Plasmodium falciparum pfhrp2 and pfhrp3 Gene Deletions and Relatedness to Other Global Isolates, Djibouti, 2019-2020.

Deletions of pfhrp2 and paralogue pfhrp3 (pfhrp2/3) genes threaten Plasmodium falciparum diagnosis by rapid diagnostic test. We examined 1,002 samples from suspected malaria patients in Djibouti City, Djibouti, to investigate pfhrp2/3 deletions. We performed assays for Plasmodium antigen carriage, pfhrp2/3 genotyping, and sequencing for 7 neutral microsatellites to assess relatedness. By PCR assay, 311 (31.0%) samples tested positive for P. falciparum infection, and 296 (95.2%) were successfully genotyped; 37 (12.5%) samples were pfhrp2+/pfhrp3+, 51 (17.2%) were pfhrp2+/pfhrp3-, 5 (1.7%) were pfhrp2-/pfhrp3+, and 203 (68.6%) were pfhrp2-/pfhrp3-. Histidine-rich protein 2/3 antigen concentrations were reduced with corresponding gene deletions. Djibouti P. falciparum is closely related to Ethiopia and Eritrea parasites (pairwise GST 0.68 [Ethiopia] and 0.77 [Eritrea]). P. falciparum with deletions in pfhrp2/3 genes were highly prevalent in Djibouti City in 2019-2020; they appear to have arisen de novo within the Horn of Africa and have not been imported.

Low Prevalence of Deletions of the pfhrp2 and pfhrp3 Genes in Plasmodium falciparum Parasites in Freetown, Sierra Leone in 2015.

Sierra Leone relies heavily on histidine-rich protein 2-based diagnostics for malaria because of the high transmission of Plasmodium falciparum. During the 2015 recombinant vesicular stomatitis virus (VSV)-Zaire Ebola virus envelope glycoprotein (GP) vaccine trial, 77 participants with asymptomatic Plasmodium infection were enrolled, with all but four having P. falciparum malaria. Of the 73 participants with P. falciparum malaria, one infection (1 of 73, 1.4%; 95% CI, 0.03-7.4) showed P. falciparum with a pfhrp3 single deletion, and two P. falciparum infections (2 of 73, 2.7%; 95% CI, 0.03-9.6) showed pfhrp2/pfhrp3 dual deletions. This study shows evidence of pfhrp2- and pfhrp3-deleted parasites in Freetown, Sierra Leone. Additional studies for more precise estimates of prevalence are warranted.

The use of a chimeric antigen for Plasmodium falciparum and P. vivax seroprevalence estimates from community surveys in Ethiopia and Costa Rica.

BACKGROUND: In low-transmission settings, accurate estimates of malaria transmission are needed to inform elimination targets. Detection of antimalarial antibodies provides exposure history, but previous studies have mainly relied on species-specific antigens. The use of chimeric antigens that include epitopes from multiple species of malaria parasites in population-based serological surveys could provide data for exposure to multiple Plasmodium species circulating in an area. Here, the utility of P. vivax/P. falciparum chimeric antigen for assessing serological responses was evaluated in Ethiopia, an endemic country for all four human malarias, and Costa Rica, where P. falciparum has been eliminated with reports of sporadic P. vivax cases. METHODS: A multiplex bead-based assay was used to determine the seroprevalence of IgG antibodies against a chimeric malaria antigen (PvRMC-MSP1) from blood samples collected from household surveys in Ethiopia in 2015 (n = 7,077) and Costa Rica in 2015 (n = 851). Targets specific for P. falciparum (PfMSP1) and P. vivax (PvMSP1) were also included in the serological panel. Seroprevalence in the population and seroconversion rates were compared among the three IgG targets. RESULTS: Seroprevalence in Costa Rica was 3.6% for PfMSP1, 41.5% for PvMSP1 and 46.7% for PvRMC-MSP1. In Ethiopia, seroprevalence was 27.6% for PfMSP1, 21.4% for PvMSP1, and 32.6% for PvRMC-MSP1. IgG levels in seropositive individuals were consistently higher for PvRMC-MSP1 when compared to PvMSP1 in both studies. Seroconversion rates were 0.023 for PvMSP1 and 0.03 for PvRMC-MSP1 in Costa Rica. In Ethiopia, seroconversion rates were 0.050 for PfMSP1, 0.044 for PvMSP1 and 0.106 for PvRMC-MSP1. CONCLUSIONS: Our data indicate that chimeric antigen PvRMC-MSP1 is able to capture antibodies to multiple epitopes from both prior P. falciparum and P. vivax infections, and suitable chimeric antigens can be considered for use in serosurveys with appropriate validation.

