Plasmodium chabaudi Merozoites Obtained through a Simpler Method Do Not Survive in Classically Activated Macrophages.

Malaria is caused by apicomplexan parasites of the Plasmodium genus. Plasmodium chabaudi is an excellent animal model for the study of human malaria caused by P. falciparum. Merozoites invade erythrocytes but are also found in other host cells including macrophages from the spleen and liver. Methodologies for obtaining merozoites usually involve treatment with protease inhibitors. However, merozoites obtained in this way may have their enzymatic profile altered and, therefore, are not ideal for cell-interaction assays. We report the obtainment of P. chabaudi merozoites naturally egressed from a synchronous erythrocyte population infected with schizonts forms. Merozoites had their infectivity and ultrastructure analyzed. Interaction assays were performed with mice erythrocytes and classically activated mice peritoneal macrophages, a very well-established classic model. Obtained merozoites were able to kill mice and efficiently infect erythrocytes. Interestingly, a lower merozoite:erythrocyte ratio resulted in a higher percentage of infected erythrocytes. We describe a simpler method for obtaining viable and infective merozoites. Classically activated macrophages killed merozoites, suggesting that these host cells may not serve as reservoirs for these parasites. These findings have implications for our understanding of P. chabaudi merozoite biology and may improve the comprehension of their host-parasite relationship.

Genetic diversity and natural selection of Plasmodium vivax reticulocyte invasion genes in Ecuador.

BACKGROUND: Knowledge of the diversity of invasion ligands in malaria parasites in endemic regions is essential to understand how natural selection influences genetic diversity of these ligands and their feasibility as possible targets for future vaccine development. In this study the diversity of four genes for merozoite invasion ligands was studied in Ecuadorian isolates of Plasmodium vivax. METHODS: Eighty-eight samples from P. vivax infected individuals from the Coast and Amazon region of Ecuador were obtained between 2012 and 2015. The merozoite invasion genes pvmsp-1-19, pvdbpII, pvrbp1a-2 and pvama1 were amplified, sequenced, and compared to the Sal-1 strain. Polymorphisms were mapped and genetic relationships between haplotypes were determined. RESULTS: Only one nonsynonymous polymorphism was detected in pvmsp-1-19, while 44 nonsynonymous polymorphisms were detected in pvdbpII, 56 in pvrbp1a-2 and 33 in pvama1. While haplotypes appeared to be more related within each area of study and there was less relationship between parasites of the coastal and Amazon regions of the country, diversification processes were observed in the two Amazon regions. The highest haplotypic diversity for most genes occurred in the East Amazon of the country. The high diversity observed in Ecuadorian samples is closer to Brazilian and Venezuelan isolates, but lower than reported in other endemic regions. In addition, departure from neutrality was observed in Ecuadorian pvama1. Polymorphisms for pvdbpII and pvama1 were associated to B-cell epitopes. CONCLUSIONS: pvdbpII and pvama1 genetic diversity found in Ecuadorian P. vivax was very similar to that encountered in other malaria endemic countries with varying transmission levels and segregated by geographic region. The highest diversity of P. vivax invasion genes in Ecuador was found in the Amazonian region. Although selection appeared to have small effect on pvdbpII and pvrbp1a-2, pvama1 was influenced by significant balancing selection.

Plasmodium vivax MSP1-42 kD Variant Proteins Detected Naturally Induced IgG Antibodies in Patients Regardless of the Infecting Parasite Phenotype in Mesoamerica.

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.

The Cellular and Molecular Interaction Between Erythrocytes and Plasmodium falciparum Merozoites.

Plasmodium falciparum is the most lethal human malaria parasite, partly due to its genetic variability and ability to use multiple invasion routes via its binding to host cell surface receptors. The parasite extensively modifies infected red blood cell architecture to promote its survival which leads to increased cell membrane rigidity, adhesiveness and permeability. Merozoites are initially released from infected hepatocytes and efficiently enter red blood cells in a well-orchestrated process that involves specific interactions between parasite ligands and erythrocyte receptors; symptoms of the disease occur during the life-cycle's blood stage due to capillary blockage and massive erythrocyte lysis. Several studies have focused on elucidating molecular merozoite/erythrocyte interactions and host cell modifications; however, further in-depth analysis is required for understanding the parasite's biology and thus provide the fundamental tools for developing prophylactic or therapeutic alternatives to mitigate or eliminate Plasmodium falciparum-related malaria. This review focuses on the cellular and molecular events during Plasmodium falciparum merozoite invasion of red blood cells and the alterations that occur in an erythrocyte once it has become infected.

Spatiotemporal Changes in Plasmodium vivax msp142 Haplotypes in Southern Mexico: From the Control to the Pre-Elimination Phase.

For 20 years, Plasmodium vivax has been the only prevalent malaria species in Mexico, and cases have declined significantly and continuously. Spatiotemporal genetic studies can be helpful for understanding parasite dynamics and developing strategies to weaken malaria transmission, thus facilitating the elimination of the parasite. The aim of the current contribution was to analyze P. vivax-infected blood samples from patients in southern Mexico during the control (1993-2007) and pre-elimination phases (2008-2011). Nucleotide and haplotype changes in the pvmsp142 fragment were evaluated over time. The majority of multiple genotype infections occurred in the 1990s, when the 198 single nucleotide sequences exhibited 57 segregating sites, 64 mutations, and 17 haplotypes. Nucleotide and genetic diversity parameters showed subtle fluctuations from across time, in contrast to the reduced haplotype diversity and the increase in the R2 index and Tajima's D value from 2008 to 2011. The haplotype network consisted of four haplogroups, the geographical distribution of which varied slightly over time. Haplogroup-specific B-cell epitopes were predicted. Since only high-frequency and divergent haplotypes persisted, there was a contraction of the parasite population. Given that 84% of haplotypes were exclusive to Mesoamerica, P. vivax flow is likely circumscribed to this region, representing important information for parasite surveillance.

