Plasmodium falciparum AMA1 and CSP antigen diversity in parasite isolates from southern Ghana.

Introduction: Diversity in malarial antigens is an immune evasion mechanism that gives malaria parasites an edge over the host. Immune responses against one variant of a polymorphic antigen are usually not fully effective against other variants due to altered epitopes. This study aimed to evaluate diversity in the Plasmodium falciparum antigens apical membrane antigen 1 (PfAMA1) and circumsporozoite protein (PfCSP) from circulating parasites in a malaria-endemic community in southern Ghana and to determine the effects of polymorphisms on antibody response specificity. Methods: The study involved 300 subjects, whose P. falciparum infection status was determined by microscopy and PCR. Diversity within the two antigens was evaluated by msp2 gene typing and molecular gene sequencing, while the host plasma levels of antibodies against PfAMA1, PfCSP, and two synthetic 24mer peptides from the conserved central repeat region of PfCSP, were measured by ELISA. Results: Of the 300 subjects, 171 (57%) had P. falciparum infection, with 165 of the 171 (96.5%) being positive for either or both of the msp2 allelic families. Gene sequencing of DNA from 55 clonally infected samples identified a total of 56 non-synonymous single nucleotide polymorphisms (SNPs) for the Pfama1 gene and these resulted in 44 polymorphic positions, including two novel positions (363 and 365). Sequencing of the Pfcsp gene from 69 clonal DNA samples identified 50 non-synonymous SNPs that resulted in 42 polymorphic positions, with half (21) of these polymorphic positions being novel. Of the measured antibodies, only anti-PfCSP antibodies varied considerably between PCR parasite-positive and parasite-negative persons. Discussion: These data confirm the presence of a considerable amount of unique, previously unreported amino acid changes, especially within PfCSP. Drivers for this diversity in the Pfcsp gene do not immediately seem apparent, as immune pressure will be expected to drive a similar level of diversity in the Pfama1 gene.

Putative molecular markers of Plasmodium falciparum resistance to antimalarial drugs in malaria parasites from Ghana.

Introduction: Antimalarial drugs including artemisinin-based combination therapy (ACT) regimens and sulphadoxine-pyrimethamine (SP) are used in Ghana for malaria therapeutics and prophylaxis respectively. The genetic basis of Plasmodium falciparum development of drug resistance involves single nucleotide polymorphisms in genes encoding proteins for multiple cellular and metabolic processes. The prevalence of single nucleotide polymorphisms in nine P. falciparum genes linked to ACT and SP resistance in the malaria parasite population was determined. Methods: Archived filter paper blood blot samples from patients aged 9 years and below with uncomplicated malaria reporting at 10 sentinel sites located in three ecological zones for the Malaria Therapeutic Efficacy Studies were used. The samples used were collected from 2007-2018 malaria transmission seasons and mutations in the genes were detected using PCR and Sanger sequencing. Results: In all 1,142 samples were used for the study. For falcipain-2 gene (pffp2), Sanger sequencing was successful for 872 samples and were further analysed. The prevalence of the mutants was 45% (392/872) with pffp2 markers V51I and S59F occurring in 15.0% (128/872) and 3.0% (26/872) of the samples respectively. Prevalence of other P. falciparum gene mutations: coronin (pfcoronin) was 44.8% (37/90); cysteine desulfurase (pfnfs) was 73.9% (68/92); apicoplast ribosomal protein S10 (pfarps10) was 36.8% (35/95); ferredoxin (pffd) was 8.8% (8/91); multidrug resistance protein-1 (pfmrp1) was 95.2.0% (80/84); multidrug resistance protein-2 (pfmrp2) was 91.4% (32/35); dihydrofolate reductase (pfdhfr) was 99.0% (84/85); dihydropteroate synthase (pfdhps) was 72% (68/95). Discussion: The observation of numerous mutations in these genes of interest in the Ghanaian isolates, some of which have been implicated in delayed parasite clearance is of great interest. The presence of these genotypes may account for the decline in the efficacies of ACT regimens being used to treat uncomplicated malaria in the country. The need for continuous monitoring of these genetic markers to give first-hand information on parasite susceptibility to antimalarial drugs to inform policy makers and stakeholders in malaria elimination in the country is further discussed.

Immunodominant T cell peptides from four candidate malarial antigens as biomarkers of protective immunity against malaria.

