Clinical formulation development of Plasmodium falciparum malaria vaccine candidates based on Pfs48/45, Pfs230, and PfCSP.

Two malaria transmission-blocking vaccine (TBV) candidates, R0.6C and ProC6C, have completed preclinical development including the selection of adjuvants, Alhydrogel® with or without the saponin based adjuvant Matrix-M™. Here, we report on the final drug product (formulation) design of R0.6C and ProC6C and evaluate their safety and biochemical stability in preparation for preclinical and clinical pharmacy handling. The point-of-injection stability studies demonstrated that both the R0.6C and ProC6C antigens are stable on Alhydrogel in the presence or absence of Matrix-M for up to 24 h at room temperature. As this is the first study to combine Alhydrogel and Matrix-M for clinical use, we also evaluated their potential interactions. Matrix-M adsorbs to Alhydrogel, while not displacing the > 95 % adsorbed protein. The R0.6C and ProC6C formulations were found to be safe and well tolerated in repeated dose toxicity studies in rabbits generating high levels of functional antibodies that blocked infection of mosquitoes. Further, the R0.6C and ProC6C drug products were found to be stable for minimally 24 months when stored at 2-8 °C, with studies ongoing through 36 months. Together, this data demonstrates the safety and suitability of the L. lactis expression system as well as supports the clinical testing of the R0.6C and ProC6C malaria vaccine candidates in First-In-Human clinical trials.

Production and Purification of Plasmodium Circumsporozoite Protein in Lactococcus lactis.

Malaria is a vector-borne disease caused by Plasmodium parasites of which Plasmodium falciparum contributed to an estimated 247 million cases worldwide in 2021 (WHO malaria report 2022). The P. falciparum Circumsporozoite protein (PfCSP) covers the surface of the sporozoite which is critical to cell invasion in the human host. PfCSP is the leading pre-erythrocytic vaccine candidate and forms the basis of the RTS'S (Mosquirix®) malaria vaccine. However, high-yield production of full-length PfCSP with proper folding has been challenging. Here, we describe expression and purification of full-length PfCSP (containing 4 NVDP and 38 NANP repeats) with proper conformation by a simple three-step procedure in the Lactococcus lactis expression system.

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).

Human blood neutrophils generate ROS through FcγR-signaling to mediate protection against febrile P. falciparum malaria.

Blood phagocytes, such as neutrophils and monocytes, generate reactive oxygen species (ROS) as a part of host defense response against infections. We investigated the mechanism of Fcγ-Receptor (FcγR) mediated ROS production in these cells to understand how they contribute to anti-malarial immunity. Plasmodium falciparum merozoites opsonized with naturally occurring IgG triggered both intracellular and extracellular ROS generation in blood phagocytes, with neutrophils being the main contributors. Using specific inhibitors, we show that both FcγRIIIB and FcγRIIA acted synergistically to induce ROS production in neutrophils, and that NADPH oxidase 2 and the PI3K intracellular signal transduction pathway were involved in this process. High levels of neutrophil ROS were also associated with protection against febrile malaria in two geographically diverse malaria endemic regions from Ghana and India, stressing the importance of the cooperation between anti-malarial IgG and neutrophils in triggering ROS-mediated parasite killing as a mechanism for naturally acquired immunity against malaria.

High opsonic phagocytosis activity and growth inhibition of merozoites are associated with RON4 antibody levels and protect against febrile malaria in Ghanaian children.

Background: Naturally acquired immunity to malaria may involve different immune mechanisms working in concert, however, their respective contributions and potential antigenic targets have not been clearly established. Here, we assessed the roles of opsonic phagocytosis and antibody-mediated merozoite growth inhibition in Plasmodium falciparum (P. falciparum) infection outcomes in Ghanaian children. Methods: The levels of merozoite opsonic phagocytosis, growth inhibition activities and six P. falciparum antigen-specific IgG of plasma samples from children (n=238, aged 0.5 to 13 years) were measured at baseline prior to the malaria seasons in southern Ghana. The children were then actively and passively followed up for febrile malaria and asymptomatic P. falciparum infection detection in a 50-week longitudinal cohort. P. falciparum infection outcome was modelled as a function of the measured immune parameters while accounting for important demographic factors. Results: High plasma activity of opsonic phagocytosis [adjusted odds ratio (aOR)= 0.16; 95%CI= 0.05 - 0.50, p = 0.002], and growth inhibition (aOR=0.15; 95% CI = 0.04-0.47; p = 0.001) were individually associated with protection against febrile malaria. There was no evidence of correlation (b= 0.13; 95% CI= -0.04-0.30; p=0.14) between the two assays. IgG antibodies against MSPDBL1 correlated with opsonic phagocytosis (OP) while IgG against PfRh2a correlated with growth inhibition. Notably, IgG antibodies against RON4 correlated with both assays. Conclusion: Opsonic phagocytosis and growth inhibition are protective immune mechanisms against malaria that may be acting independently to confer overall protection. Vaccines incorporating RON4 may benefit from both immune mechanisms.

