Merozoite Surface Protein Research Articles

Genetic diversity of plasmodium falciparum isolates in Minna, North Central Nigeria inferred by PCR genotyping of Merozoite surface protein 1 and 2.

North Central Nigeria is one region in Nigeria with a significant incidence of malaria caused majorly by Plasmodium falciparum. This study utilizes the msp1 and msp2 genes of P. falciparum to examine its diversity and multiplicity of infection (MOI). Blood samples were collected from 247 children across selected healthcare facilities in Minna, from infants and children aged 6months to 17years. Of the total collection, 143 (58%) of the children were infected with P. falciparum with parasite density≥1000μl, and from which fifty (50) samples was randomly selected and presented for PCR for the characterization of msp1 and msp2 gene using nested-PCR method. Overall, 57 msp1 genotypes, including K1, MAD20 and RO33 were identified, ranging from (250-1000bp), (100-500bp) and (400-500bp), respectively. In addition, 54 different msp2 genotypes of FC27 and 3D7 alleles ranging from (100-900bp) and (100-800bp), respectively were selected. A monoclonal infection of 39% and a polyclonal infection of 61% was recorded, however, a particularity about this study is the polyclonal nature of RO33. Determination of gene diversity revealed MAD20 as the predominant allele for msp1 with a mean MOI of 1.35 and FC27 for msp2 with 1.72 MOI. The overall MOI recorded for the study was 1.60. There was, however, no statistical significance difference between MOI and age of the child (P>0.05). Meanwhile, findings from this study revealed P. falciparum populations were not genetically diverse with Heterozygosity (He) index of 0.0636. However, a significant level gene diversity within the antigenic markers of msp1 and msp2 was observed with He index of 0.714 and 0.830, respectively. This study has demonstrated the potential of gene diversity and MOI of P. falciparum, as important markers for assessing differences in malaria transmission intensity. Continuous malaria genetic surveillance is therefore recommended as a fundamental tool for monitoring changes in gene types and for intervention programs' effectiveness.

Dynamic changes in genetic diversity, drug resistance mutations, and treatment outcomes of falciparum malaria from the low-transmission to the pre-elimination phase on the islands of S\xe3o Tom\xe9 and Pr\xedncipe

BackgroundWith effective vector control and case management, substantial progress has been made towards eliminating malaria on the islands of São Tomé and Príncipe (STP). This study assessed the dynamic changes in the genetic diversity of Plasmodium falciparum, the anti-malarial drug resistance mutations, and malaria treatment outcomes between 2010 and 2016 to provide insights for the prevention of malaria rebounding.MethodsPolymorphic regions of merozoite surface proteins 1 and 2 (msp1 and msp2) were sequenced in 118 dried blood spots (DBSs) collected from malaria patients who had visited the Central Hospital in 2010–2016. Mutations in the multi-drug resistance I (pfmdr1), chloroquine resistance transporter (pfcrt), and kelch 13 (pfk13) genes were analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) and sequencing in 111 DBSs. A total of 7482 cases that completed a 28-day follow-up were evaluated for treatment outcomes based on the microscopic results. Regression models were used to characterize factors associated with levels of parasite density and treatment failures.ResultsParasite strains in STP showed significant changes during and after the peak incidence in 2012. The prevalent allelic type in msp1 changed from K1 to MAD20, and that in msp2 changed from 3D7/IC to FC27. The dominant alleles of drug-resistance markers were pfmdr1 86Y, 184F, D1246, and pfcrt 76 T (Y-F-D-T, 51.4%). The average parasite density in malaria cases declined threefold from low-transmission (2010–2013) to pre-elimination period (2014–2016). Logistic regression models showed that patients with younger age (OR for age = 0.97–0.98, p < 0.001), higher initial parasite density (log10-transformed, OR = 1.44, p < 0.001), and receiving quinine treatment (compared to artemisinin-based combination therapy, OR = 1.91–1.96, p < 0.001) were more likely to experience treatment failures during follow-up.ConclusionsPlasmodium falciparum in STP had experienced changes in prevalent strains, and increased mutation frequencies in drug-resistance genes from the low-transmission to the pre-elimination settings. Notably, patients with younger age and receiving quinine treatment were more likely to show parasitological treatment failure during follow-up. Therapeutic efficacy should be carefully monitored to inform future treatment policy in STP.