PCR-RFLP Based genetic diversity of Plasmodium vivax genotypes in district Mardan, Pakistan / Diversidade genética baseada em PCR RFLP de genótipos de Plasmodium vivax no distrito de Mardan, Paquistão

Plasmodium vivax is the most common human malaria parasite in Asian countries including Pakistan. Present study was designed to explore the genetic diversity of plasmodium vivax genotypes based on Pvmsp-3α and Pvmsp-3βgenes using allelic specific nested PCR and RFLP assays markers from field isolates in district Mardan, Pakistan. Blood samples of 200 P. vivax malarial patients were collected after taking their written informed consent. Genetic diversity in nested PCR products was determined by Restriction Fragment Length Polymorphism (RFLP) utilizing Alu1 and PstI restriction enzymes for alpha and beta gene products digestion, respectively. For analysis the genetic diversity of the sub allelic variants of Pvmsp3α and Pvmsp3β genes, Chi-Square test was performed by utilizing Minitab programming software 18. The P value 0.05 was considered as statistically significant. For Pvmsp 3α genes after gel electrophoresis of digested products, four distinct genotypes were obtained from total of 50 samples; type A: 35 (70%) (1.5-2.0 kb), 12 of type B (24%) (1.5-1.7 kb), 2 of type C (4%) (0.5-1.5) and one for type D (2%) (0.5-0.65 kb) which could be characterized into 9 allelic pattern (A1-A4, B1-B3, C1, D), in which A3 remained the most predominant. For Pvmsp-3βgenes, three distinct genotypes were obtained from 50 samples; 40(80%) of type A (1.5-2.5 kb), 9 (18%) of type B (1.0-1.5kb) and 1(2%) of type C (0.65 kb) which could be characterized into 6 allelic patterns (A1-A3, B1-B2, and C1). Most dominant one in Type A was A1 alleles which were noted (46%), while in Type B, the most dominant were B1 (10%).This study is the first ever report of molecular epidemiology and genetic variation in Pvmsp-3α and Pvmsp-3β genes of P. vivax isolates by using PCR/RFLP from District Mardan and [...].(AU)

O Plasmodium vivax é o parasita da malária humana mais comum nos países asiáticos, incluindo o Paquistão. O presente estudo foi desenhado para explorar a diversidade genética de genótipos de Plasmodium vivax baseados nos genes Pvmsp-3α e Pvmsp-3β, usando marcadores de ensaios alélicos nested PCR e RFLP de isolados de campo no distrito de Mardan, Paquistão. Amostras de sangue de 200 pacientes com malária por P. vivax foram coletadas após assinatura do termo de consentimento livre e esclarecido. A diversidade genética em produtos de PCR nested foi determinada por polimorfismo de fragmento de restrição (RFLP) utilizando as enzimas de restrição Alu1 e PstI para a digestão dos produtos dos genes alfa e beta, respectivamente. Para análise da diversidade genética das variantes subalélicas dos genes Pvmsp3α e Pvmsp3β, o teste Qui-quadrado foi realizado utilizando o software de programação Minitab 18. O valor P = 0,05 foi considerado estatisticamente significativo. Para os genes Pvmsp 3α, após eletroforese em gel de produtos digeridos, quatro genótipos distintos foram obtidos de um total de 50 amostras; tipo A: 35 (70%) (1,5-2,0 kb), 12 do tipo B (24%) (1,5-1,7 kb), 2 do tipo C (4%) (0,5-1,5) e um para o tipo D (2%) (0,5-0,65 kb), que podem ser caracterizados em nove padrões alélicos (A1-A4, B1-B3, C1, D), em que A3 permaneceu como o mais predominante. Para Pvmsp-3βgenes, três genótipos distintos foram obtidos a partir de 50 amostras; 40 (80%) do tipo A (1,5-2,5 kb), 9 (18%) do tipo B (1,0-1,5 kb) e 1 (2%) do tipo C (0,65 kb), que podem ser caracterizados em seis padrões alélicos (A1-A3, B1-B2 e C1). Os mais dominantes no tipo A foram o alelo A1, observados em 46%, enquanto, no tipo B, os mais dominantes foram B1 (10%). Este estudo é o primeiro relato de epidemiologia molecular e variação genética em Pvmsp-3α. Os genes Pvmsp-3β de isolados de P. vivax utilizando PCR/RFLP do Distrito Mardan mostraram um nível notável de diversidade genética nos genes estudados [...].(AU)

PCR-RFLP Based genetic diversity of Plasmodium vivax genotypes in district Mardan, Pakistan / Diversidade genética baseada em PCR RFLP de genótipos de Plasmodium vivax no distrito de Mardan, Paquistão