Cloning and Recombinant Protein Expression in Lactococcus lactis.

The Lactococcus lactis, a Gram-positive bacteria, is an ideal expression host for the overproduction of heterologous proteins in a properly folded and functional form. L. lactis has been identified as an efficient cell factory, generally recognized as safe (GRAS), has a long history of safe use in food production, and is known to have probiotic properties. Key desirable features of L. lactis include the following: (1) rapid growth to high cell densities, not requiring aeration which facilitates large-scale fermentation; (2) its Gram-positive nature precludes the presence of contaminating endotoxins; (3) the capacity to secrete stable recombinant protein into the growth medium with few proteases resulting in a properly folded, full-length protein; and (4) the availability of diverse expression vectors facilitating various cloning options. We have previously described production of several recombinant proteins with varying degrees of predicted structural complexities using the L. lactis pH-dependent P170 promoter. The purpose of this chapter is to provide a detailed protocol for facilitating wider application of L. lactis as a reliable platform for expression of heterologous recombinant proteins in soluble form. Here, we present details of the various steps involved such as cloning of the target gene in appropriate expression plasmid vector, determination of the expression levels of the heterologous protein, and initial purification of the expressed soluble recombinant protein of interest.

B-Cell Epitope Mapping of the Plasmodium falciparum Malaria Vaccine Candidate GMZ2.6c in a Naturally Exposed Population of the Brazilian Amazon.

The GMZ2.6c malaria vaccine candidate is a multi-stage P. falciparum chimeric protein that contains a fragment of the sexual-stage Pfs48/45-6C protein genetically fused to GMZ2, an asexual-stage vaccine construction consisting of the N-terminal region of the glutamate-rich protein (GLURP) and the C-terminal region of the merozoite surface protein-3 (MSP-3). Previous studies showed that GMZ2.6c is widely recognized by antibodies from Brazilian exposed individuals and that its components are immunogenic in natural infection by P. falciparum. In addition, anti-GMZ2.6c antibodies increase with exposure to infection and may contribute to parasite immunity. Therefore, identifying epitopes of proteins recognized by antibodies may be an important tool for understanding protective immunity. Herein, we identify and validate the B-cell epitopes of GMZ2.6c as immunogenic and immunodominant in individuals exposed to malaria living in endemic areas of the Brazilian Amazon. Specific IgG antibodies and subclasses against MSP-3, GLURP, and Pfs48/45 epitopes were detected by ELISA using synthetic peptides corresponding to B-cell epitopes previously described for MSP-3 and GLURP or identified by BepiPred for Pfs48/45. The results showed that the immunodominant epitopes were P11 from GLURP and MSP-3c and DG210 from MSP-3. The IgG1 and IgG3 subclasses were preferentially induced against these epitopes, supporting previous studies that these proteins are targets for cytophilic antibodies, important for the acquisition of protective immunity. Most individuals presented detectable IgG antibodies against Pfs48/45a and/or Pfs48/45b, validating the prediction of linear B-cell epitopes. The higher frequency and antibody levels against different epitopes from GLURP, MSP-3, and Pfs48/45 provide additional information that may suggest the relevance of GMZ2.6c as a multi-stage malaria vaccine candidate.

Susceptibility to malaria in fulani, Bariba, Otamari and gando individuals living in sympatry in Benin: Role of opsonizing antibodies to Plasmodium falciparum merozoites.