Investigation of Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions and performance of a rapid diagnostic test for identifying asymptomatic malaria infection in northern Ethiopia, 2015.

BACKGROUND: Rapid diagnostic tests (RDTs) are widely used for malaria diagnosis of both symptomatic and asymptomatic infections. Although RDTs are a reliable and practical diagnostic tool, the sensitivity of histidine-rich protein 2 (HRP2)-based RDTs can be reduced if pfhrp2 or pfhrp3 (pfhrp2/3) gene deletions exist in the Plasmodium falciparum parasite population. This study evaluated dried blood spot (DBS) samples collected from a national household survey to investigate the presence of pfhrp2/3 deletions and the performance of the RDT used in the cross-sectional survey in a low transmission setting. METHODS: The 2015 Ethiopia Malaria Indicator Survey tested household members by RDT and collected DBS samples. DBS (n = 2648) from three regions in northern Ethiopia were tested by multiplex bead-based antigen detection assay after completion of the survey. The multiplex assay detected pan-Plasmodium lactate dehydrogenase (LDH), pAldolase, and HRP2 antigens in samples. Samples suspected for pfhrp2/3 gene deletions (pLDH and/or pAldolase positive but low or absent HRP2) were further investigated by molecular assays for gene deletions. Antigen results were also compared to each individual's RDT results. Dose-response logistic regression models were fit to estimate RDT level of detection (LOD) antigen concentrations at which 50, 75, 90, and 95% of the RDTs returned a positive result during this survey. RESULTS: Out of 2,648 samples assayed, 29 were positive for pLDH or pAldolase antigens but low or absent for HRP2 signal, and 15 of these samples (51.7%) were successfully genotyped for pfhrp2/3. Of these 15 P. falciparum infections, eight showed single deletions in pfhrp3, one showed a single pfhrp2 deletion, and six were pfhrp2/3 double-deletions. Six pfhrp2 deletions were observed in Tigray and one in Amhara. Twenty-five were positive for HRP2 by the survey RDT while the more sensitive bead assay detected 30 HRP2-positive samples. A lower concentration of HRP2 antigen generated a positive test result by RDT compared to pLDH (95% LOD: 16.9 ng/mL vs. 319.2 ng/mL, respectively). CONCLUSIONS: There is evidence of dual pfhrp2/3 gene deletions in the Tigray and Amhara regions of Ethiopia in 2015. As the prevalence of malaria was very low (< 2%), it is difficult to make strong conclusions on RDT performance, but these results challenge the utility of biomarkers in household surveys in very low transmission settings.

Symptomatic Plasmodium vivax Infection in Rwanda.

We report a Plasmodium vivax infection in a Rwandan child misdiagnosed with Plasmodium falciparum and administered artemether-lumefantrine. Antigen detection revealed an absence of P falciparum histidine-rich protein 2 (HRP2) and presence of Plasmodium vivax lactate dehydrogenase. Nested and real-time polymerase chain reactions verified that the sample only contained P vivax deoxyribonucleic acid.

Plasmodium falciparum pfhrp2 and pfhrp3 Gene Deletions from Persons with Symptomatic Malaria Infection in Ethiopia, Kenya, Madagascar, and Rwanda.

Histidine-rich protein 2 (HRP2)-based rapid diagnostic tests detect Plasmodium falciparum malaria and are used throughout sub-Saharan Africa. However, deletions in the pfhrp2 and related pfhrp3 (pfhrp2/3) genes threaten use of these tests. Therapeutic efficacy studies (TESs) enroll persons with symptomatic P. falciparum infection. We screened TES samples collected during 2016-2018 in Ethiopia, Kenya, Rwanda, and Madagascar for HRP2/3, pan-Plasmodium lactate dehydrogenase, and pan-Plasmodium aldolase antigen levels and selected samples with low levels of HRP2/3 for pfhrp2/3 genotyping. We observed deletion of pfhrp3 in samples from all countries except Kenya. Single-gene deletions in pfhrp2 were observed in 1.4% (95% CI 0.2%-4.8%) of Ethiopia samples and in 0.6% (95% CI 0.2%-1.6%) of Madagascar samples, and dual pfhrp2/3 deletions were noted in 2.0% (95% CI 0.4%-5.9%) of Ethiopia samples. Although this study was not powered for precise prevalence estimates, evaluating TES samples revealed a low prevalence of pfhrp2/3 deletions in most sites.

Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions among patients in the DRC enrolled from 2017 to 2018.

Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (HRP2) and HRP3 are widely used throughout sub-Saharan Africa (SSA) to diagnose Plasmodium falciparum malaria. However, multiple SSA countries have reported pfhrp2 and pfhrp3 (pfhrp2/3) gene deletions. Blood samples (n = 1109) collected from patients with P. falciparum infection from six health facilities throughout the Democratic Republic of the Congo (DRC) from March 2017 to January 2018 were evaluated for pfhrp2/3 deletions. Samples were assayed for HRP2, pan-Plasmodium LDH (pLDH) and aldolase (pAldolase) antigens by bead-based multiplex antigen assay. Samples with low HRP2 concentration compared to pLDH and pAldolase antigens were selected for further pfhrp2/3 genotyping PCRs. The majority of blood samples (93.3%, 1035/1109) had high concentrations of the HRP2 antigen. Single deletions of pfhrp2 were identified in 0.27% (3/1109) of screened samples, with one sample from each of the Kapolowe, Mikalayi, and Rutshuru study sites. A pfhrp3 single deletion (0.09%, 1/1109) was found in the Kapolowe site. Dual pfhrp2 and pfhrp3 deletions were not observed. Due to, the low numbers of pfhrp2 deletions and the sporadic locations of these deletions, the use of HRP2-based RDTs appears to still be appropriate for these locations in DRC.

Missed Plasmodium falciparum and Plasmodium vivax Mixed Infections in Ethiopia Threaten Malaria Elimination.

Plasmodium falciparum and Plasmodium vivax are co-endemic in Ethiopia. This study investigated whether mixed infections were missed by microscopy from a 2017 therapeutic efficacy study at two health facilities in Ethiopia. All patients (N = 304) were initially classified as having single-species P. falciparum (n = 148 samples) or P. vivax infections (n = 156). Dried blood spots were tested for Plasmodium antigens by bead-based multiplex assay for pan-Plasmodium aldolase, pan-Plasmodium lactate dehydrogenase, P. vivax lactate dehydrogenase, and histidine-rich protein 2. Of 304 blood samples, 13 (4.3%) contained both P. falciparum and P. vivax antigens and were analyzed by polymerase chain reaction for species-specific DNA. Of these 13 samples, five were confirmed by polymerase chain reaction for P. falciparum/P. vivax co-infection. One sample, initially classified as P. vivax by microscopy, was found to only have Plasmodium ovale DNA. Plasmodium falciparum/P. vivax mixed infections can be missed by microscopy even in the context of a therapeutic efficacy study with multiple trained readers.

Purification of native histidine-rich protein 2 (nHRP2) from Plasmodium falciparum culture supernatant, infected RBCs, and parasite lysate.

BACKGROUND: Despite the widespread use of histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs), purified native HRP2 antigen is not standardly used in research applications or assessment of RDTs used in the field. METHODS: This report describes the purification of native HRP2 (nHRP2) from the HB3 Plasmodium falciparum culture strain. As this culture strain lacks pfhrp3 from its genome, it is an excellent source of HRP2 protein only and does not produce the closely-related HRP3. The nHRP2 protein was isolated from culture supernatant, infected red blood cells (iRBCs), and whole parasite lysate using nickel-metal chelate chromatography. Biochemical characterization of nHRP2 from HB3 culture was conducted by SDS-PAGE and western blotting, and nHRP2 was assayed by RDT, ELISA, and bead-based immunoassay. RESULTS: Purified nHRP2 was identified by SDS-PAGE and western blot as a - 60 kDa protein that bound anti-HRP-2 monoclonal antibodies. Mouse anti-HRP2 monoclonal antibody was found to produce high optical density readings between dilutions of 1:100 and 1:3,200 by ELISA with assay signal observed up to a 1:200,000 dilution. nHRP2 yield from HB3 culture by bead-based immunoassay revealed that both culture supernatant and iRBC lysate were practical sources of large quantities of this antigen, producing a total yield of 292.4 µg of nHRP2 from two pooled culture preparations. Assessment of nHRP2 recognition by RDTs revealed that Carestart Pf HRP2 and HRP2/pLDH RDTs detected purified nHRP2 when applied at concentrations between 20.6 and 2060 ng/mL, performing within a log-fold dilution of commercially-available recombinant HRP2. The band intensity observed for the nHRP2 dilutions was equivalent to that observed for P. falciparum culture strain dilutions of 3D7 and US06 F Nigeria XII between 12.5 and 1000 parasites/µL. CONCLUSIONS: Purified nHRP2 could be a valuable reagent for laboratory applications as well as assessment of new and existing RDTs prior to their use in clinical settings. These results establish that it is possible to extract microgram quantities of the native HRP2 antigen from HB3 culture and that this purified protein is well recognized by existing monoclonal antibody lines and RDTs.