Plasmodium vivax is the most common human malaria parasite in Asian countries including Pakistan. Present study was designed to explore the genetic diversity of plasmodium vivax genotypes based on Pvmsp-3α and Pvmsp-3βgenes using allelic specific nested PCR and RFLP assays markers from field isolates in district Mardan, Pakistan. Blood samples of 200 P. vivax malarial patients were collected after taking their written informed consent. Genetic diversity in nested PCR products was determined by Restriction Fragment Length Polymorphism (RFLP) utilizing Alu1 and PstI restriction enzymes for alpha and beta gene products digestion, respectively. For analysis the genetic diversity of the sub allelic variants of Pvmsp3α and Pvmsp3β genes, Chi-Square test was performed by utilizing Minitab programming software 18. The P value 0.05 was considered as statistically significant. For Pvmsp 3α genes after gel electrophoresis of digested products, four distinct genotypes were obtained from total of 50 samples; type A: 35 (70%) (1.5-2.0 kb), 12 of type B (24%) (1.5-1.7 kb), 2 of type C (4%) (0.5-1.5) and one for type D (2%) (0.5-0.65 kb) which could be characterized into 9 allelic pattern (A1-A4, B1-B3, C1, D), in which A3 remained the most predominant. For Pvmsp-3βgenes, three distinct genotypes were obtained from 50 samples; 40(80%) of type A (1.5-2.5 kb), 9 (18%) of type B (1.0-1.5kb) and 1(2%) of type C (0.65 kb) which could be characterized into 6 allelic patterns (A1-A3, B1-B2, and C1). Most dominant one in Type A was A1 alleles which were noted (46%), while in Type B, the most dominant were B1 (10%).This study is the first ever report of molecular epidemiology and genetic variation in Pvmsp-3α and Pvmsp-3β genes of P. vivax isolates by using PCR/RFLP from District Mardan and [...].

O Plasmodium vivax é o parasita da malária humana mais comum nos países asiáticos, incluindo o Paquistão. O presente estudo foi desenhado para explorar a diversidade genética de genótipos de Plasmodium vivax baseados nos genes Pvmsp-3α e Pvmsp-3β, usando marcadores de ensaios alélicos nested PCR e RFLP de isolados de campo no distrito de Mardan, Paquistão. Amostras de sangue de 200 pacientes com malária por P. vivax foram coletadas após assinatura do termo de consentimento livre e esclarecido. A diversidade genética em produtos de PCR nested foi determinada por polimorfismo de fragmento de restrição (RFLP) utilizando as enzimas de restrição Alu1 e PstI para a digestão dos produtos dos genes alfa e beta, respectivamente. Para análise da diversidade genética das variantes subalélicas dos genes Pvmsp3α e Pvmsp3β, o teste Qui-quadrado foi realizado utilizando o software de programação Minitab 18. O valor P = 0,05 foi considerado estatisticamente significativo. Para os genes Pvmsp 3α, após eletroforese em gel de produtos digeridos, quatro genótipos distintos foram obtidos de um total de 50 amostras; tipo A: 35 (70%) (1,5-2,0 kb), 12 do tipo B (24%) (1,5-1,7 kb), 2 do tipo C (4%) (0,5-1,5) e um para o tipo D (2%) (0,5-0,65 kb), que podem ser caracterizados em nove padrões alélicos (A1-A4, B1-B3, C1, D), em que A3 permaneceu como o mais predominante. Para Pvmsp-3βgenes, três genótipos distintos foram obtidos a partir de 50 amostras; 40 (80%) do tipo A (1,5-2,5 kb), 9 (18%) do tipo B (1,0-1,5 kb) e 1 (2%) do tipo C (0,65 kb), que podem ser caracterizados em seis padrões alélicos (A1-A3, B1-B2 e C1). Os mais dominantes no tipo A foram o alelo A1, observados em 46%, enquanto, no tipo B, os mais dominantes foram B1 (10%). Este estudo é o primeiro relato de epidemiologia molecular e variação genética em Pvmsp-3α. Os genes Pvmsp-3β de isolados de P. vivax utilizando PCR/RFLP do Distrito Mardan mostraram um nível notável de diversidade genética nos genes estudados [...].

PCR-RFLP Based genetic diversity of Plasmodium vivax genotypes in district Mardan, Pakistan

Abstract Plasmodium vivax is the most common human malaria parasite in Asian countries including Pakistan. Present study was designed to explore the genetic diversity of plasmodium vivax genotypes based on Pvmsp-3 and Pvmsp-3genes using allelic specific nested PCR and RFLP assays markers from field isolates in district Mardan, Pakistan. Blood samples of 200 P. vivax malarial patients were collected after taking their written informed consent. Genetic diversity in nested PCR products was determined by Restriction Fragment Length Polymorphism (RFLP) utilizing Alu1 and PstI restriction enzymes for alpha and beta gene products digestion, respectively. For analysis the genetic diversity of the sub allelic variants of Pvmsp3 and Pvmsp3 genes, Chi-Square test was performed by utilizing Minitab programming software 18. The P value 0.05 was considered as statistically significant. For Pvmsp-3 genes after gel electrophoresis of digested products, four distinct genotypes were obtained from total of 50 samples; type A: 35 (70%) (1.5-2.0 kb), 12 of type B (24%) (1.5-1.7 kb), 2 of type C (4%) (0.5-1.5) and one for type D (2%) (0.5-0.65 kb) which could be characterized into 9 allelic pattern (A1-A4, B1-B3, C1, D), in which A3 remained the most predominant. For Pvmsp-3genes, three distinct genotypes were obtained from 50 samples; 40(80%) of type A (1.5-2.5 kb), 9 (18%) of type B (1.0-1.5kb) and 1(2%) of type C (0.65 kb) which could be characterized into 6 allelic patterns (A1-A3, B1-B2, and C1). Most dominant one in Type A was A1 alleles which were noted (46%), while in Type B, the most dominant were B1 (10%).This study is the first ever report of molecular epidemiology and genetic variation in Pvmsp-3 and Pvmsp-3 genes of P. vivax isolates by using PCR/RFLP from District Mardan and showed a remarkable level of genetic diversity in the studied genes of circulating parasites in the study area. The results of this study will contribute in future studies about the genetic structure of parasite and vaccine development against the malaria.