Objectives: Fulani in Africa are known to be less susceptible to Plasmodium falciparum (Pf) malaria. This study explored a potential involvement of antibody-mediated merozoite phagocytosis mechanism in this natural protection against malaria. Methods: Before the start of the malaria transmission season (MTS) in Benin, the functionality of antibodies against Pf merozoites was determined by the opsonic phagocytosis (OP) assay in plasma samples from Fulani, Bariba, Otamari and Gando groups. These individuals were actively followed-up for malaria detection from the beginning to the end of MTS. Anti-GLURP Immunoglobulin G antibody quantification, malaria Rapid Diagnostic Test (RDT) and spleen palpation were performed before and after MTS. Results: In Bariba, Otamari and Gando, but not in Fulani, plasma from adults promoted higher levels of OP than the children (P = 0.003; P = 0.012; P = 0.031 and P = 0.122). A high proportion of Fulani children had higher OP and anti-GLURP (P < 0.0001) antibody levels as compared to non-Fulani children; whereas this was not observed for Fulani adults (P = 0.223). High OP levels before MTS were significantly related to negative RDT after MTS (P = 0.011). Conclusion: Our results highlight the ability of opsonizing antibodies to potentially enhance natural protection of young Fulani individuals against Pf malaria in Benin.

Fc Gamma Receptor IIIB NA1/NA2/SH Polymorphisms Are Associated with Malaria Susceptibility and Antibody Levels to P. falciparum Merozoite Antigens in Beninese Children.

This paper aimed to investigate the influence of polymorphisms in the FCGR2A gene encoding R131H FcgRIIA variants and in the FCGR3B gene (108G > C, 114C > T, 194 A > G, 233C > A, 244 G > A and 316G > A) encoding FcgRIIIB-NA1, -NA2 and -SH variants on malaria susceptibility and antibody responses against P. falciparum merozoite antigens in Beninese children. An active malaria follow-up was conducted in infants from birth to 24 months of age in Allada, Benin. FCGR3B exon 3 was sequenced and FCGR2A exon 4 was genotyped. Antibodies directed to GLURP and MSP3 were quantified by ELISA. Association studies were performed using mixed-effect models. Individual carriage of FCGR3B 194 AA genotype was associated with a high number of malaria infections and a low level of IgG1 against MSP3 and GLURP-R0. High parasitemia and increased malaria infections were observed in infants carrying the FCGR3B*05 108C-114T-194A-233C-244A-316A haplotype. A reduced risk of malaria infections and low parasitemia were related to the carriages of the FCGR3B 108C-114T-194G-233C-244G-316A (FCGR3B*06), FCGR3B 108C−114T−194G−233A−244A−316A (FCGR3B*03 encoding for FcgRIIIB-SH) haplotypes and FCGR3B 297 TT genotype. Our results highlight the impact of FCGR3B polymorphisms on the individual susceptibility to malaria and antibody responses against MSP3 and GLURP in Beninese children.

Naturally acquired antibodies from Beninese infants promote Plasmodium falciparum merozoite-phagocytosis by human blood leukocytes: implications for control of asymptomatic malaria infections.

BACKGROUND: Immunoglobulin G (IgG) antibodies are thought to play important roles in the protection against Plasmodium falciparum (P. falciparum) malaria. A longitudinal cohort study performed in the Southern part of Benin, identified a group of infants who were able to control asymptomatic malaria infections (CAIG). METHODS: IgG antibodies against distinct merozoite antigens were quantified in plasma from Beninese infants. Functionality of these antibodies was assessed by the merozoite-phagocytosis assay using THP-1 cells and primary neutrophils as effector cells. Gm allotypes were determined by a serological method of haemagglutination inhibition. RESULTS: Purified IgG from infants in CAIG promoted higher levels of merozoite-phagocytosis than did IgG from children who were unable to control asymptomatic infections (Ologit multivariate regression model, Coef. = 0.06, 95% CI 0.02;0.10, P = 0.002). High level of merozoite-phagocytosis activity was significantly associated with high levels of IgG against AMA1 (Coef. = 1.76, 95% CI 0.39;3.14, P = 0.012) and GLURP-R2 (Coef. = 12.24, 95% CI 1.35;23.12, P = 0.028). Moreover, infants of the G3m5,6,10,11,13,14,24 phenotype showed higher merozoite-phagocytosis activity (Generalized linear model multivariate regression, Coef. = 7.46, 95% CI 0.31;14.61, P = 0.041) than those presenting other G3m phenotypes. CONCLUSION: The results of the present study confirm the importance of antibodies to merozoite surface antigens in the control of asymptomatic malaria infection in Beninese infants. The study also demonstrated that G3m phenotypes impact the functional activity of IgG. This last point could have a considerable impact in the research of candidate vaccines against malaria parasites or other pathogens.

