Search Results (31)

Background/Objectives: 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, which is an asexual stage vaccine construct consisting of conserved domains of Glutamate-Rich Protein (GLURP) and Merozoite Surface Protein-3 (MSP-3). Previous studies showed that GMZ2.6c is widely recognized by antibodies from individuals living in endemic areas of Brazil and that levels of anti-GMZ2.6c increase with malaria exposure and may contribute to immunity against the parasite. As cell-mediated responses are crucial for parasite control and protection, identifying antigens that elicit antigen-specific T cell recall in naturally exposed populations is the key to vaccine development. This study aimed to evaluate the cellular immune response against GMZ2.6c and its components (MSP-3, GLURP, and Pfs48/45) and to identify promiscuous T cell epitopes in individuals exposed to malaria in the Brazilian Amazon, considering the impact of active P. falciparum infection on antigen-specific T cell recall. Methods: This study was carried out using peripheral blood mononuclear cells (PBMCs) from individuals with active P. falciparum infection (PFI) and non-infected individuals exposed to malaria (NI) from Cruzeiro do Sul and Mâncio Lima, Acre State, and Guajará, Amazonas State. The PBMCs were stimulated with GMZ2.6c and its components, and cellular activation, CD4⁺ and CD8⁺ memory T cell subsets, and cytokine production were evaluated by flow cytometry. IFN-γ-secreting T cells were quantified by ELISpot using predicted T cell epitopes. Results: The individuals infected by P. falciparum displayed more CD8⁺ T cell activation in response to MSP-3 and Pfs48/45 and an increase in CD4⁺ T_CM cells and a reduction in CD4⁺ T_EM cells following stimulation with Pfs48/45 and GMZ2.6c. The PBMCs from both groups showed elevated production of IL-6 and TNF after stimulation with GMZ2.6c, MSP-3, and Pfs48/45, but only the non-infected individuals had high levels of IL-10. T cell epitope prediction identified sequences within MSP-3, GLURP, and Pfs48/45 that elicited IFN-γ responses in both the non-infected and P. falciparum-infected individuals. Conclusions: Individuals exhibit cellular immune responses to MSP-3 and Pfs48/45 that are recalled following GMZ2.6c stimulation. P. falciparum infection may modulate immune response, inducing a prominent pro-inflammatory response. Conversely, in the absence of the parasite, the individuals displayed balanced Th1/Th2 cytokine production. Several promiscuous T cell epitopes were able to recall IFN-γ responses. Further studies are needed to fully ascertain the potential of GMZ2.6c as a protective candidate vaccine against malaria. Full article

Understanding Plasmodium falciparum population genetic diversity is crucial to assess the impact of malaria control interventions. This study investigated P. falciparum genetic diversity using merozoite surface protein 1 (msp1), msp2 and glutamate-rich protein (glurp) in Korhogo district, Northern Côte d’Ivoire. DNA was extracted from dried blood spots (DBSs) collected in the health district of Korhogo between 2019 and 2020. The msp1, msp2, and glurp genes were amplified by polymerase chain reaction (PCR), and amplicon sizes were determined by capillary electrophoresis. Out of 179 samples randomly selected and genotyped, 82% were successfully amplified for msp1, 85% for msp2, and 75% for glurp. For msp1, the K1 allelic family had 20 genotypes, MAD20 had 23, and RO33 had only one genotype. For msp2, there were 59 and 33 genotypes for 3D7 and FC27, respectively, and for glurp, 45 genotypes were detected. The parasite population was highly diverse with an expected heterozygosity (H_E) of ≥0.9 for all 3 markers. Our study showed high genetic diversity of msp1, msp2, and glurp in P. falciparum isolates from Korhogo district, Northern Côte d’Ivoire. These data could provide baseline information on P. falciparum genetic diversity for further epidemiological studies, needed to assess interventions implemented in this area. Full article

