IgG subclasses pattern and high-avidity antibody to the C-terminal region of merozoite surface protein 1 of Plasmodium vivax in an unstable hypoendemic region in Iran

The C-terminal region of Plasmodium vivax merozoite surface protein 1 (PvMSP-1₁₉) is a leading vaccine candidate for inclusion in a polyvalent malaria vaccine. In the present study, the IgG subclasses profile and the avidity of IgG to PvMSP-1₁₉ were evaluated in individuals (n = 94) naturally exposed to P. vivax parasite in malaria endemic areas in Chabahar districts, Iran. In individuals with patent P. vivax malaria, 86.1% was sero-positive to PvMSP-1₁₉ and IgG1 (81.9%) was the predominant subclass. In addition, to determine the persistence of specific IgG, IgG1 and IgG3 antibodies to PvMSP-1₁₉, the frequency of antibodies was determined in the infected subjects (n = 74) after treatment with standard chloroquine and it was detected that the frequency of responders was significantly reduced to 51.3%, 51% and 16.2%, respectively. The antigen-binding avidity of IgG antibodies to PvMSP-1₁₉ was measured in sero-positive sera and the high-avidity of IgG, IgG1 and IgG3 was found in 66.6%, 61% and 47% of the infected subjects with P. vivax, respectively. The present result shows that individuals who exposed to vivax malaria in the endemic region in Iran develop antibodies with high-avidity to PvMSP-1₁₉. These results could help to understand the interactions between the host and P. vivax parasite in development of MSP-1₁₉-based vaccine.     

Role of MyD88-adaptor-like gene polymorphism rs8177374 in modulation of malaria severity in the Pakistani population

Introduction

The present study was designed to investigate the association between rs8177374 polymorphism and malaria symptoms due to exposure of Plasmodium vivax and Plasmodium falciparum.

Fy(a)/Fy(b) antigen polymorphism in human erythrocyte Duffy antigen affects susceptibility to Plasmodium vivax malaria

Plasmodium vivax (Pv) is a major cause of human malaria and is increasing in public health importance compared with falciparum malaria. Pv is unique among human malarias in that invasion of erythrocytes is almost solely dependent on the red cell's surface receptor, known as the Duffy blood-group antigen (Fy). Fy is an important minor blood-group antigen that has two immunologically distinct alleles, referred to as Fy(a) or Fy(b), resulting from a single-point mutation. This mutation occurs within the binding domain of the parasite's red cell invasion ligand. Whether this polymorphism affects susceptibility to clinical vivax malaria is unknown. Here we show that Fy(a), compared with Fy(b), significantly diminishes binding of Pv Duffy binding protein (PvDBP) at the erythrocyte surface, and is associated with a reduced risk of clinical Pv in humans. Erythrocytes expressing Fy(a) had 41-50% lower binding compared with Fy(b) cells and showed an increased ability of naturally occurring or artificially induced antibodies to block binding of PvDBP to their surface. Individuals with the Fy(a+b-) phenotype demonstrated a 30-80% reduced risk of clinical vivax, but not falciparum malaria in a prospective cohort study in the Brazilian Amazon. The Fy(a+b-) phenotype, predominant in Southeast Asian and many American populations, would confer a selective advantage against vivax malaria. Our results also suggest that efficacy of a PvDBP-based vaccine may differ among populations with different Fy phenotypes.     

Rapid Dissemination of Newly Introduced Plasmodium vivax Genotypes in South Korea

Reemerged Plasmodium vivax malaria in South Korea has not yet been eradicated despite continuous governmental efforts. It has rather become an endemic disease. Our study aimed to determine the genetic diversity in P. vivax merozoite surface protein-1 (PvMSP-1) and circumsporozoite protein (PvCSP) genes over an extended period after its reemergence to its current status. Sequence analysis of PvMSP-1 gene sequences from the 632 P. vivax isolates during 1996–2007 indicates that most isolates recently obtained were different from isolates obtained in the initial reemergence period. There was initially only one subtype (recombinant) present but its subtypes have varied since 2000; six MSP-1 subtypes were recently found. A similar variation was observed by CSP gene analysis; a new CSP subtype was found. Understanding genetic variation patterns of the parasite may help to analyze trends and assess extent of endemic malaria in South Korea.     