Humoral antimalaria immune response in Nigerian children exposed to helminth and malaria parasites.

Background: Malaria and helminthic parasites are endemic in tropical countries, and co-infections might influence host-parasite interactions. In this community-based cross-sectional study, the effect that the presence of soil-transmitted helminths (STH) (Hookworm, Hymenolepis nana) and Schistosoma haematobium infections could have on the immunoglobulin (Ig) candidate protein of the malaria vaccine GMZ2 levels was evaluated. Methods: Blood, stool, and urine samples were collected from 5-15-year-old children to diagnose P. falciparum (Pf), STH, and Schistosoma haematobium, respectively. Identification and quantification of the parasite load of STH and S. haematobium were achieved by light microscopy. A polymerase chain reaction was carried out to detect submicroscopic infections of P. falciparum. Plasma levels of GMZ2 specific IgG and its subclasses were quantified by ELISA. Results: The median level of total IgG in individuals with co-infection with Pf/H. nana was significantly lower in the mono-infected group with Pf (p = 0.0121) or study participants without infection (p=0.0217). Similarly, the median level of IgG1 was statistically lower in Pf/H. nana group compared to Pf-group (p=0.0137). Equally, the Pf/H. nana infected individuals posted a lower level of IgG1 compared to Pf-group (p=0.0137) and IgG4 compared to the Pf-group (p=0.0144). Spearman rank correlation analyses indicated positive relationships between the densities of H. nana (ρ=0.25, p=0.015) and S. haematobium (ρ=0.36, p<0.0001). Conclusions: Hookworm and H. nana infections are associated with reduced GMZ2 specific IgG levels. This study shows the possible manipulation of immune responses by helminths for their survival and transmission, which may have serious implications for vaccine development and deployment in helminth-endemic regions.

Placental Malaria is Associated with Higher LILRB2 Expression in Monocyte Subsets and Lower Anti-Malarial IgG Antibodies During Infancy.

Background: Placental malaria (PM) is associated with a higher susceptibility of infants to Plasmodium falciparum (Pf) malaria. A hypothesis of immune tolerance has been suggested but no clear explanation has been provided so far. Our goal was to investigate the involvement of inhibitory receptors LILRB1 and LILRB2, known to drive immune evasion upon ligation with pathogen and/or host ligands, in PM-induced immune tolerance. Method: Infants of women with or without PM were enrolled in Allada, southern Benin, and followed-up for 24 months. Antibodies with specificity for five blood stage parasite antigens were quantified by ELISA, and the frequency of immune cell subsets was quantified by flow cytometry. LILRB1 or LILRB2 expression was assessed on cells collected at 18 and 24 months of age. Findings: Infants born to women with PM had a higher risk of developing symptomatic malaria than those born to women without PM (IRR=1.53, p=0.040), and such infants displayed a lower frequency of non-classical monocytes (OR=0.74, p=0.01) that overexpressed LILRB2 (OR=1.36, p=0.002). Moreover, infants born to women with PM had lower levels of cytophilic IgG and higher levels of IL-10 during active infection. Interpretation: Modulation of IgG and IL-10 levels could impair monocyte functions (opsonisation/phagocytosis) in infants born to women with PM, possibly contributing to their higher susceptibility to malaria. The long-lasting effect of PM on infants' monocytes was notable, raising questions about the capacity of ligands such as Rifins or HLA-I molecules to bind to LILRB1 and LILRB2 and to modulate immune responses, and about the reprogramming of neonatal monocytes/macrophages.