Genetic diversity and immunogenicity of the merozoite surface protein 1 C-terminal 19-kDa fragment of Plasmodium ovale imported from Africa into China

BackgroundMerozoite surface protein 1 (MSP1) plays an essential role in erythrocyte invasion by malaria parasites. The C-terminal 19-kDa region of MSP1 has long been considered one of the major candidate antigens for a malaria blood-stage vaccine against Plasmodium falciparum. However, there is limited information on the C-terminal 19-kDa region of Plasmodium ovale MSP1 (PoMSP119). This study aims to analyze the genetic diversity and immunogenicity of PoMSP119.MethodsA total of 37 clinical Plasmodium ovale isolates including Plasmodium ovale curtisi and Plasmodium ovale wallikeri imported from Africa into China and collected during the period 2012–2016 were used. Genomic DNA was used to amplify P. ovale curtisi (poc) msp119 (pocmsp119) and P. ovale wallikeri (pow) msp119 (powmsp119) genes by polymerase chain reaction. The genetic diversity of pomsp119 was analyzed using the GeneDoc version 6 programs. Recombinant PoMSP119 (rPoMSP119)-glutathione S-transferase (GST) proteins were expressed in an Escherichia coli expression system and analyzed by western blot. Immune responses in BALB/c mice immunized with rPoMSP119-GST were determined using enzyme-linked immunosorbent assay. In addition, antigen-specific T cell responses were assessed by lymphocyte proliferation assays. A total of 49 serum samples from healthy individuals and individuals infected with P. ovale were used for the evaluation of natural immune responses by using protein microarrays.ResultsSequences of pomsp119 were found to be thoroughly conserved in all the clinical isolates. rPoMSP119 proteins were efficiently expressed and purified as ~ 37-kDa proteins. High antibody responses in mice immunized with rPoMSP119-GST were observed. rPoMSP119-GST induced high avidity indexes, with an average of 92.57% and 85.32% for rPocMSP119 and rPowMSP119, respectively. Cross-reactivity between rPocMSP119 and rPowMSP119 was observed. Cellular immune responses to rPocMSP119 (69.51%) and rPowMSP119 (52.17%) induced in rPocMSP119- and rPowMSP119-immunized mice were found in the splenocyte proliferation assays. The sensitivity and specificity of rPoMSP119-GST proteins for the detection of natural immune responses in patients infected with P. ovale were 89.96% and 75%, respectively.ConclusionsThis study revealed highly conserved gene sequences of pomsp119. In addition, naturally acquired humoral immune responses against rPoMSP1 were observed in P. ovale infections, and high immunogenicity of rPoMSP119 in mice was also identified. These instructive findings should encourage further testing of PoMSP119 for rational vaccine design.Graphical abstract

Genetic Diversity of Polymorphic Marker Merozoite Surface Protein 1 (Msp-1) and 2 (Msp-2) Genes of Plasmodium falciparum Isolates From Malaria Endemic Region of Pakistan.

Background: Understanding the genetic diversity of Plasmodium species through polymorphic studies can assist in designing more effective control strategies of malaria like new drug formulation and development of a vaccine. Pakistan is moderate endemic for Plasmodium falciparum, but little is known about the genetic diversity of this parasite. This study aimed to investigate the molecular diversity of P. falciparum based on msp-1 and msp-2 genes in the malaria-endemic regions of Khyber Pakhtunkhwa, Pakistan. Methods: A total of 199/723 blood samples, tested positive by microscopy for falciparum malaria, were collected from four districts (Dera Ismail Khan, Karak, Mardan, and Peshawar) of Khyber Pakhtunkhwa. Nested PCR amplification technique was employed to target block 2 of msp-1 and the central domain of msp-2 genes, including their respective allelic families K1, MAD20, RO33, FC27, and 3D7/IC, and to detect the extent of genetic diversity of P. falciparum clinical isolates. Results: Among the 199 microscopy-positive P. falciparum samples, a total of 192 were confirmed using PCR. Ninety-seven amplicons were observed for msp-1 and 95 for msp-2. A total of 33 genotypes, 17 for msp-1 (eight K1, six MAD20, and three RO33) and 16 for msp-2 (nine FC27 and seven 3D7/IC), were identified. The specific allelic frequency of the K1 family was higher (44.3%) than that of MAD20 (33.0%) and RO33 (23.0%) for msp-1, while the FC27 allelic family was dominant (60.0%) compared with 3D7/IC (40.0%) for msp-2. No polyclonal infection was observed in msp-1 and msp-2. The expected heterozygosity was 0.98 and 0.97 for msp-1 and msp-2, respectively. Conclusion: It was concluded that the P. falciparum populations are highly polymorphic, and diverse allelic variants of msp-1 and msp-2 are present in Khyber Pakhtunkhwa, Pakistan.

