Circumsporozoite Protein Research Articles

Plasmodium spp. in macaques, Macaca fascicularis, in Malaysia, and their potential role in zoonotic malaria transmission.

Macaques, Macaca fascicularis, are a known reservoir of Plasmodium knowlesi, the agent of simian malaria which is the predominant zoonotic species affecting humans in Malaysia and other Southeast Asian countries. Recently, a naturally acquired human infection of another simian malaria parasite, P. cynomolgi has been reported. Thus, it is crucial to study the distribution of simian Plasmodium infections with particular attention to the macaques. Four hundred and nineteen (419) long-tailed macaques (Macaca fascicularis) were trapped in selected areas where human cases of P. knowlesi and P. cynomolgi have been reported. Nested polymerase chain reaction (PCR) was conducted to identify the Plasmodium spp., and circumsporozoite protein (CSP) genes of P. knowlesi samples were sequenced. Plasmodium cynomolgi infection was shown to be the most prevalent among the macaque population (68.4%). Although 50.6% of analyzed samples contained single infections either with P. knowlesi, P. cynomolgi, P. inui, P. coatneyi, or P. fieldi, mixed infections with double, triple, quadruple, and all 5 species were also detected. Infection with P. cynomolgi and P. knowlesi were the highest among Malaysian macaques in areas where humans and macaques are in close contact. The risk of zoonotic infection in these areas needs to be addressed since the number of zoonotic malaria cases is on the rise. With the elimination of human malaria, the risk of humans being infected with simian malaria is very high and steps should be taken to mitigate this issue.

Towards large-scale identification of HLA-restricted T cell epitopes from four vaccine candidate antigens in a malaria endemic community in Ghana

Sterile protection against clinical malaria has been achieved in animal models and experimental human challenge studies involving immunization with radiation attenuated Plasmodium falciparum sporozoite vaccines as well as by live sporozoites under chloroquine prophylaxis. Parasite-specific IFN-γ and granzyme B-secreting CD8 + T cells have been identified as key mediators of protection. Although the exact parasite targets of protective CD8 + T cell responses are not fully defined, responses against a handful of vaccine candidate antigens have been associated with protection. Identifying the T cell targets in these antigens will facilitate the development of simpler, cost-effective, and efficacious next generation multi-epitope vaccines. The aim of this study was to identify immunodominant portions of four malaria vaccine candidate antigens using peripheral blood mononuclear cells (PBMCs) from adults with life-long exposure to malaria parasites. Cryopreserved PBMCs from 291 HLA-typed subjects were stimulated with pools of overlapping 15mer peptides spanning the entire sequences of P. falciparum circumsporozoite protein (CSP, 9 pools), apical membrane antigen 1 (AMA1, 12 pools), thrombospondin related anonymous protein (TRAP, 6 pools) and cell traversal for ookinetes and sporozoites (CelTOS, 4 pools) in FluoroSpot assays. 125 of 291 subjects made IFN-γ responses to 30 of the 31 peptide pools tested and 22 of 291 made granzyme B responses, with 20 making dual responses. The most frequent responses were to the CSP C-terminal region and the least frequent responses were to TRAP and CelTOS. There was no association between FluoroSpot responses and active malaria infection, detected by either microscopy, RDT, or PCR.In conclusion, CSP and AMA1 have relatively higher numbers of epitopes that trigger IFN-γ and granzyme B-secreting T cells in adults with life-long malaria parasite exposure compared to the other two antigens tested, and highlights the continued relevance of these two antigens as vaccine candidates.

Protective effects of combining monoclonal antibodies and vaccines against the Plasmodium falciparum circumsporozoite protein.