Malaria remains a significant public health challenge, particularly in endemic regions. The extensive genetic diversity of Plasmodium falciparum (Pf) complicates outbreak prediction and transmission control. One of its most polymorphic markers, merozoite surface protein 2 (MSP2), presents a potential target for molecular surveillance. This cross-sectional study, conducted at King Faisal Hospital Rwanda (KFHR) from October 2021 to June 2023, assessed MSP2’s utility in malaria prediction. PfMSP2 was sequenced, and selected amplicons were cloned, expressed in bacteria, and purified. These antigens were tested against sera from malaria patients and geographically diverse healthy individuals, with complementary surveys contextualizing serological findings. Of the 75 processed monoallelic clinical isolates, 3D7 strains predominated over FC27. Three MSP2-derived biomarkers were produced, eliciting significantly low IgG responses in malaria patients and Belgian controls, but a complex pattern emerged in healthy individuals, with significant differences between Rwandan and Cameroonian samples. IgG3 was the predominant subclass in individuals with high IgG responses. Notably, Rwandan individuals with weak humoral responses to the tested antigens but also other with high responses experienced malaria episodes in the subsequent year. These findings highlight MSP2 polymorphism as a valuable tool for malaria surveillance and outbreak prediction. Integrating genotyping and serology could enable precise, community-specific malaria risk assessments, strengthening control strategies. Full article

Resistance to current antimalarial drugs is steadily increasing, and new drugs are required. Drug efficacy trials remain the gold standard to assess the effectiveness of a given drug. The World Health Organization (WHO)’s recommendation for the optimal duration of follow-up for assessing antimalarial efficacy is a minimum of 28 days. However, assessing antimalarial drug efficacy in highly endemic regions can be challenging due to the potential risks of acquiring a new infection in the follow-up period, and thus, it may underestimate the efficacy of the given drugs. A new treatment should be introduced if treatment failure rates exceed 10%. Overestimation occurs as a result of retaining a drug with a clinical efficacy of less than 90% with increases in morbidity and mortality, while underestimation may occur due to a misclassification of new infections as treatment failures with tremendous clinical and economic implications. Therefore, molecular genotyping is necessary to distinguish true new infections from treatment failures to ensure accuracy in determining antimalarial efficacy. There are three genetic markers that are commonly used in antimalarial efficiency trials to discriminate between treatment failures and new infections. These include merozoite surface protein 1 (msp-1), merozoite surface protein 2 (msp-2), and glutamate-rich protein (glurp). The genotyping of P. falciparum by nested polymerase chain reaction (n-PCR) targeting these markers is discussed with the inherent limitations and uncertainties associated with the PCR technique and limitations enforced by the parasite’s biology itself. Full article

Malaria parasites increase their host erythrocyte’s permeability to obtain essential nutrients from plasma and facilitate intracellular growth. In the human Plasmodium falciparum pathogen, this increase is mediated by the plasmodial surface anion channel (PSAC) and has been linked to CLAG3, a protein integral to the host erythrocyte membrane and encoded by a member of the conserved clag multigene family. Whether paralogs encoded by other clag genes also insert at the host membrane is unknown; their contributions to PSAC formation and other roles served are also unexplored. Here, we generated transfectant lines carrying epitope-tagged versions of each CLAG. Each paralog is colocalized with CLAG3, with concordant trafficking via merozoite rhoptries to the host erythrocyte membrane of newly invaded erythrocytes. Each also exists within infected cells in at least two forms: an alkaline-extractable soluble form and a form integral to the host membrane. Like CLAG3, CLAG2 has a variant region cleaved by extracellular proteases, but CLAG8 and CLAG9 are protease resistant. Paralog knockout lines, generated through CRISPR/Cas9 transfection, exhibited uncompromised growth in PGIM, a modified medium with higher physiological nutrient levels; this finding is in marked contrast to a recently reported CLAG3 knockout parasite. CLAG2 and CLAG8 knockout lines exhibited compensatory increases in the transcription of the remaining clags and associated rhoph genes, yielding increased PSAC-mediated uptake for specific solutes. We also report on the distinct transport properties of these knockout lines. Similar membrane topologies at the host membrane are consistent with each CLAG paralog contributing to PSAC, but other roles require further examination. Full article