False positivity of circumsporozoite protein (CSP)–ELISA in zoophilic anophelines in Bangladesh

Circumsporozoite protein enzyme-linked immunosorbent assays (CSP–ELISAs) are widely used for malaria vector identification throughout the world. However, several studies have reported false-positive results when using this method. The present study was conducted to estimate the frequency of false positives among anopheline species in malaria endemic areas of Bangladesh. In total, 4724 Anopheles females belonging to 25 species were collected and tested for Plasmodium falciparum, Plasmodium vivax-210, and P. vivax-247 CSP. Initially, 144 samples tested positive using routine CSP–ELISA, but the number of positive results declined to 85 (59%) when the samples were tested after heating at 100° C for 10 min to remove false-positive specimens. Ten species, Anopheles annularis, Anopheles baimaii, Anopheles barbirostris, Anopheles jeyporiensis, Anopheles karwari, Anopheles kochi, Anopheles minimus s.l., Anopheles peditaeniatus, Anopheles philippinensis, and Anopheles vagus were CSP-positive. The highest and lowest infection rates were found in An. baimaii (4/25, 16.0%) and An. jeyporiensis (1/139, 0.67%), respectively. A significant correlation was found (regression analysis, R² = 0.49, F = 8.25, P < 0.05) between human blood index results and the true CSP-positive ratios in 15 Anopheles species. We confirmed that false-positive reactions occurred more frequently in zoophilic species. The relatively high proportion of false positives (40%) that was found in this study should warn malaria epidemiologists working in the field to be cautious when interpreting ELISA results.     

Reactivity of sera from cases of Plasmodium vivax malaria towards three recombinant antigens based on the surface proteins of the parasite

Sera collected in South Korea, from 61 cases of Plasmodium vivax malaria and, as controls, 40 healthy volunteers, were tested in ELISA for IgG or IgM reacting with any of three recombinant P. vivax proteins. The antigens used, representing the parasite's major merozoite surface protein (MSP), circumsporozoite surface protein (CSP) and Duffy-binding protein (DBP), had all been expressed in an Escherichia coli system and purified. The ELISA results were recorded as optical densities (OD). The highest ratio observed between the mean OD for a malaria serum and that for a control serum was that for IgG against MSP, although CSP gave a higher ratio than MSP or DBP in the IgM ELISA. In the ELISA for IgG, the OD for MSP were found to be correlated with those for DBP (r = 0.53; P < 0.5) but the OD for CSP were not correlated with those for MSP or DBP. As the most intense reactions observed were those between the IgG from the malaria sera and the recombinant MSP, the latter antigen may be useful in diagnostic tests and as a component of any vaccine used to protect against P. vivax malaria.     

Influence of HLA-DRB-1 alleles on the production of antibody against CSP, MSP-1, AMA-1, and DBP in Brazilian individuals naturally infected with Plasmodium vivax

We evaluated the influence of allelic frequency of the human leukocyte antigen (HLA) -DRB1 on the acquisition of antibody response against malaria sporozoite and merozoite peptides in patients with Plasmodium vivax malaria acquired in endemic areas of Brazil. IgG antibodies were detected by enzyme-linked immunosorbent assay against four peptides of circumsporozoite protein (CSP) (amino, carboxyl, and VK210 and VK247 repeats) and peptides of merozoite surface protein 1 (MSP-1), apical membrane antigen 1 (AMA-1), and Duffy-binding protein (DBP). We found an association between HLA-DR3 and HLA-DR5 alleles and lack of antibody response to CSP amino terminal, as well as an association between HLA-DR3 and the highest antibody response to MSP1 (Pv200L). In conclusion, we suggest a potential regulatory role of the HLA-DRB1 alleles in the production of antibodies to a conserved region of P. vivax CSP and MSP1 in Brazilian population exposed to malaria.     