Combinations of monoclonal antibodies (mAbs) against different epitopes on the same antigen synergistically neutralize many viruses. However, there are limited studies assessing whether combining human mAbs against distinct regions of the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) enhances in vivo protection against malaria compared to each mAb alone or whether passive transfer of PfCSP mAbs would improve protection following vaccination against PfCSP. Here, we isolated a panel of human mAbs against the subdominant C-terminal domain of PfCSP (C-CSP) from a volunteer immunized with radiation-attenuated Pf sporozoites. These C-CSP-specific mAbs had limited binding to sporozoites in vitro that was increased by combination with neutralizing human "repeat" mAbs against the NPDP/NVDP/NANP tetrapeptides in the central repeat region of PfCSP. Nevertheless, passive transfer of repeat- and C-CSP-specific mAb combinations did not provide enhanced protection against in vivo sporozoite challenge compared to repeat mAbs alone. Furthermore, combining potent repeat-specific mAbs (CIS43, L9, and 317) that respectively target the three tetrapeptides (NPDP/NVDP/NANP) did not provide additional protection against in vivo sporozoite challenge. However, administration of either CIS43, L9, or 317 (but not C-CSP-specific mAbs) to mice that had been immunized with R21, a PfCSP-based virus-like particle vaccine that induces polyclonal antibodies against the repeat region and C-CSP, provided enhanced protection against sporozoite challenge when compared to vaccine or mAbs alone. Collectively, this study shows that while combining mAbs against the repeat and C-terminal regions of PfCSP provide no additional protection in vivo, repeat mAbs do provide increased protection when combined with vaccine-induced polyclonal antibodies. These data should inform the implementation of PfCSP human mAbs alone or following vaccination to prevent malaria infection.

Antibody Targets and Properties for Complement-Fixation Against the Circumsporozoite Protein in Malaria Immunity.

The Plasmodium falciparum circumsporozoite protein (CSP) forms the basis of leading subunit malaria vaccine candidates. However, the mechanisms and specific targets of immunity are poorly defined. Recent findings suggest that antibody-mediated complement-fixation and activation play an important role in immunity. Here, we investigated the regions of CSP targeted by functional complement-fixing antibodies and the antibody properties associated with this activity. We quantified IgG, IgM, and functional complement-fixing antibody responses to different regions of CSP among Kenyan adults naturally exposed to malaria (n=102) and using a series of rabbit vaccination studies. Individuals who acquired functional complement-fixing antibodies had higher IgG, IgM and IgG1 and IgG3 to CSP. Acquired complement-fixing antibodies targeted the N-terminal, central-repeat, and C-terminal regions of CSP, and positive responders had greater antibody breadth compared to those who were negative for complement-fixing antibodies (p<0.05). Using rabbit vaccinations as a model, we confirmed that IgG specific to the central-repeat and non-repeat regions of CSP could effectively fix complement. However, vaccination with near full length CSP in rabbits poorly induced antibodies to the N-terminal region compared to naturally-acquired immunity in humans. Poor induction of N-terminal antibodies was also observed in a vaccination study performed in mice. IgG and IgM to all three regions of CSP play a role in mediating complement-fixation, which has important implications for malaria vaccine development.

Identification of immunodominant epitopes in allelic variants VK210 and VK247 of Plasmodium Vivax Circumsporozoite immunogen.

Plasmodium vivax-induced malaria is among the leading causes of morbidity and mortality in sub-tropical and tropical regions and infect 2.85 billion people globally. The continual rise and propagation of resistance against anti-malarial drugs is a prerequisite to develop a potent vaccine candidate for Plasmodium vivax (P. vivax). Circumsporozoite protein (CSP) is an important immunogen of malaria parasite that has the conserved CSP structure as an immune dominant B-cell epitope. In current study, we focused on designing multi-epitope vaccines (MEVs) using various immunoinformatics tools against Pakistani based allelic variants VK210 and VK247 of P. vivax CSP (PvCSP) gene. Antigenicity, allergic potential and physicochemical parameters of both PvCSP variants were assessed for the designed MEVs and they were within acceptable range suitable for post experimental investigations. The three-dimensional structures of both MEVs have been predicted ab initio, optimized, and validated by using different online servers. The both MEVs candidates were stable and free from aggregation-prone regions. The stability of both MEVs had been improved by a disulfide engineering approach. To estimate the binding energy and stability of the MEVs, molecular docking simulation and binding free energy calculations with TLR-4 immune receptor have been conducted. The docking score of PvCSP210 and PvCSP247 for TLR-4 was -6.34kJ/mol and-2.3kJ/mol, respectively. For PvCSP210-TLR4 system, mean RMSD was 4.96Å while PvCSP247-TLR4 system, average RMSD was 4.49Å. The binding free energy of PvCSP210-TLR4 complex and PvCSP247-TLR4 complex was -50.49/-117.15kcal/mol (MMGBSA/MMPSA) and -52.94/-96.26kcal/mol (MMGBSA/MMPSA), respectively. The expression of both MEVs produced in Escherichia coli K12 expression system by in silico cloning was significant. Immune simulation revealed that the proposed MEVs induce strong humoral and cellular immunological responses, in addition to significant production of interleukins and cytokines. In conclusions, we believed that the MEVs proposed in current research, using combine approach of immunoinformatics, structural biology and biophysical approaches, could induce protective and effective immune responses against P. vivax and the experimental validation of our findings could contribute to the development of potential malaria vaccine.