Natural infections with different Plasmodium species induce antibodies reactive to a chimeric Plasmodium vivax recombinant protein.

BACKGROUND: As malaria incidence and transmission in a region decreases, it becomes increasingly difficult to identify areas of active transmission. Improved methods for identifying and monitoring foci of active malaria transmission are needed in areas of low parasite prevalence in order to achieve malaria elimination. Serological assays can provide population-level infection history to inform elimination campaigns. METHODS: A bead-based multiplex antibody detection assay was used to evaluate a chimeric Plasmodium vivax MSP1 protein (PvRMC-MSP1), designed to be broadly immunogenic for use in vaccine studies, to act as a pan-malaria serological tool based on its ability to capture IgG in plasma samples obtained from naturally exposed individuals. Samples from 236 US travellers with PCR confirmed infection status from all four major Plasmodium species infecting humans, Plasmodium falciparum (n = 181), Plasmodium vivax (n = 38), Plasmodium malariae (n = 4), and Plasmodium ovale (n = 13) were tested for IgG capture using PvRMC-MSP1 as well as the four recombinant MSP1-19 kD isoforms representative of these Plasmodium species. RESULTS: Regardless of infecting Plasmodium species, a large proportion of plasma samples from infected US travellers provided a high assay signal to the PvRMC-MSP1 chimeric protein, with 115 high responders out of 236 samples assessed (48.7%). When grouped by active infection, 38.7% P. falciparum-, 92.1% of P. vivax-, 75.0% P. malariae-, and 53.4% of P. ovale-infected individuals displayed high assay signals in response to PvRMC-MSP1. It was also determined that plasma from P. vivax-infected individuals produced increased assay signals in response to the PvRMC-MSP1 chimera as compared to the recombinant PvMSP1 for 89.5% (34 out of 38) of individuals. PvRMC-MSP1 also showed improved ability to capture IgG antibodies from P. falciparum-infected individuals when compared to the capture by recombinant PvMSP1, with high assay signals observed for 38.7% of P. falciparum-infected travellers in response to PvRMC-MSP1 IgG capture compared to just 1.1% who were high responders to capture by the recombinant PvMSP1 protein. CONCLUSIONS: These results support further study of designed antigens as an approach for increasing sensitivity or broadening binding capacity to improve existing serological tools for determining population-level exposure to Plasmodium species. Including both broad-reacting and Plasmodium species-specific antigen-coated beads in an assay panel could provide a nuanced view of population-level exposure histories, an extensive IgG profile, and detailed seroestimates. A more sensitive serological tool for detection of P. vivax exposure would aid malaria elimination campaigns in co-endemic areas and regions where P. vivax is the dominant parasite.

Nanopatterning protein antigens to refocus the immune response.

Vaccines for many important diseases remain elusive, and those for others need to be updated frequently. Vaccine efficacy has been hindered by existing sequence diversity in proteins and by newly-acquired mutations that enable escape from vaccine-induced immune responses. To address these limitations, we developed an approach for nanopatterning protein antigens that combines the site-specific incorporation of non-canonical amino acids with chemical modification to focus the immune response on conserved protein regions. We demonstrated the approach using green fluorescent protein (GFP) as a model antigen and with a promising malaria vaccine candidate, Merozoite surface protein 119 (MSP119). Immunization of mice with nanopatterned MSP119 elicited antibodies that recognized MSP119 from heterologous strains, differing in sequence at as many as 21 of 96 residues. Nanopatterning should enable the elicitation of broadly protective antibodies against a wide range of pathogens and toxins.

A Multi-Stage Plasmodium vivax Malaria Vaccine Candidate Able to Induce Long-Lived Antibody Responses Against Blood Stage Parasites and Robust Transmission-Blocking Activity.