Improved Detectability of Plasmodium falciparum Clones with Repeated Sampling in Incident and Chronic Infections in Burkina Faso.

ABSTRACT.We evaluated the detectability of Plasmodium falciparum clones when assessed on 3 consecutive days in incident and chronic infections in naturally exposed children living in an area of intense malaria transmission in Burkina Faso. The median number of clones by merozoite surface protein 2 (MSP2) genotyping was 3 (interquartile range [IQR] 2–5) in incident infections compared with 6 (IQR 4–8) in chronic infections (P < 0.0001). When all clones detected on days 1-3 were considered as true complexity of infection, sampling on day 1 detected only 69.4% (109/157) or 68.3% (228/334) of all clones in incident and chronic infections, respectively. Our findings demonstrate that a large proportion of clones are missed by single time-point sampling. In addition, because of the high complexity of infection early in incident infections, our data suggest many infections may be caused by genetically complex inocula.

Detection of high frequency of MAD20 allelic variants of Plasmodium falciparum merozoite surface protein 1 gene from Adama and its surroundings, Oromia, Ethiopia

BackgroundOne of the major challenges in developing an effective vaccine against asexual stages of Plasmodium falciparum is genetic polymorphism within parasite population. Understanding the genetic polymorphism like block 2 region of merozoite surface protein-1 (msp-1) gene of P. falciparum enlighten mechanisms underlining disease pathology, identification of the parasite clone profile from the isolates, transmission intensity and potential deficiencies of the ongoing malaria control and elimination efforts in the locality. Detailed understanding of local genetic polymorphism is an input to pave the way for better management, control and elimination of malaria. The aim of this study was to detect the most frequent allelic variant of the msp-1 gene of P. falciparum clinical isolates from selected health facilities in Adama town and its surroundings, Oromia, Ethiopia.MethodsOne hundred thirty-nine clinical isolates were successfully amplified for msp-1 gene using specific primers. Nested PCR amplification was conducted targeting K1, MAD20, and R033 alleles followed by gel electrophoresis for fragment analysis. Based on the detection of a PCR fragment, infections were classified as monoclonal or multiple infections.Results19 different size polymorphism of msp-1 gene were identified in the study, with 67(48%) MAD20, 18 (13%) K-1 and 18 (13%) RO33 allelic family. Whereas, the multiple infections were 21(15%), 8 (5.8%), 4(2.9%), 3(2.2%) for MAD20 + K-1, MAD20 + RO33, K-1 + RO33, and MAD20 + K-1, RO33, respectively. The overall Multiplicity of infection (MOI) was 1.3 and the expected heterozygosity (He) was 0.39 indicating slightly low falciparum malaria transmission.ConclusionThe status of msp-1 allele size polymorphism, MOI and He observed in the study revealed the presence of slightly low genetic diversity of P. falciparum clinical isolates. However, highly frequent MAD20 allelic variant was detected from clinical isolates in the study area. Moreover, the driving force that led to high predominance of MAD20 allelic variant revealed in such malaria declining region demands further research.

Conjugation strategies on functionalized iron oxide nanoparticles as a malaria vaccine delivery system

Vaccination has been used effectively to protect from infectious diseases and non-infectious diseases such as cancer and allergies. Different forms of particulate arrangements, including nanoparticles, virus-like particles (VLPs), and virosomes, have been built recently depending on the type of pathogen to be targeted. The ability to conjugate the recombinant Plasmodium yoelii, 19-kDa C-terminal fragment of merozoite surface protein 1 (PyMSP119) on the surface of superparamagnetic magnetite nanoparticles (SPIONs) was explored as a new technique of enhancing vaccination against malaria. Different conjugation strategies were performed to correlate the effects of nanoparticle chemistry surfaces to bind later with the malaria protein. (SPIONs) were prepared by chemical coprecipitation method and coated with 3-aminopropyltriethoxysilane (APTS) alone (as a surface coater), or with both APTS and polyethylene glycol (PEG) (as a shield to protect the malaria protein from proteolytic enzymes) by using a modified silanisation method. X-ray powder diffraction (XRD, Philips Model) patterns indicated that the SPIONs were of high purity with an inverse spinal structure. Fourier Transform Infrared Spectroscopy (FTIR) was collected using PerkinElmer Spectrum 100 Series; spectra of uncoated and coated magnetite nanoparticles confirmed that the silane layer had been coated on the surface Fe3O4. The SPIONs were superparamagnetic as investigated by Vibrating Sample Magnetometry (VSM, Princeton Applied Research, model ISS) and relatively stable in aqueous phase at room temperature and could also be quickly recovered from suspension using an external magnet. Introduce the carboxyl groups onto the SPIONs surfaces, resulting in a relatively high protein binding capacity onto the nanoparticle surfaces. The bare particles had a mean size of around 20 nm with a relatively narrow size distribution. 82% of African Green Monkey fibroblast (COS-7) were alive in nanoparticle suspension using the MTT assay method. The quantity of protein explicitly bound to particles was determined using Sodium Dodecyl Sulfate (SDS) - Polyacrylamide Gel Electrophoresis (PAGE). SDS–PAGE. When the conjugation blend was prepared in EDC, there was approximately 100% binding between PyMSP119 and the Fe3O4-COOH particles because no protein band was apparent at the expected molecular weight for PyMSP119 (45 kDa). The current study investigates the theory that the gradual, persistent release of the malaria antigen may stimulate and maintain an elevated level of immune response for an extended period in vivo, which will be the scope of future work.