Spatiotemporal Dynamic of the RTS,S/AS01 Malaria Vaccine Target Antigens in Senegal.

The RTS,S/AS01 malaria vaccine confers only moderate protection against malaria. Evidence suggests that the effectiveness of the RTS,S/AS01 vaccine depends upon the parasite population genetics, specifically regarding the circumsporozoite protein haplotypes in the population. We investigated Plasmodium falciparum circumsporozoite protein (PfCSP) gene sequences from two endemic sites in 2018 in Senegal. The PfCSP sequences were compared with those retrieved from the Pf3k genome database. In the central repeat region of PfCSP, the distribution of haplotypes differed significantly between the two study sites (Fisher's exact test, P < 0.001). No 3D7 vaccine strain haplotype was observed in this locus. In the C-terminal region, there was no significant difference in haplotypes distribution between Kedougou and Diourbel (Fischer's exact test, P = 0.122). The 3D7 haplotype frequency was 8.4% in early samples (2001-2011), but then it contracted in the subsequent years. The extensive plasticity of the P. falciparum genes coding the RTS,S/AS01 vaccine target antigens may influence the immune responses to circulating alleles. Monitoring the genetic diversity baseline and its dynamics over time and space would be instrumental in rationally improving the malaria RTS,S/AS01 vaccine and/or its implementation schedule.

How to train your antibody to fight malaria

The Plasmodium falciparum circumsporozoite protein (PfCSP) is targeted by neutralizing antibodies and is a focus of malaria vaccine development. In this issue of Immunity, Kratochvil etal. (2021) combine vaccination and bioinformatics strategies to develop a best-in-class PfCSP-targeting antibody.

Vaccination in a humanized mouse model elicits highly protective PfCSP-targeting anti-malarial antibodies

Repeat antigens, such as the Plasmodium falciparum circumsporozoite protein (PfCSP), use both sequence degeneracy and structural diversity to evade the immune response. A few PfCSP-directed antibodies have been identified that are effective at preventing malaria infection, including CIS43, but how these repeat-targeting antibodies might be improved has been unclear. Here, we engineered a humanized mouse model in which B cells expressed inferred human germline CIS43 (iGL-CIS43) B cell receptors and used both vaccination and bioinformatic analysis to obtain variant CIS43 antibodies with improved protective capacity. One such antibody, iGL-CIS43.D3, was significantly more potent than the current best-in-class PfCSP-directed antibody. We found that vaccination with a junctional epitope peptide was more effective than full-length PfCSP at recruiting iGL-CIS43 B cells to germinal centers. Structure-function analysis revealed multiple somatic hypermutations that combinatorically improved protection. This mouse model can thus be used to understand vaccine immunogens and to develop highly potent anti-malarial antibodies.

Assessment of genetic diversity of Plasmodium falciparum circumsporozoite protein in Sudan: the RTS,S leading malaria vaccine candidate