Haptoglobin phenotypes and iron status in children living in a malaria endemic area of Kenyan coast

Malaria infection may be affected by host genetic factors as well as nutritional status. Iron status and the phenotype of haptoglobin, a heme-binding acute phase reactant may be determinants of malaria parasitemia. A combination of cross sectional studies and longitudinal follow-up were used to describe the association between iron status, C-reactive protein, malaria infections and host genetic factors including; haptoglobin (Hp) phenotypes, in children below 9 years in a malaria endemic area in Coastal Kenya. The prevalence of 0.45 and 0.41, respectively for Hp 1-1 and Hp 2-1 phenotypes was significantly higher than 0.14 for Hp 2-2 phenotype (n = 162). Children with Hp 2-2 phenotype showed significantly higher iron storage compared to those with Hp 1-1 and Hp 2-1 phenotypes when children with malaria parasites and high C-reactive protein (>9 mg/L) were excluded from the analysis. There were no significant differences in malaria parasite densities among Hp phenotypes but children with Hp 2-2 had lower number of clinical malaria episodes (P = 0.045). Taken together, this study shows that the presence of malaria may complicate the interpretation of iron status in children based on their Hp-phenotypes. Further studies will be required to address possible interactions among the various genetic factors and iron status in a malaria endemic setting.     

Anti-gamete antibodies block transmission of human vivax malaria to mosquitoes

Antibodies were raised in rabbits by immunizing against fresh unfixed or cryopreserved female gametes of the human malaria pathogen Plasmodium vivax. The antibodies were shown to react with the surface of gametes by the indirect immunofluorescent test. When parasite isolates from P. vivax infected individuals were fed through a membrane to Anopheles tessellatus mosquitoes in the presence of immune rabbit sera, they completely blocked the infectivity of the parasite isolates to the vector. Immunoglobulins separated from these sera also blocked infectivity to the same extent as did the immune sera indicating that antibodies were responsible for the transmission blocking effect of the sera. This study indicated that P. vivax like other malaria parasites is highly susceptible to anti gamete transmission blocking immunity.     

Evaluation of the NOW Malaria Immunochromatographic Test for Quantitative Diagnosis of Falciparum and Vivax Malaria Parasite Density

The NOW® Malaria Test, an immunochromatographic test (ICT), was evaluated to determine its ability to quantitatively detect malaria parasites using 100 blood samples from Thailand, including 50 Plasmodium falciparum (Pf) infections and 50 P. vivax (Pv) infections. Intensities of the thickness of the visible bands of the positive ICT were compared with the parasite densities. In cases of Pf infection, the intensities of both HRP-2 bands (T1 bands: Pf specific bands) and aldolase bands (T2 bands: pan-Plasmodium bands) correlated with the parasite densities. The intensities of T2 bands in Pf positive samples showed better correlation with the parasite densities than the T1 bands. In the cases of Pv infection, the intensities of T2 bands were also well correlated with parasite density. These results suggest that the ICT is useful not only for rapid detection of malaria parasites but also for estimating parasite density.     

Antibodies to a merozoite surface protein promote multiple invasion of red blood cells by malaria parasites