Guiding the Immune Response to a Conserved Epitope in MSP2, an Intrinsically Disordered Malaria Vaccine Candidate

The malaria vaccine candidate merozoite surface protein 2 (MSP2) has shown promise in clinical trials and is in part responsible for a reduction in parasite densities. However, strain-specific reductions in parasitaemia suggested that polymorphic regions of MSP2 are immuno-dominant. One strategy to bypass the hurdle of strain-specificity is to bias the immune response towards the conserved regions. Two mouse monoclonal antibodies, 4D11 and 9H4, recognise the conserved C-terminal region of MSP2. Although they bind overlapping epitopes, 4D11 reacts more strongly with native MSP2, suggesting that its epitope is more accessible on the parasite surface. In this study, a structure-based vaccine design approach was applied to the intrinsically disordered antigen, MSP2, using a crystal structure of 4D11 Fv in complex with its minimal binding epitope. Molecular dynamics simulations and surface plasmon resonance informed the design of a series of constrained peptides that mimicked the 4D11-bound epitope structure. These peptides were conjugated to keyhole limpet hemocyanin and used to immunise mice, with high to moderate antibody titres being generated in all groups. The specificities of antibody responses revealed that a single point mutation can focus the antibody response towards a more favourable epitope. This structure-based approach to peptide vaccine design may be useful not only for MSP2-based malaria vaccines, but also for other intrinsically disordered antigens.

Microfluidic device based on electrodeposited Nanoporous Gold/Carbon Nanotubes for Plasmodium vivax detection

A portable microfluidic electrochemical immunosensor for Plasmodium vivax antibodies determination was developed. A gold microelectrode placed inside the central channel of the microfluidic device was used as immobilization platform for a specific fragment (19-kDa) derived from the P. vivax merozoite surface protein 1, known as PvMSP119. The gold microelectrode surface was modified by using a dynamic hydrogen bubble template (DHBT) method in the presence of multiwalled carbon nanotubes. The synthesized nanocomposite presented exceptional properties, like high specific surface area, remarkable biocompatibility, and excellent electrochemical activity. The material was characterized by scanning electron microscopy, energy dispersive spectrometry, x-ray diffraction, and cyclic voltammetry. The antibodies (anti-PvMSP119) present in the sample bind to the PvMSP119 immobilized on the microelectrode surface, which is then labeled with an anti-IgG antibody marked with horseradish peroxidase (HRP-anti-IgG). Finally, the substrate solution (H2O2 + catechol) is added, and the enzymatic product (quinone) is reduced on the NPAu electrode at +0.2 V (vs. Ag/AgCl). The current obtained is directly proportional to the anti-PvMSP119 concentration in the sample. The detection limit of the microfluidic electrochemical immunosensor was 0.6 ng mL−1, much lower compared to the ELISA detection limit of 15 ng mL−1. This is the first microfluidic electrochemical immunosensor device suitable for point-of-care determination of anti-PvMSP119 in human serum samples.

Temporal Changes in the Genetic Diversity of Plasmodium vivax Merozoite Surface Protein-1 in Myanmar.