BackgroundThe currently used malaria vaccine, RTS,S, is designed based on the Plasmodium falciparum circumsporozoite protein (PfCSP). The pfcsp gene, besides having different polymorphic patterns, can vary between P. falciparum isolates due to geographical origin and host immune response. Such aspects are essential when considering the deployment of the RTS,S vaccine in a certain region. Therefore, this study assessed the genetic diversity of P. falciparum in Sudan based on the pfcsp gene by investigating the diversity at the N-terminal, central repeat, and the C-terminal regions.MethodsA cross-sectional molecular study was conducted; P. falciparum isolates were collected from different health centres in Khartoum State between January and December 2019. During the study period, a total of 261 febrile patients were recruited. Malaria diagnosis was made by expert microscopists using Giemsa-stained thick and thin blood films. DNA samples were examined by the semi-nested polymerase chain reaction (PCR). Single clonal infection of the confirmed P. falciparum cases, were used to amplify the pfcsp gene. The amplified amplicons of pfcsp have been sequenced using the Sanger dideoxy method. The obtained sequences of pfcsp nucleotide diversity parameters including the numbers of haplotypes (Hap), haplotypes diversity (Hapd), the average number of nucleotide differences between two sequences (p), and the numbers of segregating sites (S) were obtained. The haplotype networks were constructed using the online tcsBU software. Natural selection theory was also tested on pfcsp using Fuand Li’s D, Fuand Li’s F statistics, and Tajima’s D test using DnaSP.ResultsIn comparison with the different pfcsp reference strains, the Sudanese isolates showed high similarity with other African isolates. The results of the N-terminal region showed the presence of 2 different haplotypes with a Hapd of 0.425 ± 0.00727. The presence of the unique insertion of NNNGDNGREGKDEDKRDGNN was reported. The KLKQP motif was conserved in all the studied isolates. At the central repeat region, 11 haplotypes were seen with a Hapd of 0.779 ± 0.00097. The analysis of the genetic diversity in the C-terminal region showed the presence of 10 haplotypes with a Hapd of 0.457 ± 0.073. Several non-synonymous amino acids changes were also seen at the Th2R and the Th3R T-cell epitope regions including T317K, E317K, Q318E, K321N, I322K, T322K, R322K, K324Q, I327L, G352N, S354P, R355K, N356D, Q357E, and E361A.ConclusionsIn this study, the results indicated a high conservation at the pfcsp gene. This may further contribute in understanding the genetic polymorphisms of P. falciparum prior to the deployment of the RTS,S vaccine in Sudan.

The P. falciparum CSP repeat region contains three distinct epitopes required for protection by antibodies in vivo.

Rare and potent monoclonal antibodies (mAbs) against the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) on infective sporozoites (SPZ) preferentially bind the PfCSP junctional tetrapeptide NPDP or NVDP minor repeats while cross-reacting with NANP central repeats in vitro. The extent to which each of these epitopes is required for protection in vivo is unknown. Here, we assessed whether junction-, minor repeat- and central repeat-preferring human mAbs (CIS43, L9 and 317 respectively) bound and protected against in vivo challenge with transgenic P. berghei (Pb) SPZ expressing either PfCSP with the junction and minor repeats knocked out (KO), or PbCSP with the junction and minor repeats knocked in (KI). In vivo protection studies showed that the junction and minor repeats are necessary and sufficient for CIS43 and L9 to neutralize KO and KI SPZ, respectively. In contrast, 317 required major repeats for in vivo protection. These data establish that human mAbs can prevent malaria infection by targeting three different protective epitopes (NPDP, NVDP, NANP) in the PfCSP repeat region. This report will inform vaccine development and the use of mAbs to passively prevent malaria.

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.

The effect of irrigation on malaria vector bionomics and transmission intensity in western Ethiopia

BackgroundIrrigation schemes may result in subsequent changes in malaria disease dynamics. Understanding the mechanisms and effects of irrigation on malaria vector bionomics and transmission intensity is essential to develop new or alternative surveillance and control strategies to reduce or control malaria risk. This study was designed to assess the effect of rice irrigation on malaria vector bionomics and transmission intensity in the Gambella Region, Ethiopia.MethodsComparative cross-sectional study was conducted in Abobo District of the Gambella Region, Ethiopia. Accordingly, clusters (kebeles) were classified into nearby and faraway clusters depending on their proximity to the irrigation scheme. Adult mosquito survey was conducted in February, August and November 2018 from three nearby and three faraway clusters using Centers for Disease Control and Prevention (CDC) light traps (LTs). During the November survey, human landing catch (HLC) and pyrethrum spray catch (PSC) were also conducted. The collected mosquitoes were morphologically identified to species and tested for Plasmodium infection using circumsporozoite protein enzyme-linked immunosorbent assay (CSP-ELISA). Furthermore, species-specific polymerase chain reaction (PCR) was performed to identify member species of the Anopheles gambiae complex. Chi-square and t-tests were used to analyze the data using the SPSS version 20 software package.ResultsA total of 4319 female anopheline mosquitoes comprising An. gambiae sensu lato, An. funestus group, An. pharoensis, An. coustani complex and An. squamosus were collected. Overall, 84.5% and 15.5% of the anopheline mosquitoes were collected from the nearby and faraway clusters, respectively. Anopheles gambiae s.l. was the predominant (56.2%) anopheline species in the area followed by An. pharoensis (15.7%). The density of anopheline mosquitoes was significantly higher in the nearby clusters in both HLCs [t(3) = 5.14, P = 0.0143] and CDC LT catches [t(271.97) = 7.446, P < 0.0001). The overall sporozoite rate of anopheline species from the nearby clusters was 10-fold higher compared to the faraway clusters.ConclusionsSignificantly higher mosquito population density was observed in areas close to the irrigation sites. Sporozoite infection rate in the mosquito population was also markedly higher from the nearby clusters. Therefore, the irrigation scheme could increase the risk of malaria in the area.Graphical abstract

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay.