A malaria vaccine with high efficacy and capable of inducing sterile immunity against malaria within genetically diverse populations is urgently needed to complement ongoing disease control and elimination efforts. Parasite-specific IFN-γ and granzyme B-secreting CD8 + T cells have been identified as key mediators of protection and the rapid identification of malaria antigen targets that elicit these responses will fast-track the development of simpler, cost-effective interventions. This study extends our previous work which used peripheral blood mononuclear cells (PBMCs) from adults with life-long exposure to malaria parasites to identify immunodominant antigen-specific peptide pools composed of overlapping 15mer sequences spanning full length proteins of four malarial antigens. Our current study aimed to identify CD8 + T cell epitopes within these previously identified positive peptide pools. Cryopreserved PBMCs from 109 HLA-typed subjects were stimulated with predicted 9-11mer CD8 + T cell epitopes from P. falciparum circumsporozoite protein (CSP), apical membrane antigen 1 (AMA1), thrombospondin related anonymous protein (TRAP) and cell traversal for ookinetes and sporozoites (CelTOS) in FluoroSpot assays. A total of 135 epitopes out of 297 tested peptides from the four antigens were experimentally identified as positive for IFN-γ and/or granzyme B production in 65 of the 109 subjects. Forty-three of 135 epitopes (32 %) were promiscuous for HLA binding, with 31 of these promiscuous epitopes (72 %) being presented by HLA alleles that fall within at least two different HLA supertypes. Furthermore, about 52 % of identified epitopes were conserved when the respective sequences were aligned with those from 16 highly diverse P. falciparum parasite strains. In summary, we have identified a number of conserved epitopes, immune responses to which could be effective against multiple P. falciparum parasite strains in genetically diverse populations.

Novel pfk13 polymorphisms in Plasmodium falciparum population in Ghana.

The molecular determinants of Plasmodium falciparum artemisinin resistance are the single nucleotide polymorphisms in the parasite's kelch propeller domain, pfk13. Validated and candidate markers are under surveillance in malaria endemic countries using artemisinin-based combination therapy. However, pfk13 mutations which may confer parasite artemisinin resistance in Africa remains elusive. It has therefore become imperative to report all observed pfk13 gene polymorphisms in malaria therapeutic efficacy studies for functional characterization. We herein report all novel pfk13 mutations observed only in the Ghanaian parasite population. In all, 977 archived samples from children aged 12 years and below with uncomplicated malaria from 2007 to 2017 were used. PCR/Sanger sequencing analysis revealed 78% (763/977) of the samples analyzed were wild type (WT) for pfk13 gene. Of the 214 (22%) mutants, 78 were novel mutations observed only in Ghana. The novel SNPs include R404G, P413H, N458D/H/I, C473W/S, R529I, M579T/Y, C580R/V, D584L, N585H/I, Q661G/L. Some of the mutations were sites and ecological zones specific. There was low nucleotide diversity and purifying selection at the pfk13 locus in Ghanaian parasite population. With increasing drug pressure and its consequent parasite resistance, documenting these mutations as baseline data is crucial for future molecular surveillance of P. falciparum resistance to artemisinin in Ghana.

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.

Genetic deletions and high diversity of Plasmodium falciparum histidine-rich proteins 2 and 3 genes in parasite populations in Ghana.

Rapid diagnostic tests (RDTs) are used to diagnose malaria in Ghana and other malaria endemic countries. Plasmodium falciparum histidine-rich protein 2 (PFHRP2) based RDTs are widely used, however the occurrence of deletions of the pfhrp2 gene in some parasites have resulted in false negative test results. Monoclonal antibodies of PFHRP2 cross reacts with PFHRP3 because they share structural similarities and this complements the detection of the parasites by RDT. These two genes were investigated in Ghanaian P. falciparum parasite population to detect deletions and the polymorphisms in exon 2 of the pfhrp2 and pfhrp3 genes. Parasite isolates (2,540) from children ≤ 12 years with uncomplicated malaria from 2015 to 2020 transmission seasons were used. Both genes were amplified using nested PCR and negative results indicated the presence of the deletion of genes. Amplified genes were sequenced for the detection of the amino acid repeats. Deletions were observed in 30.7% (780/2,540) and 17.2% (438/2,540) of the samples for pfhrp2 and pfhrp3 respectively with increasing trends over the three time periods (χ2 -10.305, p = 0.001). A total of 1,632 amplicons were sequenced for each gene, analysis was done on 1,124 and 1,307 good quality sequences for pfhrp2 and pfhrp3 respectively. Pfhrp2 repeat polymorphisms were dominantly of types 2 (AHHAHHAAD) and 7 (AHHAAD) with large numbers of variants. A novel variant of type 14 (AHHANHATD) was seen for pfhrp2. For the pfhrp3 repeat types, 16 (AHHAAN), 17 (AHHDG) and 18 (AHHDD) were the dominant types observed. Variants of type 16 (AHHAAH) and (AHHASH) were also dominant. Repeat types 1, 2, 3, 4, 5, 6, 7, 8, 11, 13, 15, 16, and 19 were observed be shared by both genes. The haplotype diversity of both genes ranged between 0.872 and 1 indicating high diversity of the polymorphisms in the isolates. The implication of the findings of the frequencies of the pfhrp2 and pfhrp3 deletions as well as the variants of the main epitopes of the monoclonal antibodies for the RDT (types 2 and 7) in our isolates is an indication of decreased sensitivity of the RDTs in diagnosing malaria infections in Ghana.