Despite a significant decline in the incidence of malaria in Myanmar recently, malaria is still an important public health concern in the country. Although Plasmodium falciparum is associated with the highest incidence of malaria in Myanmar, the proportion of P. vivax cases has shown a gradual increase in recent years. The genetic diversity of P. vivax merozoite surface protein-1 block 5-6 (pvmsp-1 ICB 5-6) in the P. vivax population of Myanmar was analyzed to obtain a comprehensive insight into its genetic heterogeneity and evolutionary history. High levels of genetic diversity of pvmsp-1 ICB 5-6 were identified in the P. vivax isolates collected from Myanmar between 2013 and 2015. Thirty-nine distinct haplotypes of pvmsp-1 ICB 5-6 (13 for Sal I type, 20 for recombinant type, and 6 for Belem type) were found at the amino acid level. Comparative analyses of the genetic diversity of pvmsp-1 ICB 5-6 sequences in the recent (2013–2015) and the past (2004) P. vivax populations in Myanmar revealed genetic expansion of the pvmsp-1 ICB 5-6 in recent years, albeit with a declined incidence. The recent increase in the genetic heterogeneity of Myanmar pvmsp-1 ICB 5-6 is attributed to a combination of factors, including accumulated mutations and recombination. These results suggest that the size of the P. vivax population in Myanmar is sufficient to enable the generation and maintenance of genetic diversity, warranting continuous molecular surveillance of genetic variation in Myanmar P. vivax.

Application of 23 Novel Serological Markers for Identifying Recent Exposure to Plasmodium vivax Parasites in an Endemic Population of Western Thailand.

Thailand is aiming for malaria elimination by the year 2030. However, the high proportion of asymptomatic infections and the presence of the hidden hypnozoite stage of Plasmodium vivax are impeding these efforts. We hypothesized that a validated surveillance tool utilizing serological markers of recent exposure to P. vivax infection could help to identify areas of ongoing transmission. The objective of this exploratory study was to assess the ability of P. vivax serological exposure markers to detect residual transmission “hot-spots” in Western Thailand. Total IgG levels were measured against a panel of 23 candidate P. vivax serological exposure markers using a multiplexed bead-based assay. A total of 4,255 plasma samples from a cross-sectional survey conducted in 2012 of endemic areas in the Kanchanaburi and Ratchaburi provinces were assayed. We compared IgG levels with multiple epidemiological factors that are associated with an increased risk of P. vivax infection in Thailand, including age, gender, and spatial location, as well as Plasmodium infection status itself. IgG levels to all proteins were significantly higher in the presence of a P. vivax infection (n = 144) (T-test, p < 0.0001). Overall seropositivity rates varied from 2.5% (PVX_097625, merozoite surface protein 8) to 16.8% (PVX_082670, merozoite surface protein 7), with 43% of individuals seropositive to at least 1 protein. Higher IgG levels were associated with older age (>18 years, p < 0.05) and males (17/23 proteins, p < 0.05), supporting the paradigm that men have a higher risk of infection than females in this setting. We used a Random Forests algorithm to predict which individuals had exposure to P. vivax parasites in the last 9-months, based on their IgG antibody levels to a panel of eight previously validated P. vivax proteins. Spatial clustering was observed at the village and regional level, with a moderate correlation between PCR prevalence and sero-prevalence as predicted by the algorithm. Our data provides proof-of-concept for application of such surrogate markers as evidence of recent exposure in low transmission areas. These data can be used to better identify geographical areas with asymptomatic infection burdens that can be targeted in elimination campaigns.

Low MSP-1 haplotype diversity in the West Palearctic population of the avian malaria parasite Plasmodium relictum

BackgroundAlthough avian Plasmodium species are widespread and common across the globe, limited data exist on how genetically variable their populations are. Here, the hypothesis that the avian blood parasite Plasmodium relictum exhibits very low genetic diversity in its Western Palearctic transmission area (from Morocco to Sweden in the north and Transcaucasia in the east) was tested.MethodsThe genetic diversity of Plasmodium relictum was investigated by sequencing a portion (block 14) of the fast-evolving merozoite surface protein 1 (MSP1) gene in 75 different P. relictum infections from 36 host species. Furthermore, the full-length MSP1 sequences representing the common block 14 allele was sequenced in order to investigate if additional variation could be found outside block 14.ResultsThe majority (72 of 75) of the sequenced infections shared the same MSP1 allele. This common allele has previously been found to be the dominant allele transmitted in Europe.ConclusionThe results corroborate earlier findings derived from a limited dataset that the globally transmitted malaria parasite P. relictum exhibits very low genetic diversity in its Western Palearctic transmission area. This is likely the result of a recent introduction event or a selective sweep.

Structure of the merozoite surface protein 1 from Plasmodium falciparum.