40 Years of Pfs48/45 Research as a Transmission-Blocking Vaccine Target of Plasmodium falciparum Malaria.

In the early 1980s, Richard Carter was among the first researchers to identify the sexual stage-specific Pfs48/45 protein, leading to the identification of target epitopes. Carter predicted its tertiary conformation while involved in a number of studies on naturally acquired sexual stage-specific antibodies. Pfs48/45 is a cysteine-rich surface protein of sexual stages of Plasmodium falciparum that plays a critical role in male gamete fertility. Antibodies against Pfs48/45 prevent parasite development in the mosquito vector, and therefore prevent the spread of malaria in the population. Since the gene was sequenced in the early 1990s, Pfs48/45 has been considered a prime target candidate for a malaria transmission-blocking vaccine. However, major manufacturing challenges-in particular, difficulty realizing satisfactory yields of a properly folded protein for the induction of functional antibodies-delayed clinical development significantly. These challenges were met roughly 20 years later. The first clinical trial with a Pfs48/45 subunit vaccine (R0.6C) was started in the Netherlands in early 2021. The excellent contributions to the long and winding path of Pfs48/45 research by Richard Carter are well recognized and are an integrated part of his seminal contributions to unraveling Plasmodium sexual stage biology.

Heterologous Expression and Evaluation of Novel Plasmodium falciparum Transmission Blocking Vaccine Candidates.

Malaria transmission blocking vaccines (TBV) aim to induce antibodies that can interrupt Plasmodium falciparum development in the mosquito midgut and thereby prevent onward malaria transmission. A limited number of TBV candidates have been identified and only three (Pfs25, Pfs230 and Pfs48/45) have entered clinical testing. While one of these candidates may emerge as a highly potent TBV candidate, it is premature to determine if they will generate sufficiently potent and sustained responses. It is therefore important to explore novel candidate antigens. We recently analyzed sera from naturally exposed individuals and found that the presence and/or intensity of antibodies against 12 novel putative surface expressed gametocyte antigens was associated with transmission reducing activity. In this study, protein fragments of these novel TBV candidates were designed and heterologously expressed in Drosophila melanogaster S2 cells and Lactococcus lactis. Eleven protein fragments, covering seven TBV candidates, were successfully produced. All tested antigens were recognized by antibodies from individuals living in malaria-endemic areas, indicating that native epitopes are present. All antigens induced antigen-specific antibody responses in mice. Two antigens induced antibodies that recognized a native protein in gametocyte extract, and antibodies elicited by four antigens recognized whole gametocytes. In particular, we found that antigen Pf3D7_0305300, a putative transporter, is abundantly expressed on the surface of gametocytes. However, none of the seven novel TBV candidates expressed here induced an antibody response that reduced parasite development in the mosquito midgut as assessed in the standard membrane feeding assay. Altogether, the antigen fragments used in this study did not prove to be promising transmission blocking vaccine constructs, but led to the identification of two gametocyte surface proteins that may provide new leads for studying gametocyte biology.

GMZ2 Vaccine-Induced Antibody Responses, Naturally Acquired Immunity and the Incidence of Malaria in Burkinabe Children.

GMZ2 is a malaria vaccine candidate evaluated in a phase 2b multi-centre trial. Here we assessed antibody responses and the association of naturally acquired immunity with incidence of malaria in one of the trial sites, Banfora in Burkina Faso. The analysis included 453 (GMZ2 = 230, rabies = 223) children aged 12-60 months old. Children were followed-up for clinical malaria episodes for 12 months after final vaccine administration. Antibody levels against GMZ2 and eleven non-GMZ2 antigens were measured on days 0 and 84 (one month after final vaccine dose). Vaccine efficacy (VE) differed by age group (interaction, (12-35 months compared to 36-60 months), p = 0.0615). During the twelve months of follow-up, VE was 1% (95% confidence interval [CI] -17%, 17%) and 23% ([CI] 3%, 40%) in the 12 - 35 and 36 - 60 months old children, respectively. In the GMZ2 group, day 84 anti-GMZ2 IgG levels were associated with reduced incidence of febrile malaria during the follow up periods of 1-6 months (hazard ratio (HR) = 0.87, 95%CI = (0.77, 0.98)) and 7-12 months (HR = 0.84, 95%CI = (0.71, 0.98)) in the 36-60 months old but not in 12-35 months old children. Multivariate analysis involving day 84 IgG levels to eleven non-vaccine antigens, identified MSP3-K1 and GLURP-R2 to be associated with reduced incidence of malaria during the 12 months of follow up. The inclusion of these antigens might improve GMZ2 vaccine efficacy.

Cellular and antibody response in GMZ2-vaccinated Gabonese volunteers in a controlled human malaria infection trial.