Elevated Levels of the Endothelial Molecules ICAM-1, VEGF-A, and VEGFR2 in Microscopic Asymptomatic Malaria.

BACKGROUND: In malaria, clinical disease has been associated with increased levels of endothelial activation due to the sequestration of infected erythrocytes. However, the levels and impact of endothelial activation and pro-angiogenic molecules such as vascular endothelial growth factor (VEGF)-A and its receptor vascular endothelial growth factor receptor 2 (VEGFR2) in asymptomatic malaria have not been well characterized. METHODS: Blood samples were obtained from community children for malaria diagnosis using microscopy and polymerase chain reaction. A multiplex immunoassay was used to determine the levels of intracellular adhesion molecule (ICAM)-1, vascular endothelial growth factor (VEGF)-A, and VEGFR2 in the plasma of children with microscopic or submicroscopic asymptomatic parasitemia and compared with levels in uninfected controls. RESULTS: Levels of ICAM-1, VEGF-A, and VEGFR2 were significantly increased in children with microscopic asymptomatic parasitemia compared with uninfected controls. Also, levels of VEGF-A were found to be inversely associated with age. Additionally, a receiver operating characteristic analysis revealed that plasma levels of ICAM-1 (area under the curve [AUC], 0.72) showed a moderate potential in discriminating between children with microscopic malaria from uninfected controls when compared with VEGF-A (AUC, 0.67) and VEGFR2 (AUC, 0.69). CONCLUSIONS: These data imply that endothelial activation and pro-angiogenic growth factors could be one of the early host responders during microscopic asymptomatic malaria and may play a significant role in disease pathogenesis.

Prevalence of Plasmodium falciparum delayed clearance associated polymorphisms in adaptor protein complex 2 mu subunit (pfap2mu) and ubiquitin specific protease 1 (pfubp1) genes in Ghanaian isolates.

BACKGROUND: Plasmodium falciparum delayed clearance with the use of artemisinin-based combination therapy (ACTs) has been reported in some African countries. Single nucleotide polymorphisms (SNPs) in two genes, P. falciparum adaptor protein complex 2 mu subunit (pfap2mu) and ubiquitin specific protease 1 (pfubp1), have been linked to delayed clearance with ACT use in Kenya and recurrent imported malaria in Britain. With over 12 years of ACT use in Ghana, this study investigated the prevalence of SNPs in the pfap2mu and pfubp1 in Ghanaian clinical P. falciparum isolates to provide baseline data for antimalarial drug resistance surveillance in the country. METHODS: Filter paper blood blots collected in 2015-2016 from children aged below 9 years presenting with uncomplicated malaria at hospitals in three sentinel sites Begoro, Cape Coast and Navrongo were used. Parasite DNA was extracted from 120 samples followed by nested polymerase chain reaction (nPCR). Sanger sequencing was performed to detect and identify SNPs in pfap2mu and pfubp1 genes. RESULTS: In all, 11.1% (9/81) of the isolates carried the wildtype genotypes for both genes. A total of 164 pfap2mu mutations were detected in 67 isolates whilst 271 pfubp1 mutations were observed in 72 isolates. The majority of the mutations were non-synonymous (NS): 78% (128/164) for pfap2mu and 92.3% (250/271) for pfubp1. Five unique samples had a total of 215 pfap2mu SNPs, ranging between 15 and 63 SNPs per sample. Genotypes reportedly associated with ART resistance detected in this study included pfap2mu S160N (7.4%, 6/81) and pfubp1 E1528D (7.4%, 6/81) as well as D1525E (4.9%, 4/81). There was no significant difference in the prevalence of the SNPs between the three ecologically distinct study sites (pfap2mu: χ2 = 6.905, df = 2, P = 0.546; pfubp1: χ2 = 4.883, df = 2, P = 0.769). CONCLUSIONS: The detection of pfap2mu and pfubp1 genotypes associated with ACT delayed parasite clearance is evidence of gradual nascent emergence of resistance in Ghana. The results will serve as baseline data for surveillance and the selection of the genotypes with drug pressure over time. The pfap2mu S160N, pfubp1 E1528D and D1525E must be monitored in Ghanaian isolates in ACT susceptibility studies, especially when cure rates of ACTs, particularly AL, is less than 100%.