The merozoite surface protein 1 (MSP-1) is the most abundant protein on the surface of the erythrocyte-invading Plasmodium merozoite, the causative agent of malaria. MSP-1 is essential for merozoite formation, entry into and escape from erythrocytes, and is a promising vaccine candidate. Here, we present monomeric and dimeric structures of full-length MSP-1. MSP-1 adopts an unusual fold with a large central cavity. Its fold includes several coiled-coils and shows structural homology to proteins associated with membrane and cytoskeleton interactions. MSP-1 formed dimers through these domains in a concentration-dependent manner. Dimerization is affected by the presence of the erythrocyte cytoskeleton protein spectrin, which may compete for the dimerization interface. Our work provides structural insights into the possible mode of interaction of MSP-1 with erythrocytes and establishes a framework for future investigations into the role of MSP-1 in Plasmodium infection and immunity.

Diversity and natural selection of Merozoite surface Protein-1 in three species of human malaria parasites: Contribution from South-East Asian isolates

The present study aimed to examine the genetic diversity of human malaria parasites (i.e., P. falciparum, P. vivax and P. knowlesi) in Malaysia and southern Thailand targeting the 19-kDa C-terminal region of Merozoite Surface Protein-1 (MSP-119). This region is essential for the recognition and invasion of erythrocytes and it is considered one of the leading candidates for asexual blood stage vaccines. However, the genetic data of MSP-119 among human malaria parasites in Malaysia is limited and there is also a need to update the current sequence diversity of this gene region among the Thailand isolates. In this study, genomic DNA was extracted from 384 microscopy-positive blood samples collected from patients who attended the hospitals or clinics in Malaysia and malaria clinics in Thailand from the year 2008 to 2016. The MSP-119 was amplified using PCR followed by bidirectional sequencing. DNA sequences identified in the present study were subjected to Median-joining network analysis with sequences of MSP-119 obtained from GenBank. DNA sequence analysis revealed that PfMSP-119 of Malaysian and Thailand isolates was not genetically conserved as high number of haplotypes were detected and positive selection was prevalent in PfMSP-119, hence questioning its suitability to be used as a vaccine candidate. A novel haplotype (Q/TNG/L) was also detected in Thailand P. falciparum isolate. In contrast, PvMSP-119 was highly conserved, however for the first time, a non-synonymous substitution (A1657S) was reported among Malaysian isolates. As for PkMSP-119, the presence of purifying selection and low nucleotide diversity indicated that it might be a potential vaccine target for P. knowlesi.

Plasmodium vivax vaccine candidate MSP1 displays conserved B-cell epitope despite high genetic diversity

The polymorphic nature of merozoite surface protein 1(MSP1) raises doubts whether it may serve as a vaccine target against Plasmodium vivax malaria. This study analyses the impact of genetic variability on the epitope organization of different Pvmsp1 blocks. Ten blood samples collected from P. vivax infected malaria patients from West Bengal, India were used to analyze sequence and antigenic diversities of block 2 region of Pvmsp1. An additional 48 block 2 sequences from other countries were also analyzed. Global genetic framework of Pvmsp1 block 2 was represented by 12 indel clusters & 33 haplotypes (haplotype diversiy=0.965±0.024). Parasite sequences pertaining to other Pvmsp1 modules, namely block 6 and 10 displayed 14 & 29 (haplotype diversiy=0.975±0.003) and 22 & 30 indel clusters and haplotypes (haplotype diversiy=0.947±0.004), respectively. In spite of this remarkable genetic diversity, a small number of conserved epitopes were detected in all three PvMSP1 blocks. This novel finding substantiates that MSP1 could serve as a promising vaccine candidate against vivax malaria.

Development of a real-time PCR method for rapid diagnosis of canine babesiosis and anaplasmosis

BackgroundCanine babesiosis and anaplasmosis, caused by Babesia canis and Anaplasma phagocytophilum, respectively, are significant tick-borne diseases in Baltic countries. Both diseases can be diagnosed on the basis of clinicopathological findings, by direct pathogen detection in blood smears or by indirect pathogen detection; however, because of high selectivity and specificity, molecular methods may be advantageous. The goal of this study was to develop a duplex real-time polymerase chain reaction (RT-PCR) method for the detection of B. canis and A. phagocytophilum in canine clinical samples.MethodsSequence-based polymorphism analysis of genes encoding B. canis-specific merozoite surface protein Bc28.1 (Bc28.1) and A. phagocytophilum malate dehydrogenase (mdh) was performed on pathogen isolates present in Latvian domestic dogs. The obtained results were used to design a species-specific duplex RT-PCR assay.ResultsThe presence of three B. canis Bc28.1 gene sequence types was revealed in canine samples with a nonuniform geographical distribution, and two types of A. phagocytophilum mdh genes were detected. The novel duplex RT-PCR assay provided correct classification of samples positive and negative for B. canis and A. phagocytophilum. The analytical sensitivity of this assay was ten gene copies/ reaction for both pathogens.ConclusionsA novel duplex RT-PCR molecular method was developed for the detection of B. canis and A. phagocytophilum in canine clinical samples. Sequence variability of Bc28.1 and mdh genes indicated the genetic variability of B. canis and A. phagocytophilum isolates occurring in Latvian domestic dogs.Graphical