Efficacy data on two malaria vaccines, RTS,S and R21, targeting Plasmodium falciparum circumsporozoite protein (PfCSP), are encouraging. Efficacy may be improved by induction of additional antibodies to neutralizing epitopes outside of the central immunodominant repeat domain of PfCSP. We designed four rPfCSP-based vaccines in an effort to improve the diversity of the antibody response. We also evaluated P. falciparum merozoite surface protein 8 (PfMSP8) as a malaria-specific carrier protein as an alternative to hepatitis B surface antigen. We measured the magnitude, specificity, subclass, avidity, durability, and efficacy of vaccine-induced antibodies in outbred CD1 mice. In comparison to N-terminal- or C-terminal-focused constructs, immunization with near full-length vaccines, rPfCSP (#1) or the chimeric rPfCSP/8 (#2), markedly increased the breadth of B cell epitopes recognized covering the N-terminal domain, junctional region, and central repeat. Both rPfCSP (#1) and rPfCSP/8 (#2) also elicited a high proportion of antibodies to conformation-dependent epitopes in the C-terminus of PfCSP. Fusion of PfCSP to PfMSP8 shifted the specificity of the T cell response away from PfCSP toward PfMSP8 epitopes. Challenge studies with transgenic Plasmodium yoelii sporozoites expressing PfCSP demonstrated high and consistent sterile protection following rPfCSP/8 (#2) immunization. Of note, antibodies to conformational C-terminal epitopes were not required for protection. These results indicate that inclusion of the N-terminal domain of PfCSP can drive responses to protective, repeat, and non-repeat B cell epitopes and that PfMSP8 is an effective carrier for induction of high-titer, durable anti-PfCSP antibodies. Full article

Vaccines are highly effective tools against infectious diseases and are also considered necessary in the fight against malaria. Vaccine-induced immunity is frequently mediated by antibodies. We have recently conducted a first-in-human clinical trial featuring SumayaVac-1, a malaria vaccine based on the recombinant, full-length merozoite surface protein 1 (MSP1_FL) formulated with GLA-SE as an adjuvant. Vaccination with MSP1_FL was safe and elicited sustainable IgG antibody titers that exceeded those observed in semi-immune populations from Africa. Moreover, IgG antibodies stimulated various Fc-mediated effector mechanisms associated with protection against malaria. However, these functionalities gradually waned. Here, we show that the initial two doses of SumayaVac-1 primarily induced the cytophilic subclasses IgG1 and IgG3. Unexpectedly, a shift in the IgG subclass composition occurred following the third and fourth vaccinations. Specifically, there was a progressive transition to IgG4 antibodies, which displayed a reduced capacity to engage in Fc-mediated effector functions and also exhibited increased avidity. In summary, our analysis of antibody responses to MSP1_FL vaccination unveils a temporal shift towards noninflammatory IgG4 antibodies. These findings underscore the importance of considering the impact of IgG subclass composition on vaccine-induced immunity, particularly concerning Fc-mediated effector functions. This knowledge is pivotal in guiding the design of optimal vaccination strategies against malaria, informing decision making for future endeavors in this critical field. Full article