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Adaptation of ELISA detection of Plasmodium falciparum and Plasmodium vivax circumsporozoite proteins in mosquitoes to a multiplex bead-based immunoassay

BackgroundPlasmodium spp. sporozoite rates in mosquitoes are used to better understand malaria transmission intensity, the relative importance of vector species and the impact of interventions. These rates are typically estimated using an enzyme-linked immunosorbent assay (ELISA) utilizing antibodies against the circumsporozoite protein of Plasmodium falciparum, Plasmodium vivax VK210 (P. vivax210) or P. vivax VK247 (P. vivax247), employing assays that were developed over three decades ago. The ELISA method requires a separate assay plate for each analyte tested and can be time consuming as well as requiring sample volumes not always available. The bead-based multiplex platform allows simultaneous measurement of multiple analytes and may improve the lower limit of detection for sporozoites.MethodsRecombinant positive controls for P. falciparum, P. vivax210 and P. vivax247 and previously developed circumsporozoite (cs) ELISA antibodies were used to optimize conditions for the circumsporozoite multiplex bead assay (csMBA) and to determine the detection range of the csMBA. After optimizing assay conditions, known amounts of sporozoites were used to determine the lower limit of detection for the csELISA and csMBA and alternate cut-off measures were applied to demonstrate how cut-off criteria can impact lower limits of detection. Sporozoite rates from 1275 mosquitoes collected in Madagascar and 255 mosquitoes collected in Guinea were estimated and compared using the established csELISA and newly optimized csMBA. All mosquitoes were tested (initial test), and those that were positive were retested (retest). When sufficient sample volume remained, an aliquot of homogenate was boiled and retested (boiled retest), to denature any heat-unstable cross-reactive proteins.ResultsFollowing optimization of the csMBA, the lower limit of detection was 25 sporozoites per mosquito equivalent for P. falciparum, P. vivax210 and P. vivax247 whereas the lower limits of detection for csELISA were found to be 1400 sporozoites for P. falciparum, 425 for P. vivax210 and 1650 for P. vivax247. Combined sporozoite rates after re-testing of samples that initially tested positive for Madagascar mosquitoes by csELISA and csMBA were 1.4 and 10.3%, respectively, and for Guinea mosquitoes 2% by both assays. Boiling of samples followed by csMBA resulted in a decrease in the Madagascar sporozoite rate to 2.8–4.4% while the Guinea csMBA sporozoite rate remained at 2.0%. Using an alternative csMBA cut-off value of median fluorescence intensity (MFI) of 100 yielded a sporozoite rate after confirmational testing of 3.7% for Madagascar samples and 2.0% for Guinea samples. Whether using csMBA or csELISA, the following steps may help minimize false positives: specimens are appropriately stored and bisected anterior to the thorax-abdomen junction, aliquots of homogenate are boiled and retested following initial testing, and an appropriate cut-off value is determined.ConclusionsThe csMBA is a cost-comparable and time saving alternative to the csELISA and may help eliminate false negatives due to a lower limit of detection, thus increasing sensitivity over the csELISA. The csMBA expands the potential analyses that can be done with a small volume of sample by allowing multiplex testing where analytes in addition to P. falciparum, P. vivax210 and P. vivax247 can be added following optimization.

A comparison of PCR and ELISA methods to detect different stages of Plasmodium vivax in Anopheles arabiensis