Plasmodium falciparum-specific B cell responses in malaria-susceptible children and malaria-immune adults

Abstract Malaria is a vector-borne disease caused by parasites of the Plasmodium genus that infect millions of people annually, with P. falciparum being the most common and deadliest species. Development of immunological protection against clinical malaria requires chronic or repetitive parasite exposure, is mediated by IgG, and is correlated with antibodies against proteins expressed by merozoites, the free form of the parasite that invades erythrocytes. The goal of our study was to better understand the development of this protective antibody response, using merozoite surface protein 1 (MSP1) as a model antigen. MSP1 is highly abundant on the merozoite surface, highly immunogenic, and highly variable between parasite strains. Using B cell tetramers, we isolated MSP1-specific IgM+ and IgG+ memory B cells (MBCs) from immune adults and non-immune children living in Tororo, Uganda, a region of high transmission. In children, MSP1-specific B cells were mainly unswitched (IgM+) classical MBCs (cMBCs; CD21+ CD27+), while adults showed enrichment for class-switched (IgG+) cMBCs. Sequence analysis of the heavy chain variable regions revealed that MSP1-specific MBCs from adults carried higher rates of somatic hypermutation and showed extensive clonal expansion compared to those from children. When expressed as IgG, antibodies from IgM+ MBCs showed low avidity to MSP1, while those from IgG+ MBCs displayed strong reactivity with recombinant MSP1 and whole merozoites and inhibited parasite growth. These results suggest that extensive evolution of B cell responses takes place as a result of life-long P. falciparum exposure, which may be essential for the development of protection against malaria.

Inclusion of an Optimized Plasmodium falciparum Merozoite Surface Protein 2-Based Antigen in a Trivalent, Multistage Malaria Vaccine.

Plasmodium falciparum merozoite surface protein (PfMSP)2 is a target of parasite-neutralizing Abs. Inclusion of recombinant PfMSP2 (rPfMSP2) as a component of a multivalent malaria vaccine is of interest, but presents challenges. Previously, we used the highly immunogenic PfMSP8 as a carrier to enhance production and/or immunogenicity of malaria vaccine targets. In this study, we exploited the benefits of rPfMSP8 as a carrier to optimize a rPfMSP2-based subunit vaccine. rPfMSP2 and chimeric rPfMSP2/8 vaccines produced in Escherichia coli were evaluated in comparative immunogenicity studies in inbred (CB6F1/J) and outbred (CD1) mice, varying the dose and adjuvant. Immunization of mice with both rPfMSP2-based vaccines elicited high-titer anti-PfMSP2 Abs that recognized the major allelic variants of PfMSP2. Vaccine-induced T cells recognized epitopes present in both PfMSP2 and the PfMSP8 carrier. Competition assays revealed differences in Ab specificities induced by the two rPfMSP2-based vaccines, with evidence of epitope masking by rPfMSP2-associated fibrils. In contrast to aluminum hydroxide (Alum) as adjuvant, formulation of rPfMSP2 vaccines with glucopyranosyl lipid adjuvant-stable emulsion, a synthetic TLR4 agonist, elicited Th1-associated cytokines, shifting production of Abs to cytophilic IgG subclasses. The rPfMSP2/8 + glucopyranosyl lipid adjuvant-stable emulsion formulation induced significantly higher Ab titers with superior durability and capacity to opsonize P. falciparum merozoites for phagocytosis. Immunization with a trivalent vaccine including PfMSP2/8, PfMSP1/8, and the P. falciparum 25 kDa sexual stage antigen fused to PfMSP8 (Pfs25/8) induced high levels of Abs specific for epitopes in each targeted domain, with no evidence of antigenic competition. These results are highly encouraging for the addition of rPfMSP2/8 as a component of an efficacious, multivalent, multistage malaria vaccine.