Resumo O Plasmodium vivax é o parasita da malária humana mais comum nos países asiáticos, incluindo o Paquistão. O presente estudo foi desenhado para explorar a diversidade genética de genótipos de Plasmodium vivax baseados nos genes Pvmsp-3 e Pvmsp-3, usando marcadores de ensaios alélicos nested PCR e RFLP de isolados de campo no distrito de Mardan, Paquistão. Amostras de sangue de 200 pacientes com malária por P. vivax foram coletadas após assinatura do termo de consentimento livre e esclarecido. A diversidade genética em produtos de PCR nested foi determinada por polimorfismo de fragmento de restrição (RFLP) utilizando as enzimas de restrição Alu1 e PstI para a digestão dos produtos dos genes alfa e beta, respectivamente. Para análise da diversidade genética das variantes subalélicas dos genes Pvmsp3 e Pvmsp3, o teste Qui-quadrado foi realizado utilizando o software de programação Minitab 18. O valor P = 0,05 foi considerado estatisticamente significativo. Para os genes Pvmsp-3, após eletroforese em gel de produtos digeridos, quatro genótipos distintos foram obtidos de um total de 50 amostras; tipo A: 35 (70%) (1,5-2,0 kb), 12 do tipo B (24%) (1,5-1,7 kb), 2 do tipo C (4%) (0,5-1,5) e um para o tipo D (2%) (0,5-0,65 kb), que podem ser caracterizados em nove padrões alélicos (A1-A4, B1-B3, C1, D), em que A3 permaneceu como o mais predominante. Para Pvmsp-3genes, três genótipos distintos foram obtidos a partir de 50 amostras; 40 (80%) do tipo A (1,5-2,5 kb), 9 (18%) do tipo B (1,0-1,5 kb) e 1 (2%) do tipo C (0,65 kb), que podem ser caracterizados em seis padrões alélicos (A1-A3, B1-B2 e C1). Os mais dominantes no tipo A foram o alelo A1, observados em 46%, enquanto, no tipo B, os mais dominantes foram B1 (10%). Este estudo é o primeiro relato de epidemiologia molecular e variação genética em Pvmsp-3. Os genes Pvmsp-3 de isolados de P. vivax utilizando PCR/RFLP do Distrito Mardan mostraram um nível notável de diversidade genética nos genes estudados de parasitas circulantes na área de estudo. Os resultados desse estudo contribuirão em estudos futuros sobre a estrutura genética do parasita e o desenvolvimento de vacinas contra a malária.

PCR-RFLP Based genetic diversity of Plasmodium vivax genotypes in district Mardan, Pakistan / Diversidade genética baseada em PCR-RFLP de genótipos de Plasmodium vivax no distrito de Mardan, Paquistão

Plasmodium vivax is the most common human malaria parasite in Asian countries including Pakistan. Present study was designed to explore the genetic diversity of plasmodium vivax genotypes based on Pvmsp-3α and Pvmsp-3ßgenes using allelic specific nested PCR and RFLP assays markers from field isolates in district Mardan, Pakistan. Blood samples of 200 P. vivax malarial patients were collected after taking their written informed consent. Genetic diversity in nested PCR products was determined by Restriction Fragment Length Polymorphism (RFLP) utilizing Alu1 and PstI restriction enzymes for alpha and beta gene products digestion, respectively. For analysis the genetic diversity of the sub allelic variants of Pvmsp3α and Pvmsp3ß genes, Chi-Square test was performed by utilizing Minitab programming software 18. The P value 0.05 was considered as statistically significant. For Pvmsp3α genes after gel electrophoresis of digested products, four distinct genotypes were obtained from total of 50 samples; type A: 35 (70%) (1.5-2.0 kb), 12 of type B (24%) (1.5-1.7 kb), 2 of type C (4%) (0.5-1.5) and one for type D (2%) (0.5-0.65 kb) which could be characterized into 9 allelic pattern (A1-A4, B1-B3, C1, D), in which A3 remained the most predominant. For Pvmsp-3ßgenes, three distinct genotypes were obtained from 50 samples; 40(80%) of type A (1.5-2.5 kb), 9 (18%) of type B (1.0-1.5kb) and 1(2%) of type C (0.65 kb) which could be characterized into 6 allelic patterns (A1-A3, B1-B2, and C1). Most dominant one in Type A was A1 alleles which were noted (46%), while in Type B, the most dominant were B1 (10%).This study is the first ever report of molecular epidemiology and genetic variation in Pvmsp-3α and Pvmsp-3ß genes of P. vivax isolates by using PCR/RFLP from District Mardan and showed a remarkable level of genetic diversity in the studied genes of circulating parasites in the study area. The results of this study will contribute in future studies about the genetic structure of parasite and vaccine development against the malaria.