BackgroundIn characterizing malaria epidemiology, measuring mosquito infectiousness informs the entomological inoculation rate, an important metric of malaria transmission. PCR-based methods have been touted as more sensitive than the current “gold-standard” circumsporozoite (CSP) ELISA. Wider application of PCR-based methods has been limited by lack of specificity for the infectious sporozoite stage. We compared a PCR method for detecting the parasite’s mitochondrial (mt) cytochrome oxidase I (COX-I) gene with ELISA for detecting circumsporozoite protein for identification of different life stages of the parasite during development within a mosquito.MethodsA PCR-based method targeting the Plasmodium mt COX-I gene was compared with the CSP ELISA method to assess infectivity in Anopheles arabiensis colony mosquitoes fed on blood from patients infected with Plasmodium vivax. Mosquitoes were tested at six post-infection time points (days 0.5, 1, 6, 9, 12, 15). The head and thorax and the abdomen for each specimen were tested separately with each method. Agreement between methods at each infection stage was measured using Cohen’s kappa measure of test association.ResultsInfection status of mosquitoes was assessed in approximately 90 head/thorax and 90 abdomen segments at each time point; in total, 538 head/thorax and 534 abdomen segments were tested. In mosquitoes bisected after 0.5, 1, and 6 days post-infection (dpi), the mt COX-I PCR detected Plasmodium DNA in both the abdomen (88, 78, and 67%, respectively) and head/thorax segments (69, 60, and 44%, respectively), whilst CSP ELISA detected sporozoites in only one abdomen on day 6 post-infection. PCR was also more sensitive than ELISA for detection of Plasmodium in mosquitoes bisected after 9, 12, and 15 dpi in both the head and thorax and abdomen. There was fair agreement between methods for time points 9–15 dpi (κ = 0.312, 95% CI: 0.230–0.394).ConclusionsThe mt COX-I PCR is a highly sensitive, robust method for detecting Plasmodium DNA in mosquitoes, but its limited Plasmodium life-stage specificity cannot be overcome by bisection of the head and thorax from the abdomen prior to PCR. Thus, the mt COX-I PCR is a poor candidate for identifying infectious mosquitoes.Graphical

A universal vaccine candidate against Plasmodium vivax malaria confers protective immunity against the three PvCSP alleles

Malaria is a highly prevalent parasitic disease in regions with tropical and subtropical climates worldwide. Among the species of Plasmodium causing human malaria, P. vivax is the second most prevalent and the most geographically widespread species. A major target of a pre-erythrocytic vaccine is the P. vivax circumsporozoite protein (PvCSP). In previous studies, we fused two recombinant proteins representing three allelic variants of PvCSP (VK210, VK247 and P. vivax-like) to the mumps virus nucleocapsid protein to enhance immune responses against PvCSP. The objective of the present study was to evaluate the protective efficacy of these recombinants in mice challenged with transgenic P. berghei parasites expressing PvCSP allelic variants. Formulations containing Poly (I:C) or Montanide ISA720 as adjuvants elicited high and long-lasting IgG antibody titers specific to each PvCSP allelic variant. Immunized mice were challenged with two existing chimeric P. berghei parasite lines expressing PvCSP-VK210 and PvCSP-VK247. We also developed a novel chimeric line expressing the third allelic variant, PvCSP-P. vivax-like, as a new murine immunization-challenge model. Our formulations conferred partial protection (significant delay in the time to reach 1% parasitemia) against challenge with the three chimeric parasites. Our results provide insights into the development of a vaccine targeting multiple strains of P. vivax.

Subclass and avidity of circumsporozoite protein specific antibodies associate with protection status against malaria infection

RTS,S/AS01 is an advanced pre-erythrocytic malaria vaccine candidate with demonstrated vaccine efficacy up to 86.7% in controlled human malaria infection (CHMI) studies; however, reproducible immune correlates of protection (CoP) are elusive. To identify candidates of humoral correlates of vaccine mediated protection, we measured antibody magnitude, subclass, and avidity for Plasmodium falciparum (Pf) circumsporozoite protein (CSP) by multiplex assays in two CHMI studies with varying RTS,S/AS01B vaccine dose and timing regimens. Central repeat (NANP6) IgG1 magnitude correlated best with protection status in univariate analyses and was the most predictive for protection in a multivariate model. NANP6 IgG3 magnitude, CSP IgG1 magnitude, and total serum antibody dissociation phase area-under-the-curve for NANP6, CSP, NPNA3, and N-interface binding were also associated with protection status in the regimen adjusted univariate analysis. Identification of multiple immune response features that associate with protection status, such as antibody subclasses, fine specificity and avidity reported here may accelerate development of highly efficacious vaccines against P. falciparum.

