mean levels of a pool of malaria naïve sera). We described the dynamics of antibody responses to these antigens by identifying factors predictive of antibody levels using linear regression models.ResultsOf the 25 antigens selected, seven antigens were produced successfully as recombinant proteins, with one variant antigen, giving a total of eight proteins for evaluation. Antibodies to the candidate antigens were detectable in the study population (N = 216), with seroprevalence ranging from 37.0% (95% CI: 30.6%, 43.9%) for PSOP1 to 77.8% (95% CI: 71.6%, 83.1%) for G377 (3D7 variant). Responses to AMA1 and GE were more prevalent than those to the gametocyte proteins at 87.9% (95% CI: 82.8%, 91.9%) and 88.3% (95% CI: 83.1%, 92.4%), respectively. Additionally, both antibody levels and breadth of antibody responses were associated with age and concurrent parasitaemia.ConclusionAge and concurrent parasitaemia remain important determinants of naturally acquired immunity to gametocyte antigens. Furthermore, we identify novel candidates for transmission-blocking activity evaluation.

Global diversity of the gene encoding the Pfs25 protein—a Plasmodium falciparum transmission-blocking vaccine candidate

Background Vaccines against the sexual stages of the malarial parasite Plasmodium falciparum are indispensable for controlling malaria and abrogating the spread of drug-resistant parasites. Pfs25, a surface antigen of the sexual stage of P. falciparum, is a leading candidate for transmission-blocking vaccine development. While clinical trials have reported that Pfs25-based vaccines are safe and effective in inducing transmission-blocking antibodies, the extent of the genetic diversity of Pfs25 in malaria endemic populations has rarely been studied. Thus, this study aimed to investigate the global diversity of Pfs25 in P. falciparum populations. Methods A database of 307 Pfs25 sequences of P. falciparum was established. Population genetic analyses were performed to evaluate haplotype and nucleotide diversity, analyze haplotypic distribution patterns of Pfs25 in different geographical populations, and construct a haplotype network. Neutrality tests were conducted to determine evidence of natural selection. Homology models of the Pfs25 haplotypes were constructed, subjected to molecular dynamics (MD), and analyzed in terms of flexibility and percentages of secondary structures. Results The Pfs25 gene of P. falciparum was found to have 11 unique haplotypes. Of these, haplotype 1 (H1) and H2, the major haplotypes, represented 70% and 22% of the population, respectively, and were dominant in Asia, whereas only H1 was dominant in Africa, Central America, and South America. Other haplotypes were rare and region-specific, resulting in unique distribution patterns in different geographical populations. The diversity in Pfs25 originated from ten single-nucleotide polymorphism (SNP) loci located in the epidermal growth factor (EGF)-like domains and anchor domain. Of these, an SNP at position 392 (GGA/GCA), resulting in amino acid substitution 131 (Gly/Ala), defined the two major haplotypes. The MD results showed that the structures of H1 and H2 variants were relatively similar. Limited polymorphism in Pfs25 could likely be due to negative selection. Conclusions The study successfully established a Pfs25 sequence database that can become an essential tool for monitoring vaccine efficacy, designing assays for detecting malaria carriers, and conducting epidemiological studies of P. falciparum. The discovery of the two major haplotypes, H1 and H2, and their conserved structures suggests that the current Pfs25-based vaccines could be used globally for malaria control. Graphical Abstract