O Plasmodium vivax é o parasita da malária humana mais comum nos países asiáticos, incluindo o Paquistão. O presente estudo foi desenhado para explorar a diversidade genética de genótipos de Plasmodium vivax baseados nos genes Pvmsp-3α e Pvmsp-3ß, usando marcadores de ensaios alélicos nested PCR e RFLP de isolados de campo no distrito de Mardan, Paquistão. Amostras de sangue de 200 pacientes com malária por P. vivax foram coletadas após assinatura do termo de consentimento livre e esclarecido. A diversidade genética em produtos de PCR nested foi determinada por polimorfismo de fragmento de restrição (RFLP) utilizando as enzimas de restrição Alu1 e PstI para a digestão dos produtos dos genes alfa e beta, respectivamente. Para análise da diversidade genética das variantes subalélicas dos genes Pvmsp3α e Pvmsp3ß, o teste Qui-quadrado foi realizado utilizando o software de programação Minitab 18. O valor P = 0,05 foi considerado estatisticamente significativo. Para os genes Pvmsp3α, após eletroforese em gel de produtos digeridos, quatro genótipos distintos foram obtidos de um total de 50 amostras; tipo A: 35 (70%) (1,5-2,0 kb), 12 do tipo B (24%) (1,5-1,7 kb), 2 do tipo C (4%) (0,5-1,5) e um para o tipo D (2%) (0,5-0,65 kb), que podem ser caracterizados em nove padrões alélicos (A1-A4, B1-B3, C1, D), em que A3 permaneceu como o mais predominante. Para Pvmsp-3ßgenes, três genótipos distintos foram obtidos a partir de 50 amostras; 40 (80%) do tipo A (1,5-2,5 kb), 9 (18%) do tipo B (1,0-1,5 kb) e 1 (2%) do tipo C (0,65 kb), que podem ser caracterizados em seis padrões alélicos (A1-A3, B1-B2 e C1). Os mais dominantes no tipo A foram o alelo A1, observados em 46%, enquanto, no tipo B, os mais dominantes foram B1 (10%). Este estudo é o primeiro relato de epidemiologia molecular e variação genética em Pvmsp-3α. Os genes Pvmsp-3ß de isolados de P. vivax utilizando PCR/RFLP do Distrito Mardan mostraram um nível notável de diversidade genética nos genes estudados de parasitas circulantes na área de estudo. Os resultados desse estudo contribuirão em estudos futuros sobre a estrutura genética do parasita e o desenvolvimento de vacinas contra a malária.

Protective Immunity Induced by an Eimeria tenella Whole Sporozoite Vaccine Elicits Specific B-Cell Antigens.

This study investigated protection against Eimeria tenella following the vaccination of chicks with 5.3 × 106 E. tenella whole-sporozoites emulsified in the nanoparticle adjuvant IMS 1313 N VG Montanide™ (EtSz-IMS1313). One-day-old specific pathogen-free (SPF) chicks were subcutaneously injected in the neck with EtSz-IMS1313 on the 1st and 10th days of age. Acquired immunity was assayed through a challenge with 3 × 104 homologous sporulated oocysts at 21 days of age. The anticoccidial index (ACI) calculated for every group showed the effectiveness of EtSz-IMS1313 as a vaccine with an ACI of 186; the mock-injected control showed an ACI of 18 and the unimmunized, challenged control showed an ACI of -28. In a comparison assay, antibodies from rabbits and SPF birds immunized with EtSz-IMS1313 recognized almost the same polypeptides in the blotting of E. tenella sporozoites and merozoites. However, rabbit antisera showed the clearest recognition pattern. Polypeptides of 120, 105, 94, 70, 38, and 19 kDa from both E. tenella life cycle stages were the most strongly recognized by both animal species. The E. tenella zoite-specific IgG antibodies from the rabbits demonstrated the feasibility for successful B cell antigen identification.

The molecular basis for peptide-based antimalarial vaccine development targeting erythrocyte invasion by P. falciparum.

This work describes a methodology for developing a minimal, subunit-based, multi-epitope, multi-stage, chemically-synthesised, anti-Plasmodium falciparum malaria vaccine. Some modified high activity binding peptides (mHABPs) derived from functionally relevant P. falciparum MSP, RH5 and AMA-1 conserved amino acid regions (cHABPs) for parasite binding to and invasion of red blood cells (RBC) were selected. They were highly immunogenic as assessed by indirect immunofluorescence (IFA) and Western blot (WB) assays and protective immune response-inducers against malarial challenge in the Aotus monkey experimental model. NetMHCIIpan 4.0 was used for predicting peptide-Aotus/human major histocompatibility class II (MHCII) binding affinity in silico due to the similarity between Aotus and human immune system molecules; ∼50% of Aotus MHCII allele molecules have a counterpart in the human immune system, being Aotus-specific, whilst others enabled recognition of their human counterparts. Some peptides' 1H-NMR-assessed structural conformation was determined to explain residue modifications in mHABPs inducing secondary structure changes. These directly influenced immunological behaviour, thereby highlighting the relationship with MHCII antigen presentation. The data obtained in such functional, immunological, structural and predictive approach suggested that some of these peptides could be excellent components of a fully-protective antimalarial vaccine.