BACKGROUND: Antibody and cellular memory responses following vaccination are important measures of immunogenicity. These immune markers were quantified in the framework of a vaccine trial investigating the malaria vaccine candidate GMZ2. METHODS: Fifty Gabonese adults were vaccinated with two formulations (aluminum Alhydrogel and CAF01) of GMZ2 or a control vaccine (Verorab). Vaccine efficacy was assessed using controlled human malaria infection (CHMI) by direct venous inoculation of 3200 live Plasmodium falciparum sporozoites (PfSPZ Challenge). GMZ2-stimulated T and specific B-cell responses were estimated by flow cytometry before and after vaccination. Additionally, the antibody response against 212 P. falciparum antigens was estimated before CHMI by protein microarray. RESULTS: Frequencies of pro- and anti-inflammatory CD4+ T cells stimulated with the vaccine antigen GMZ2 as well as B cell profiles did not change after vaccination. IL-10-producing CD4+ T cells and CD20+ IgG+ B cells were increased post-vaccination regardless of the intervention, thus could not be specifically attributed to any malaria vaccine regimen. In contrast, GMZ2-specific antibody response increased after the vaccination, but was not correlated to protection. Antibody responses to several P. falciparum blood and liver stage antigens (MSP1, MSP4, MSP8, PfEMP1, STARP) as well as the breadth of the malaria-specific antibody response were significantly higher in protected study participants. CONCLUSIONS: In lifelong malaria exposed adults, the main marker of protection against CHMI is a broad antibody pattern recognizing multiple stages of the plasmodial life cycle. Despite vaccination with GMZ2 using a novel formulation, expansion of the GMZ2-stimulated T cells or the GMZ2-specific B cell response was limited, and the vaccine response could not be identified as a marker of protection against malaria. Trial registration PACTR; PACTR201503001038304; Registered 17 February 2015; https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=1038.

Anti-Gametocyte Antigen Humoral Immunity and Gametocytemia During Treatment of Uncomplicated Falciparum Malaria: A Multi-National Study.

Introduction: Understanding the human immune response to Plasmodium falciparum gametocytes and its association with gametocytemia is essential for understanding the transmission of malaria as well as progressing transmission blocking vaccine candidates. Methods: In a multi-national clinical efficacy trial of artemisinin therapies (13 sites of varying transmission over South-East Asia and Africa), we measured Immunoglobulin G (IgG) responses to recombinant P. falciparum gametocyte antigens expressed on the gametocyte plasma membrane and leading transmission blocking vaccine candidates Pfs230 (Pfs230c and Pfs230D1M) and Pfs48/45 at enrolment in 1,114 participants with clinical falciparum malaria. Mixed effects linear and logistic regression were used to determine the association between gametocyte measures (gametocytemia and gametocyte density) and antibody outcomes at enrolment. Results: Microscopy detectable gametocytemia was observed in 11% (127/1,114) of participants at enrolment, and an additional 9% (95/1,114) over the follow-up period (up to day 42) (total 20% of participants [222/1,114]). IgG levels in response to Pfs230c, Pfs48/45 and Pfs230D1M varied across study sites at enrolment (p < 0.001), as did IgG seroprevalence for anti-Pfs230c and D1M IgG (p < 0.001), but not for anti-Pfs48/45 IgG (p = 0.159). In adjusted analyses, microscopy detectable gametocytemia at enrolment was associated with an increase in the odds of IgG seropositivity to the three gametocyte antigens (Pfs230c OR [95% CI], p: 1.70 [1.10, 2.62], 0.017; Pfs48/45: 1.45 [0.85, 2.46], 0.174; Pfs230D1M: 1.70 [1.03, 2.80], 0.037), as was higher gametocyte density at enrolment (per two-fold change in gametocyte density Pfs230c OR [95% CI], p: 1.09 [1.02, 1.17], 0.008; Pfs48/45: 1.05 [0.98, 1.13], 0.185; Pfs230D1M: 1.07 [0.99, 1.14], 0.071). Conclusion: Pfs230 and Pfs48/45 antibodies are naturally immunogenic targets associated with patent gametocytemia and increasing gametocyte density across multiple malaria endemic settings, including regions with emerging artemisinin-resistant P. falciparum.

Naturally acquired antibody response to a Plasmodium falciparum chimeric vaccine candidate GMZ2.6c and its components (MSP-3, GLURP, and Pfs48/45) in individuals living in Brazilian malaria-endemic areas.