Malaria vector dynamics and utilization of insecticide-treated nets in low-transmission setting in Southwest Ethiopia: implications for residual transmission

BackgroundUnderstanding the behaviour of local malaria vectors is essential as effectiveness of the commonly used vector-targeted malaria control tools heavily relies on behaviour of the major malaria vectors. This study was conducted to determine species composition, biting behaviour, host preference and infectivity of anopheline mosquitoes, and assess utilization of insecticide-treated nets (ITNs) in a low transmission setting in Southwest Ethiopia.MethodsAdult anopheline mosquitoes were collected using human landing catches (HLCs), Centers for Disease Control and Prevention (CDC) light traps (LTs) and Pyrethrum Spray Catches (PSCs) from June 2016 to May 2018 in Kishe, Jimma Zone, Southwest Ethiopia. The anopheline mosquitoes were morphologically identified. Moreover, sub-sample of An. gambiae s.l. was identified to species using polymerase chain reaction (PCR). Circum-sporozoite proteins (CSPs) and blood meal sources of the anopheline mosquitoes were tested using enzyme-linked immunosorbent assay (ELISA). In addition, a cross-sectional survey was conducted to assess ITN utilization by the inhabitants.ResultsA total of 3659 anopheline mosquitoes comprising An. coustani complex (84.4%), An. gambiae s.l. (11.3%), and An. pharoensis and An. squamosus comprising less than 5% were collected. The anopheline mosquitoes showed marked outdoor (67%) and early evening (63%) biting behaviour. An. coustani complex and An. gambiae s.l. were predominantly zoophilic and anthropophilic, respectively. None of the sampled anopheline were CSP-positive. Most of the households (97.8%) owned at least one ITN, with modest usage by the inhabitants (73.4%). ITN usage was significantly higher among under-five children (AOR = 7.9, 95% CI: 4.41–14.03), household heads and spouses (AOR = 4.8, 95% CI: 3.0–7.59), those with sufficient access to ITNs (AOR = 1.8, 95% CI: 1.39–2.35), and who were not utilizing alternative mosquito repellents (AOR = 2.2, 95% CI: 1.58–2.99).ConclusionThe anopheline mosquito species exhibited predominantly outdoor and early evening biting activity. Household ITN coverage was high with slight gap in usage. Vector control interventions should target outdoor and early biting vectors to further suppress the local mosquito population. Moreover, sensitization of the community on consistent use of ITNs is required.

Flowcytometric and ImageStream Rna-Fish Gene Expression, Quantification and Phenotypic Characterization of Blood Sporozoites and Sporozoites From Human Malaria Species.

We adapted the RNA FISH Stellaris method to specifically detect the expression of Plasmodium genes by flow cytometry and ImageStream (Flow-FISH). This new method accurately quantified the erythrocytic forms of (1) Plasmodium falciparum and Plasmodium vivax and (2) the sexual stages of P vivax from patient isolates. ImageStream analysis of liver stage sporozoites using a combination of surface circumsporozoite protein (CSP), deoxyribonucleic acid, and 18S RNA labeling proved that the new Flow-FISH is suitable for gene expression studies of transmission stages. This powerful multiparametric single-cell method offers a platform of choice for both applied and fundamental research on the biology of malaria parasites.

Live attenuated rubella vectors expressing Plasmodium falciparum circumsporozoite protein (Pf-CSP) provide a novel malaria vaccine platform in the rhesus macaque

There is an urgent need for a malaria vaccine that can prevent severe disease in young children and adults. Despite earlier work showing an immunological mechanism for preventing infection and reducing disease severity, there is currently no reliable vaccine that can provide durable protection. In part, this may reflect a limited number of ways that the host can respond to the NANP repeat sequences of circumsporozoite protein (CSP) in the parasite. In addition, it may reflect antigenic escape by the parasite from protective antibodies. To be successful, a vaccine must protect against repeated exposure to infected mosquitoes in endemic areas. We have created a series of live viral vectors based on the rubella vaccine strain that express multiple tandem repeats of NANP, and we demonstrate immunogenicity in a rhesus macaque model.We tested the vectors in a sequential immunization strategy. In the first step, the animals were primed with CSP-DNA vaccine and boosted with rubella/CSP vectors. In the second step, we gave rubella/CSP vectors again, followed by recombinant CSP protein. Following the second step, antibody titers were comparable to adult exposure to malaria in an endemic area. The antibodies were specific for native CSP protein on sporozoites, and they persisted for at least 1½ years in two out of three macaques. Given the safety profile of rubella vaccine in children, these vectors could be most useful in protecting young children, who are at greatest risk of severe malarial disease.