Influence of Qingchang Oral Liquid on Second Generation Merozoite of the Chicken Eimeria tenella

To determine the effect of Qingchang Oral Liquid (QOL) on second generation merozoite of chicken E. tenella, healthy Roman pink chickens were randomly divided into model group and QOL group (drug group), and both groups of chicks were inoculated with 5×104 sporulated oocysts by oral gavage. Then, the drug group was given QOL at a dose of 2.4 ml/kg, and the model group was given the same volume of normal saline. After 120 hours of inoculation, both groups of experimental chickens were killed at the same time, their caecum tissues were collected, the second generation merozoite were separated, the ultra-microstructure of the second generation merozoite were observed with transmission electron microscope and the mitochondrial membrane potential and apoptosis proportion of the second generation merozoite were analyzed with flow cytometer. The current results suggested that QOL could cause swelling and vacuoles of mitochondria, swelling of endoplasmic reticulum and damage of outer membrane in the second generation merozoite of E. tenella. Compared with the model group, the drug group could increase the total apoptosis rate of the second generation merozoite (p<0.01), and reduce the depolarization rate of mitochondrial membrane potential (p<0.01). Conclusion: QOL can directly affect the outer membrane and mitochondria of the second generation merozoite of E. tenella, reduce the depolarization rate of mitochondrial membrane potential of the second generation merozoite and increase the apoptosis rate of the second generation merozoite.

Influence of Qingchang Oral Liquid on Second Generation Merozoite of the Chicken Eimeria tenella

To determine the effect of Qingchang Oral Liquid (QOL) on second generation merozoite of chicken E. tenella, healthy Roman pink chickens were randomly divided into model group and QOL group (drug group), and both groups of chicks were inoculated with 5×104 sporulated oocysts by oral gavage. Then, the drug group was given QOL at a dose of 2.4 ml/kg, and the model group was given the same volume of normal saline. After 120 hours of inoculation, both groups of experimental chickens were killed at the same time, their caecum tissues were collected, the second generation merozoite were separated, the ultra-microstructure of the second generation merozoite were observed with transmission electron microscope and the mitochondrial membrane potential and apoptosis proportion of the second generation merozoite were analyzed with flow cytometer. The current results suggested that QOL could cause swelling and vacuoles of mitochondria, swelling of endoplasmic reticulum and damage of outer membrane in the second generation merozoite of E. tenella. Compared with the model group, the drug group could increase the total apoptosis rate of the second generation merozoite (p<0.01), and reduce the depolarization rate of mitochondrial membrane potential (p<0.01). Conclusion: QOL can directly affect the outer membrane and mitochondria of the second generation merozoite of E. tenella, reduce the depolarization rate of mitochondrial membrane potential of the second generation merozoite and increase the apoptosis rate of the second generation merozoite.(AU)

Plasmodium vivax Cysteine-Rich Protective Antigen Polymorphism at Exon-1 Shows Recombination and Signatures of Balancing Selection.

Plasmodium vivax Cysteine-Rich Protective Antigen (CyRPA) is a merozoite protein participating in the parasite invasion of human reticulocytes. During natural P. vivax infection, antibody responses against PvCyRPA have been detected. In children, low anti-CyRPA antibody titers correlated with clinical protection, which suggests this protein as a potential vaccine candidate. This work analyzed the genetic and amino acid diversity of pvcyrpa in Mexican and global parasites. Consensus coding sequences of pvcyrpa were obtained from seven isolates. Other sequences were extracted from a repository. Maximum likelihood phylogenetic trees, genetic diversity parameters, linkage disequilibrium (LD), and neutrality tests were analyzed, and the potential amino acid polymorphism participation in B-cell epitopes was investigated. In 22 sequences from Southern Mexico, two synonymous and 21 nonsynonymous mutations defined nine private haplotypes. These parasites had the highest LD-R2 index and the lowest nucleotide diversity compared to isolates from South America or Asia. The nucleotide diversity and Tajima's D values varied across the coding gene. The exon-1 sequence had greater diversity and Rm values than those of exon-2. Exon-1 had significant positive values for Tajima's D, ß-α values, and for the Z (HA: dN > dS) and MK tests. These patterns were similar for parasites of different origin. The polymorphic amino acid residues at PvCyRPA resembled the conformational B-cell peptides reported in PfCyRPA. Diversity at pvcyrpa exon-1 is caused by mutation and recombination. This seems to be maintained by balancing selection, likely due to selective immune pressure, all of which merit further study.

Plasmodium falciparum Blood Stage Antimalarial Vaccines: An Analysis of Ongoing Clinical Trials and New Perspectives Related to Synthetic Vaccines.