Malaria control and interventions including long-lasting insecticide-treated nets, indoor residual spraying, and intermittent preventative treatment in pregnancy have resulted in a significant reduction in the number of Plasmodium falciparum cases. Considerable efforts have been devoted to P. falciparum vaccines development with much less to P. vivax. Transmission-blocking vaccines, which can elicit antibodies targeting Plasmodium antigens expressed during sexual stage development and interrupt transmission, offer an alternative strategy to achieve malaria control. The post-fertilization antigen P25 mediates several functions essential to ookinete survival but is poorly immunogenic in humans. Previous clinical trials targeting this antigen have suggested that conjugation to a carrier protein could improve the immunogenicity of P25. Here we report the production, and characterization of a vaccine candidate composed of a chimeric P. vivax Merozoite Surface Protein 1 (cPvMSP1) genetically fused to P. vivax P25 (Pvs25) designed to enhance CD4+ T cell responses and its assessment in a murine model. We demonstrate that antibodies elicited by immunization with this chimeric protein recognize both the erythrocytic and sexual stages and are able to block the transmission of P. vivax field isolates in direct membrane-feeding assays. These findings provide support for the continued development of multi-stage transmission blocking vaccines targeting the life-cycle stage responsible for clinical disease and the sexual-stage development accountable for disease transmission simultaneously.

Inclusion of the murine IgGκ signal peptide increases the cellular immunogenicity of a simian adenoviral vectored Plasmodium vivax multistage vaccine.

INTRODUCTION: Cellular and humoral immune responses are both involved in protection against Plasmodium infections. The only malaria vaccine available, RTS,S, primarily induces short-lived antibodies and targets only a pre-erythrocytic stage antigen. Inclusion of erythrocytic stage targets and enhancing cellular immunogenicity are likely necessary for developing an effective second-generation malaria vaccine. Adenovirus vectors have been used to improve the immunogenicity of protein-based vaccines. However, the clinical assessment of adenoviral-vectored malaria vaccines candidates has shown the induction of robust Plasmodium-specific CD8+ but not CD4+ T cells. Signal peptides (SP) have been used to enhance the immunogenicity of DNA vaccines, but have not been tested in viral vector vaccine platforms. OBJECTIVES: The objective of this study was to determine if the addition of the SP derived from the murine IgGκ light chain within a recombinant adenovirus vector encoding a multistage P. vivax vaccine candidate could improve the CD4+ T cell response. METHODS: In this proof-of-concept study, we immunized CB6F1/J mice with either the recombinant simian adenovirus 36 vector containing the SP (SP-SAd36) upstream from a transgene encoding a chimeric P. vivax multistage protein or the same SAd36 vector without the SP. Mice were subsequently boosted twice with the corresponding recombinant proteins emulsified in Montanide ISA 51 VG. Immunogenicity was assessed by measurement of antibody quantity and quality, and cytokine production by T cells after the final immunization. RESULTS: The SP-SAd36 immunization regimen induced significantly higher antibody avidity against the chimeric P. vivax proteins tested and higher frequencies of IFN-γ and IL-2 CD4+ and CD8+ secreting T cells, when compared to the unmodified SAd36 vector. CONCLUSIONS: The addition of the murine IgGκ signal peptide significantly enhances the immunogenicity of a SAd36 vectored P. vivax multi-stage vaccine candidate in mice. The potential of this approach to improve upon existing viral vector vaccine platforms warrants further investigation.

A prime-boost immunization regimen based on a simian adenovirus 36 vectored multi-stage malaria vaccine induces protective immunity in mice.

Malaria remains a considerable burden on public health. In 2015, the WHO estimates there were 212 million malaria cases causing nearly 429,000 deaths globally. A highly effective malaria vaccine is needed to reduce the burden of this disease. We have developed an experimental vaccine candidate (PyCMP) based on pre-erythrocytic (CSP) and erythrocytic (MSP1) stage antigens derived from the rodent malaria parasite P. yoelii. Our protein-based vaccine construct induces protective antibodies and CD4+ T cell responses. Based on evidence that viral vectors increase CD8+ T cell-mediated immunity, we also have tested heterologous prime-boost immunization regimens that included human adenovirus serotype 5 vector (Ad5), obtaining protective CD8+ T cell responses. While Ad5 is commonly used for vaccine studies, the high prevalence of pre-existing immunity to Ad5 severely compromises its utility. Here, we report the use of the novel simian adenovirus 36 (SAd36) as a candidate for a vectored malaria vaccine since this virus is not known to infect humans, and it is not neutralized by anti-Ad5 antibodies. Our study shows that the recombinant SAd36PyCMP can enhance specific CD8+ T cell response and elicit similar antibody titers when compared to an immunization regimen including the recombinant Ad5PyCMP. The robust immune responses induced by SAd36PyCMP are translated into a lower parasite load following P. yoelii infectious challenge when compared to mice immunized with Ad5PyCMP.