The combinations of artemether-lumefantrine (AL) and artesunate-amodiaquine (ASAQ) are used as first-line treatments for uncomplicated malaria in the Ivory Coast. Different studies document the efficacy of two artemisinin-based combination therapies (ACTs) (AL and ASAQ) in the Ivory Coast. However, there is no meta-analysis examining the data set of these studies. The purpose of this work was to determine the prevalence of malaria treatment failure cases in randomized control trials with two artemisinin-based combination therapies (AL versus ASAQ) in the Ivory Coast between 2009 to 2016. This study is a meta-analysis of data from the results of four previous multicenter, open-label, randomized clinical trial studies evaluating the clinical and parasitological efficacy of artemether-lumefantrine and artesunate-amodiaquine conducted between 2009 and 2016 following World Health Organization (WHO) protocol at sentinel sites in the Ivory Coast. These drug efficacy data collected between 2009 and 2016 were analyzed. During these studies, to distinguish between recrudescence and new infection, molecular genotyping of genes encoding merozoite surface protein 1 and 2 was carried out using nested polymerase chain reaction (PCR). A total of 1575 patients enrolled in the four studies, including 768 in the AL arm and 762 in the ASAQ arm, which were fully followed either for 28 days or 42 days according to WHO protocol. The adequate clinical and parasitological response (ACPR) was higher than 95% in the two groups (intention to treat (ITT): AL = 96.59% and ASAQ = 96.81; Per Protocol (PP): AL = 99.48% and ASAQ = 99.61%) after PCR correction at day 28. Aggregate data analysis (2009–2016) showed that at day 28, the proportions of patients with recurrent infection was higher in the AL group (ITT: 3.79%, PP: 3.9%) than in the ASAQ group (ITT: 2.17%, PP: 2.23%). After PCR correction, most treatment failures were classified as new infections (AL group (ITT: 0.13%, PP: 0.13%); ASAQ group (ITT: 0.39%, PP: 0.39%). The recrudescent infections rate was high, at 0.39% compared to 0.13% for ASAQ and AL, respectively, for both ITT and PP, no significant difference. However, the Kaplan–Meier curve of cumulative treatment success showed a significant difference between the two groups after PCR from 2012–2013 (p = 0.032). Overall, ASAQ and AL have been shown to be effective drugs for the treatment of uncomplicated P. falciparum malaria in the study areas, 14 years after deployment of these drugs. Full article

Tick-borne diseases (TBDs) of livestock are endemic across various parts of tropical countries. Theileriosis is one such economically important TBD, caused by the Theileriidae family of organisms, which is transmitted by ticks. Theileria annulata, the causative agent of tropical theileriosis, contributes a significant loss to the dairy sector by causing anorexia, high fever, anemia, inflammatory changes in vital organs and icterus, thus, a loss in milk yield. Though vaccines are available, their protective efficacy is not absolute, and treatment is limited to early diagnosis of the causative agent. Routinely, microscopic identification of piroplasms in the erythrocytes (Giemsa-stained) of infected animals or schizonts in lymph node biopsies are practiced for diagnosis. PCR-based techniques (multiplex, uniplex, nested and real-time) have been reported to perform well in diagnosing active infection. Several attempts have been made using serological assays like Dot blot, ELISA and ICT, but the results were of variable sensitivity and specificity. Recombinant proteins like the Theileria annulata merozoite surface antigen (Tams1) and Theileria annulata surface protein (TaSP) have been explored as antigenic candidates for these assays. In the present study, we predicted an immunogenic peptide, i.e., TaSP-34, from the TaSP using various computational tools. The predicted peptide was custom synthesized. The diagnostic potential of the peptide was assessed by indirect plate ELISA to detect the bovine-IgM against Theileria annulata. Alongside, a recombinant truncated TaSP (rTaSP(tr)) was expressed and purified, which was used to compare the performance of the peptide as a diagnostic candidate. The IgM-based peptide ELISA was 100% sensitive and 92.77% specific as compared to PCR (Tams1 targeting), while 98.04% sensitivity and 97.44% specificity were observed in comparison with rTaSP(tr) ELISA. Almost perfect agreement between peptide ELISA and Tams1 PCR was observed with a Cohen’s kappa coefficient (κ-value) of 0.901 and agreement of 95.31%. Further, the κ-value between the peptide ELISA and rTaSP(tr) ELISA was found to be 0.95, and the agreement was 97.65%, which shows a good correlation between the two tests. The findings suggest that the TaSP-34 peptide can be an efficient and new-generation diagnostic candidate for the diagnosis of T. annulata. Furthermore, the peptide can be synthesized commercially at a larger scale and can be a cost-effective alternative for the protein-based diagnostic candidates for T. annulata. Full article