Plasmodium falciparum malaria is a disease causing high morbidity and mortality rates worldwide, mainly in sub-Saharan Africa. Candidates have been identified for vaccines targeting the parasite's blood stage; this stage is important in the development of symptoms and clinical complications. However, no vaccine that can directly affect morbidity and mortality rates is currently available. This review analyzes the formulation, methodological design, and results of active clinical trials for merozoite-stage vaccines, regarding their safety profile, immunological response (phase Ia/Ib), and protective efficacy levels (phase II). Most vaccine candidates are in phase I trials and have had an acceptable safety profile. GMZ2 has made the greatest progress in clinical trials; its efficacy has been 14% in children aged less than 5 years in a phase IIb trial. Most of the available candidates that have shown strong immunogenicity and that have been tested for their protective efficacy have provided good results when challenged with a homologous parasite strain; however, their efficacy has dropped when they have been exposed to a heterologous strain. In view of these vaccines' unpromising results, an alternative approach for selecting new candidates is needed; such line of work should be focused on how to increase an immune response induced against the highly conserved (i.e., common to all strains), functionally relevant, protein regions that the parasite uses to invade target cells. Despite binding regions tending to be conserved, they are usually poorly antigenic and/or immunogenic, being frequently discarded as vaccine candidates when the conventional immunological approach is followed. The Fundación Instituto de Inmunología de Colombia (FIDIC) has developed a logical and rational methodology based on including conserved high-activity binding peptides (cHABPs) from the main P. falciparum biologically functional proteins involved in red blood cell (RBC) invasion. Once appropriately modified (mHABPs), these minimal, subunit-based, chemically synthesized peptides can be used in a system covering the human immune system's main genetic variables (the human leukocyte antigen HLA-DR isotype) inducing a suitable, immunogenic, and protective immune response in most of the world's populations.

Vaccination against babesiosis using recombinant GPI-anchored proteins.

The increase in human babesiosis is of major concern to health authorities. In the USA, most of these cases are due to infections with Babesia microti, whereas in Europe B. divergens is the major cause of clinical disease in humans. Here we review the immunological and biological literature of glycosylphosphatidylinositol (GPI)-anchored merozoite proteins of human Babesia parasites with emphasis on their role in immunity, and provide some new bioinformatical information on B. microti GPI-Anchored Proteins (GPI-AP). Cattle can be vaccinated with soluble parasite antigens (SPA) of Babesia divergens that are released by the parasite during proliferation. The major component in SPA preparations appeared to be a 37 kDa merozoite surface protein that is anchored in the merozoite membrane by a GPI anchor. Animals could be protected by vaccination with the recombinant 37 kDa protein expressed in Escherichia coli, provided the protein had a hydrophobic terminal sequence. Based on this knowledge, a recombinant vaccine was developed against Babesia canis infection in dogs, successfully. In order to identify similar GPI-AP in B. microti, the genome was analysed. Here it is shown that B. microti encodes all proteins necessary for GPI assembly and its subsequent protein transfer. In addition, in total 21 genes encoding for GPI-AP were detected, some of which reacted particularly strongly with sera from B. microti-infected human patients. Reactivity of antibodies with GPI-anchored merozoite proteins appears to be dependent on the structural conformation of the molecule. It is suggested that the three-dimensional structure of the protein that is anchored in the membrane is different from that of the protein that has been shed from the merozoite surface. The significance of this protein's dynamics in parasite biology and immune evasion is discussed. Finally, we discuss developments in tick and Babesia vaccine research, and the role such vaccines could play in the control of human babesiosis.

Receptores del hospedero implicados en la invasión del merozoito de Plasmodium falciparum: revisión / Host receptors involved in the invasion of Plasmodium falciparum merozoite: Review / Receptores do hospedeiro envolvidos na invasão do merozoíto de Plasmodium falciparum: revisão

Introducción. La malaria es un problema de salud pública a nivel mundial y es causada por 5 especies de parásitos apicomplejos del género Plasmodium. La invasión exitosa de los merozoítos al glóbulo rojo es una etapa fundamental en el ciclo de vida del parásito, el cual usa un variado repertorio de ligandos que interactúan de forma específica con receptores presentes en la membrana del glóbulo rojo. Objetivo. Revisar las características moleculares y estructurales de los receptores expresados en la superficie de los glóbulos rojos, implicados en el proceso de invasión del merozoito de Plasmodium falciparum. Método. Revisión descriptiva sobre las características moleculares y estructurales de los receptores de la superficie del glóbulo rojo, los cuales juegan un papel fundamental durante la invasión del merozoíto de Plasmodium falciparum. Esta revisión empezó por la búsqueda de literatura publicada hasta el año 2019 en bases de datos electrónicas, especializadas en la divulgación de investigación biomédica. Se encontraron 127 documentos, de los cuales se seleccionaron 111 y se excluyeron 33 por no cumplir los criterios de inclusión; en total, se analizaron 78 referencias. Conclusión. En esta revisión se resumieron las características moleculares y estructurales de los receptores presentes en el glóbulo rojo importantes en el proceso de invasión del merozoito de P. falciparum. También, se resaltó la importancia de elucidar las diferentes vías de invasión del parásito y así, poder desarrollar alternativas profilácticas o terapéuticas que conduzcan a mitigar o eliminar la malaria

Introduction. Malaria is a public health problem worldwide. It is caused by 5 species of the Apicomplexa genus Plasmodium. The successful invasion of the erythrocyte by Plasmodium merozoites is a critical stage in the life cycle of the parasite, which uses a broad repertoire of ligands that interact in a specific way with receptors expressed on the membrane of the erythrocyte. Objective. To review the molecular and structural characteristics of the receptors expressed on the erythrocyte surface, involved in the process of merozoite invasion by Plasmodium falciparum. Method. Here, we descriptively review of the molecular and structural characteristics of the red blood cell surface receptors, which play a key role during the invasion of Plasmodum falciparum merozoite. To this purpose, we searched the literature published until 2019 in electronic databases specialized in biomedical research. 127 documents were found, of these, 111 were selected, 33 were excluded and 78 references were analyzed. Conclusion. In this review, the molecular and structural characteristics of the receptors expressed on the erythrocytes and important in the process of invasion of P. falciparum merozoites were discussed. With this, we highlight the importance of elucidating the different invasion pathways the parasite, in order to develop prophylactic or therapeutic alternatives that could lead to mitigate or eliminate malaria.