The 40-50 kDa merozoite surface antigen (MSA2) is a candidate molecule for use in a malaria vaccine. The gene for MSA2 from the 3D7 isolate of Plasmodium falciparum was amplified by polymerase chain reaction and cloned into the bacterial expression vector pGEX-3X to obtain a fusion protein of MSA2 with Schistosoma japonicum glutathione S-transferase. The recombinant fusion protein was used to immunize rabbits. After four injections, the sera had Western blotting and immunofluorescence titres of 10(-6). Immune sera, and immunoglobulin (Ig)G, F(ab)'2, F(ab) prepared from the immune sera, were assessed for their effects on the growth of 3D7 parasites in vitro by microscopy and a [3H]-hypoxanthine incorporation assay. The antibodies did not significantly inhibit red blood cell invasion and parasite growth when added to cultures as 10% v/v serum or as immunoglobulin preparations at concentrations up to 200 microg ml(-1). However, in the presence of IgG or F(ab)'2, but not F(ab), antibodies to MSA2, the proportions of red blood cells invaded by more than one merozoite increased significantly. Multiple invasion is attributed to merozoites cross-linked by bivalent antibodies, attaching to and subsequently invading the same red cell. These observations have a bearing on the evasion of host immune responses by the parasite and the use of full-length recombinant MSA2 protein in a malaria vaccine.     

Genetic diversity and natural selection of Duffy binding protein of Plasmodium vivax Korean isolates

Plasmodium vivax Duffy binding protein (PvDBP) is a micronemal type I membrane protein that plays an essential role in erythrocyte invasion of merozoites. PvDBP is a prime blood stage vaccine candidate antigen against P. vivax, but its polymorphic nature represents a major obstacle to the successful design of a protective vaccine against vivax malaria. In this study, we analyzed the genetic polymorphism and natural selection at the N-terminal cysteine-rich region of PvDBP (PvDBPII) among 70 P. vivax isolates collected from Korean patients during 2005–2010. Seventeen single nucleotide polymorphisms (SNP), which resulted in 14 non-synonymous and 3 synonymous mutations, were found in PvDBPII among the Korean P. vivax isolates. Sequence analyses revealed that 13 different PvDBPII haplotypes, which were clustered into 3 distinct clades, were identified in Korean P. vivax isolates. The difference between the rates of nonsynomyous and synonymous mutations suggested that the region has evolved under natural selection. High selective pressure preferentially acted on regions identified or predicted to be B- and T-cell epitopes and MHC binding regions of PvDBPII. Recombination may also contribute to genetic diversity of PvDBPII. Our results suggest that PvDBPII of Korean P. vivax isolates display a limited genetic polymorphism and are under selective pressure. These results have significant implications for understanding the nature of the P. vivax population circulating in Korea and provide useful information for development of malaria vaccines based on this antigen.     

Reactivity of sera from dogs living in a leishmaniasis-endemic area to the COOH-terminal region of cysteine proteinase B

Cysteine proteinases are well-known virulence factors of Leishmania spp. with demonstrated actions in both experimental mouse infection and human infection. However, studies on these enzymes in canine leishmaniasis are scarce. Here, we show, for the first time, the reactivity of sera from dogs living in an endemic area to a recombinant protein from the COOH-terminal region of cysteine B protease. In this work, enzyme-linked immunosorbent assays were performed using a 14 kDa rcyspep protein obtained through a pET28-a expression system in Escherichia coli. First, 96-well plates were coated with rcyspep (500 ng/well) and incubated with sera from dogs (1:100). Subsequently, IgG antibody detection was performed using rabbit anti-dog IgG antibodies conjugated with peroxidase. Sera from dogs (n = 114), including suspect (n = 30) and positive (n = 50) dogs from a leishmaniasis-endemic area and dogs from a nonendemic area, (n = 34), negative for leishmaniasis, were assessed. The results showed that sera from the suspect (42%) and positive (68%) groups responded differently to the antigen titers tested above the cut-off (Optical Density = 0.166). This finding suggests that the immune response detected against cyspep may be related to clinical disorders present in these animals. Collectively, the data gathered here suggest that cyspep can sensitize the immune systems of dogs from a leishmaniasis-endemic area to elicit a humoral response, an immunological parameter indicating the contribution of this protein in host-parasite interaction.     