Allelic diversity of merozoite surface protein genes (msp1 and msp2) and clinical manifestations of Plasmodium falciparum malaria cases in Aceh, Indonesia

BackgroundThe malaria control programme in Indonesia has successfully brought down malaria incidence in many parts in Indonesia, including Aceh Province. Clinical manifestation of reported malaria cases in Aceh varied widely from asymptomatic, mild uncomplicated to severe and fatal complications. The present study aims to explore the allelic diversity of merozoite surface protein 1 gene (msp1) and msp2 among the Plasmodium falciparum isolates in Aceh Province and to determine their potential correlation with the severity of malaria clinical manifestation.MethodsScreening of over 500 malaria cases admitted to the hospitals in 11 districts hospital within Aceh Province during 2013–2015, identified 90 cases of P. falciparum mono-infection without any co-morbidity. The subjects were clinically phenotyped and parasite DNA was extracted and polymerase chain reaction (PCR) amplified for the msp1 and msp2 allelic subfamilies.ResultsAnalysis of clinical manifestation revealed that fever-chill is the most frequent symptom. Based on WHO criteria showed 19 cases were classified as severe and 71 as mild malaria. Analysis of msp1 gene revealed the presence of K1 allele subfamily in 34 subjects, MAD20 in 42 subjects, RO33 in 1 subject, and mixed allelic of K1 + MAD20 in 5 subjects, K1 + RO33 in 4 subjects, and MAD20 + RO33 in 4 subjects. Analysis of msp2 gene revealed 34 subjects carried the FC27 allelic subfamily, 37 subjects carried the 3D7 and 19 subjects carried the mixed FC27 + 3D7. Analysis of multiplicity of infection revealed that msp1 alleles is slightly higher than msp2 with the mean of MOI were 2.69 and 2.27, respectively. Statistical analysis to determine the association between each clinical manifestation and msp1 and msp2 alleles revealed that liver function abnormal value was associated with the msp2 mixed alleles (odds ratio (OR):0.13; 95%CI: 0.03–0.53). Mixed msp1 of K1 + RO33 was associated with severe malaria (OR: 28.50; 95%CI: 1.59–1532.30).ConclusionThis study found a strong association between severe malaria in Aceh with subjects carrying the msp1 mixed alleles of K1 and RO33. The liver function abnormal value associated with the msp2 mixed allelic subfamilies. Further study in different geographic areas is recommended.

Effect of hookworm infection and anthelmintic treatment on naturally acquired antibody responses against the GMZ2 malaria vaccine candidate and constituent antigens

BackgroundMalaria and helminths diseases are co-endemic in most parts of sub-Saharan Africa. Immune responses from each of these pathogens interact, and these interactions may have implications on vaccines. The GMZ2 malaria vaccine candidate is a fusion protein of Plasmodium falciparum merozoite surface protein 3 (MSP3) and glutamate rich protein (GLURP R0). GMZ2 has recently showed modest efficacy in a phase IIb multicenter trial. Here, we assessed the effect of hookworm (Necator americanus) infection and anthelmintic treatment on naturally acquired antibody responses against GMZ2 and constituent antigens.MethodsThis longitudinal cross-sectional study was conducted in the Kintampo North Municipality of Ghana. Blood and stool samples were taken from 158 individuals (4–88 years old) infected with either P. falciparum alone (n = 59) or both hookworm and P. falciparum (n = 63) and uninfected endemic controls (n = 36). Stool hookworm infection was detected by the Kato-Katz method and PCR. Malaria parasitaemia was detected by RDT, light microscopy and P. falciparum-specific 18S rRNA gene PCR. Serum samples were obtained prior to hookworm treatment with a single dose of albendazole (400 mg) and 3 weeks (21 days) after treatment. Levels of IgG1, IgG3 and IgM against GMZ2, MSP3 and GLURP R0 were measured by ELISA and compared among the groups, before and after treatment.ResultsParticipants with P. falciparum and hookworm co-infection had significantly higher IgG3 levels to GMZ2 than those with only P. falciparum infection and negative control (p < 0.05) at baseline. Treatment with albendazole led to a significant reduction in IgG3 levels against both GMZ2 and GLURP R0. Similarly, IgM and IgG1 levels against MSP3 also decreased following deworming treatment.ConclusionIndividuals with co-infection had higher antibody responses to GMZ2 antigen. Treatment of hookworm/malaria co-infection resulted in a reduction in antibody responses against GMZ2 and constituent antigens after albendazole treatment. Thus, hookworm infection and treatment could have a potential implication on malaria vaccine efficacy.