Introdução. A malária é um problema de saúde pública a nível mundial, é causada por 5 espécies de parasitos do Filo Apicomplexa, do gênero Plasmodium. A invasão bem-sucedida de merozoítos nas hemácias, é uma etapa fundamental no ciclo de vida do parasita, que usa um repertório variado de ligandos que interatuam especificamente com receptores presentes na membrana dos glóbulos vermelhos. Objetivo. Revisão descritiva das características moleculares e estruturais dos receptores da superfície dos glóbulos vermelhos, que desempenham um papel fundamental durante a invasão do merozoíto de Plasmodium falciparum. Método. Revisão descritiva das características moleculares e estruturais dos receptores da superfície dos glóbulos vermelhos, que desempenham um papel fundamental durante a invasão do merozoíto de Plasmodium falciparum. Esta revisão foi baseada na pesquisa de literatura publicada até 2019 nas bases de dados eletrônicas especializadas na divulgação de pesquisas biomédicas. Foram encontrados 127 documentos, dos quais 111 foram selecionados e 33 foram excluídos por não apresentarem os critérios de inclusão, analisando um total de 78 referências. Conclusão. Nesta revisão, foram resumidas as características moleculares e estruturais dos receptores presentes nos glóbulos vermelhos, importantes no processo de invasão do merozoíto de P. falciparum. Também foi destacada a importância de elucidar as diferentes vias de invasão do parasita, a fim de desenvolver alternativas profiláticas ou terapêuticas que levem a mitigar ou eliminar a malária.

The malaria parasite Plasmodium relictum in the endemic avifauna of eastern Cuba.

Island populations are vulnerable to introduced pathogens, as evidenced by extinction or population decline of several endemic Hawaiian birds caused by the malaria parasite, Plasmodium relictum (order Haemosporida). We analyzed blood samples from 363 birds caught near Guantánamo Bay, Cuba, for the presence of haemosporidian infections. We characterized parasite lineages by determining nucleotide variation of the parasite's mitochondrial cyt b gene. Fifty-nine individuals were infected, and we identified 7 lineages of haemosporidian parasites. Fifty individuals were infected by 6 Haemoproteus sp. lineages, including a newly characterized lineage of Haem. (Parahaemoproteus) sp. CUH01. Nine individuals carried the P. relictum lineage GRW4, including 5 endemic Cuban Grassquits (Tiaris canorus) and 1 migratory Cape May Warbler (Setophaga tigrina). A sequence of the merozoite surface protein gene from one Cuban Grassquit infected with GRW4 matched that of the Hawaiian haplotype Pr9. Our results indicate that resident and migratory Cuban birds are infected with a malaria lineage that has severely affected populations of several endemic Hawaiian birds. We suggest GRW4 may be associated with the lack of several bird species on Cuba that are ubiquitous elsewhere in the West Indies. From the standpoint of avian conservation in the Caribbean Basin, it will be important to determine the distribution of haemosporidian parasites, especially P. relictum GRW4, in Cuba as well as the pathogenicity of this lineage in species that occur and are absent from Cuba.

Functionally relevant proteins in Plasmodium falciparum host cell invasion.

A totally effective, antimalarial vaccine must involve sporozoite and merozoite proteins (or their fragments) to ensure complete parasite blocking during critical invasion stages. This Special Report examines proteins involved in critical biological functions for parasite survival and highlights the conserved amino acid sequences of the most important proteins involved in sporozoite invasion of hepatocytes and merozoite invasion of red blood cells. Conserved high activity binding peptides are located in such proteins' functionally strategic sites, whose functions are related to receptor binding, nutrient and protein transport, enzyme activity and molecule-molecule interactions. They are thus excellent targets for vaccine development as they block proteins binding function involved in invasion and also their biological function.

Merozoite-Protein Loaded Liposomes Protect against Challenge in Two Murine Models of Plasmodium Infection.

Liposomes loaded with GPI-anchored proteins induce a strong and effective response against protozoan pathogens when immunized in mice. On the basis of results using Plasmodium falciparum merozoite proteins loaded on liposomes, we tested whether a similar approach would be protective in the P. yoelii XL and P. berghei NK65 models of lethal murine malaria infections. We also analyzed the enhancing capacity of additional adjuvants. As expected, merozoite protein loaded liposomes provided strong humoral responses (IgG titers of 105 against MSP119) in BALB/c and C57BL/6 host backgrounds and showed total protection in lethal challenges (100% survival) with P. yoelii, while protection against lethal challenge with P. berghei NK was at least partial (11%-20% in C57BL/6 mice and no protection in BALB/c). Thus, immunization with proteoliposomes is not only highly immunogenic but also provides a protective response in widely used murine models of malaria.