Malaria poses a significant global health challenge, resulting in approximately 600,000 deaths each year. Individuals living in regions with endemic malaria have the potential to develop partial immunity, thanks in part to the presence of anti-plasmodium antibodies. As efforts are made to optimize and implement strategies to reduce malaria transmission and ultimately eliminate the disease, it is crucial to understand how these interventions impact naturally acquired protective immunity. To shed light on this, our study focused on assessing antibody responses to a carefully curated library of P. falciparum recombinant proteins (n = 691) using samples collected from individuals residing in a low-malaria-transmission region of Thailand. We conducted the antibody assays using the AlphaScreen system, a high-throughput homogeneous proximity-based bead assay that detects protein interactions. We observed that out of the 691 variable surface and merozoite stage proteins included in the library, antibodies to 268 antigens significantly correlated with the absence of symptomatic malaria in an univariate analysis. Notably, the most prominent antigens identified were P. falciparum erythrocyte membrane protein 1 (PfEMP1) domains. These results align with our previous research conducted in Uganda, suggesting that similar antigens like PfEMP1s might play a pivotal role in determining infection outcomes in diverse populations. To further our understanding, it remains critical to conduct functional characterization of these identified proteins, exploring their potential as correlates of protection or as targets for vaccine development. Full article

Polymorphisms in the genes encoding the merozoite surface proteins msp-1 and msp-2 are widely used markers for characterizing the genetic diversity of Plasmodium falciparum. This study aimed to compare the genetic diversity of circulating parasite strains in rural and urban settings in the Republic of Congo after the introduction of artemisinin-based combination therapy (ACT) in 2006. A cross-sectional survey was conducted from March to September 2021 in rural and urban areas close to Brazzaville, during which Plasmodium infection was detected using microscopy (and nested-PCR for submicroscopic infection). The genes coding for merozoite proteins-1 and -2 were genotyped by allele-specific nested PCR. Totals of 397 (72.4%) and 151 (27.6%) P. falciparum isolates were collected in rural and urban areas, respectively. The K1/msp-1 and FC27/msp-2 allelic families were predominant both in rural (39% and 64%, respectively) and urban (45.4% and 54.5% respectively) areas. The multiplicity of infection (MOI) was higher (p = 0.0006) in rural areas (2.9) compared to urban settings (2.4). The rainy season and the positive microscopic infection were associated with an increase in MOI. These findings reveal a higher P. falciparum genetic diversity and MOI in the rural setting of the Republic of Congo, which is influenced by the season and the participant clinical status. Full article

The genetic diversity of pkmsp-1 of Malaysian Plasmodium knowlesi isolates was studied recently. However, the study only included three relatively older strains from Peninsular Malaysia and focused mainly on the conserved blocks of this gene. In this study, the full-length pkmsp-1 sequence of recent P. knowlesi isolates from Peninsular Malaysia was characterized, along with Malaysian Borneo and Thailand pkmsp-1 sequences that were retrieved from GenBank. Genomic DNA of P. knowlesi was extracted from human blood specimens and the pkmsp-1 gene was PCR-amplified, cloned, and sequenced. The sequences were analysed for genetic diversity, departure from neutrality, and geographical clustering. The pkmsp-1 gene was found to be under purifying/negative selection and grouped into three clusters via a neighbour-joining tree and neighbour net inferences. Of the four polymorphic blocks in pkmsp-1, block IV, was most polymorphic, with the highest insertion–deletion (indel) sites. Two allelic families were identified in block IV, thereby highlighting the importance of this block as a promising genotyping marker for the multiplicity of infection study of P. knowlesi malaria. A single locus marker may provide an alternate, simpler method to type P. knowlesi in a population. Full article