BACKGROUND: The GMZ2.6c malaria vaccine candidate is a multi-stage Plasmodium falciparum chimeric protein which contains a fragment of the sexual-stage Pfs48/45-6C protein genetically fused to GMZ2, a fusion protein of GLURP and MSP-3, that has been shown to be well tolerated, safe and immunogenic in clinical trials performed in a malaria-endemic area of Africa. However, there is no data available on the antigenicity or immunogenicity of GMZ2.6c in humans. Considering that circulating parasites can be genetically distinct in different malaria-endemic areas and that host genetic factors can influence the immune response to vaccine antigens, it is important to verify the antigenicity, immunogenicity and the possibility of associated protection in individuals living in malaria-endemic areas with different epidemiological scenarios. Herein, the profile of antibody response against GMZ2.6c and its components (MSP-3, GLURP and Pfs48/45) in residents of the Brazilian Amazon naturally exposed to malaria, in areas with different levels of transmission, was evaluated. METHODS: This study was performed using serum samples from 352 individuals from Cruzeiro do Sul and Mâncio Lima, in the state of Acre, and Guajará, in the state of Amazonas. Specific IgG, IgM, IgA and IgE antibodies and IgG subclasses were detected by Enzyme-Linked Immunosorbent Assay. RESULTS: The results showed that GMZ2.6c protein was widely recognized by naturally acquired antibodies from individuals of the Brazilian endemic areas with different levels of transmission. The higher prevalence of individuals with antibodies against GMZ2.6c when compared to its individual components may suggest an additive effect of GLURP, MSP-3, and Pfs48/45 when inserted in a same construct. Furthermore, naturally malaria-exposed individuals predominantly had IgG1 and IgG3 cytophilic anti-GMZ2.6c antibodies, an important fact considering that the acquisition of anti-malaria protective immunity results from a delicate balance between cytophilic/non-cytophilic antibodies. Interestingly, anti-GMZ2.6c antibodies seem to increase with exposure to malaria infection and may contribute to parasite immunity. CONCLUSIONS: The data showed that GMZ2.6c protein is widely recognized by naturally acquired antibodies from individuals living in malaria-endemic areas in Brazil and that these may contribute to parasite immunity. These data highlight the importance of GMZ2.6c as a candidate for an anti-malarial vaccine.

Association of Immunoglobulin G3 Hinge Region Length Polymorphism With Cerebral Malaria in Ghanaian Children.

Cerebral malaria (CM) may cause death or long-term neurological damage in children, and several host genetic risk factors have been reported. Malaria-specific immunoglobulin (Ig) G3 antibodies are crucial to human immune response against malaria. The hinge region of IgG3 exhibits length polymorphism (with long [L], medium [M], and short [S] alleles), which may influence its functionality. We studied IgG3 hinge region length polymorphisms in 136 Ghanaian children with malaria. Using logistic regression models, we found that children with the recessive MM allotype encoding medium IgG3 hinge region length had an increased risk of CM (adjusted odds ratio, 6.67 [95% confidence interval,1.30-34.32]; P=.004) . This has implications for future epidemiological studies on CM.

Preclinical development of a Pfs230-Pfs48/45 chimeric malaria transmission-blocking vaccine.

The Plasmodium falciparum Pfs230 and Pfs48/45 proteins are leading candidates for a malaria transmission-blocking vaccine (TBV). Previously, we showed that a Pfs230-Pfs48/45 fusion protein elicits higher levels of functional antibodies than the individual antigens, but low yields hampered progression to clinical evaluation. Here we identified a modified construct (ProC6C) with a circumsporozoite protein (CSP) repeat-linker sequence that enhances expression. A scalable and reproducible process in the Lactococcus lactis expression system was developed and ProC6C was successfully transferred for manufacturing under current Good Manufacturing Practices (cGMP). In addition, a panel of analytical assays for release and stability were developed. Intact mass spectrometry analysis and multiangle light scattering showed that the protein contained correct disulfide bonds and was monomeric. Immunogenicity studies in mice showed that the ProC6C adsorbed to Alhydrogel®, with or without Matrix-MTM, elicited functional antibodies that reduced transmission to mosquitoes and sporozoite invasion of human hepatocytes. Altogether, our data support manufacture and clinical evaluation of ProC6C as a multistage malaria-vaccine candidate.