High levels of genetic variants of Plasmodium vivax have previously been reported in Thailand. Circumsporozoite surface protein (CSP), merozoite surface protein (MSP), and microsatellite markers were used to determine the genetic polymorphisms of P. vivax. This study aimed to investigate the molecular epidemiology of P. vivax populations at the Thai–Myanmar border by genotyping the PvCSP, PvMSP-3α, and PvMSP-3β genes. Four hundred and forty P. vivax clinical isolates were collected from the Mae Sot and Sai Yok districts from 2006–2007 and 2014–2016. Polymerase chain reaction with restriction fragment length polymorphism (RFLP) was used to investigate the genetic polymorphisms of the target genes. Based on PCR band size variations, 14 different PvCSP alleles were identified: eight for VK210 and six for VK247. The VK210 genotype was the dominant variant during both sample collection periods. Based on PCR genotyping, three distinct types (A, B, and C) for both PvMSP-3α and PvMSP-3β were observed. Following RFLP, 28 and 14 allelic variants of PvMSP-3α and 36 and 20 allelic variants of PvMSP-3β with varying frequencies were identified during the first and second periods, respectively. High genetic variants of PvMSP-3 and PvCSP were found in the study area. PvMSP-3β exhibited a higher level of genetic diversity and multiple-genotype infection versus PvMSP-3α. Full article

Background: The serological tests using blood stage antigens might be helpful for detecting recent exposure to Plasmodium parasites, and seroepidemiological studies would aid in the elimination of malaria. This work produced recombinant proteins of PvMSP1₄₂ variants and evaluated their capacity to detect IgG antibodies in symptomatic patients from Mesoamerica. Methods: Three variant Pvmsp1₄₂ genes were cloned in the pHL-sec plasmid, expressed in the Expi293F™ eukaryotic system, and the recombinant proteins were purified by affinity chromatography. Using an ELISA, 174 plasma or eluted samples from patients infected with different P. vivax haplotypes were evaluated against PvMSP1₄₂ proteins and to a native blood stage antigen (NBSA). Results: The antibody IgG OD values toward PvMSP142 variants (v88, v21, and v274) were heterogeneous (n = 178; median = 0.84 IQR 0.28–1.64). The correlation of IgG levels among all proteins was very high (spearman’s rho = 0.96–0.98; p < 0.0001), but was lower between them and the NBSA (rho = 0.771; p < 0.0001). In only a few samples, higher reactivity to the homologous protein was evident. Patients with a past infection who were seropositive had higher IgG levels and lower parasitemia levels than those who did not (p < 0.0001). Conclusions: The PvMSP1₄₂ variants were similarly efficient in detecting specific IgG antibodies in P. vivax patients from Mesoamerica, regardless of the infecting parasite’s haplotype, and might be good candidates for malaria surveillance and epidemiological studies in the region. Full article

Plasmodium malaria parasites use erythrocyte-binding-like (EBL) ligands to invade erythrocytes in their vertebrate host. EBLs are released from micronemes, which are secretory organelles located at the merozoite apical end and bind to erythrocyte surface receptors. Because of their essential nature, EBLs have been studied as vaccine candidates, such as the Plasmodium vivax Duffy binding protein. Previously, we showed through using the rodent malaria parasite Plasmodium yoelii that a single amino acid substitution within the EBL C-terminal Cys-rich domain (region 6) caused mislocalization of this molecule and resulted in alteration of the infection course and virulence between the non-lethal 17X and lethal 17XL strains. In the present study, we generated a panel of transgenic P. yoelii lines in which seven of the eight conserved Cys residues in EBL region 6 were independently substituted to Ala residues to observe the consequence of these substitutions with respect to EBL localization, the infection course, and virulence. Five out of seven transgenic lines showed EBL mislocalizations and higher parasitemias. Among them, three showed increased virulence, whereas the other two did not kill the infected mice. The remaining two transgenic lines showed low parasitemias similar to their parental 17X strain, and their EBL localizations did not change. The results indicate the importance of Cys residues in EBL region 6 for EBL localization, parasite infection course, and virulence and suggest an association between EBL localization and the parasite infection course. Full article