The intradermal vaccination route – an attractive opportunity for influenza vaccination in the elderly

An age-related decline in functioning of the innate and adaptive immune systems results in increased susceptibility to infections (e.g. influenza) and decreased responses to vaccination in elderly people. A satellite symposium held during the xixth IAGG World Congress of Gerontology and Aging in Paris, 5–9 July 2009, considered the potential of intradermal vaccination to enhance immune responses in the elderly. The rich supply of capillary blood and lymphatic vessels in the dermis, along with its resident population of dendritic cells, make the skin an attractive site for vaccine delivery. Intanza^® 15 μg is a purified, inactivated, trivalent, split-virus influenza vaccine containing 15 μg haemagglutinin/strain/0.1 ml dose that is administered using a novel intradermal microneedle injection system. A randomised, open-label phase III trial in 3695 people aged 60–95 years found that antibody responses to the intradermal influenza vaccine were superior to those for the same vaccine administered intramuscularly. The systemic safety profile of the intradermal vaccine was comparable with that of the intramuscular vaccine, but rates of injection-site reactions were higher with the intradermal vaccine, reflecting the close proximity of injected vaccine to the skin surface. The increased immunogenicity of Intanza^® 15 μg in the elderly compared with the standard intramuscular influenza vaccine supports the concept of intradermal vaccination to enhance immune responses in elderly people.     

Evaluation of four rapid diagnostic tests for the diagnosis of Plasmodium vivax in Korea

Objective To evaluate 4 rapid malaria diagnostic kits (RDTs) in Korea: OptiMAL test, SD BIOLINE Malaria Ag P.f/Pan test, Humasis Malaria P.f/Pan antigen test and CareStart? Malaria Pf/Pv Combo test. Methods Hundred malaria patients with Plasmodium vivax (P. vivax) and 100 healthy volunteers were recruited. The results from earlier four RDTs were compared with the reference standard, the Giemsa‐stained traditional microscopic diagnosis. Results Compared with the reference standard, the sensitivity and specificity for Plasmodium vivax were 92.7 and 100% for SD BIOLINE Malaria Ag P.f/Pan; and 94.6% and 100% for OptiMAL; 95.5% and 100% for both Humasis Malaria P.f/Pan antigen test and CareStart? Malaria Pf/Pv Combo test. Conclusion The performances of all four malaria RDT kits were acceptable, although Humasis Malaria P.f/Pan antigen test and CareStartTM Malaria Pf/Pv Combo test gave superior performances with ROK isolates.     

Ni-NTA蛋白芯片技术检测间日疟原虫感染的体液免疫应答

目的 建立检测间日疟感染的Ni-NTA蛋白芯片技术.方法 采用无细胞蛋白合成体系表达问日疟原虫重组蛋白,建立原位纯化重组蛋白和检测间日疟原虫感染患者血清中抗体反应的Ni-NTA蛋白芯片技术.并对3个裂殖子表面蛋白(merozoite surface proteins,MSPs)MSPl-42、MSP8和MSP10的免疫应答进行分析.结果 应用Ni-NTA蛋白芯片技术检测问日疟原虫感染患者血清抗体,鉴定出具有免疫原性的15个间日疟原虫蛋白,主要包括10个MSPs、2个Cys6蛋白以及其他3个未知蛋白,结果与以往报道的抗体芯片类似.MSPl-42、MSP8和MSP10依次识别出100.0%(20/20)、90.0%(18/20)和70.0%(14/20)的间日疟原虫感染患者血清,特异性均为100%(10/10),且曲线下面积(area under the curve.AUC)达到0.87～1.00.结论 成功建立了检测间日疟原虫感染的Ni-NTA蛋白芯片技术.该方法有助于从间日疟原虫基因组中快速筛选鉴定具有免疫原性的蛋白以及应用于功能蛋白质组学研究.

Abstract：

Objective To develop Ni-NTA surface based chips to detect the humoral immune response to Plasmodium vivax infection.Methotis A set of recombinant P.vivax proteins was expressed by wheat germ cell-free system and IgG immune responses to these recombinant P. vivax proteins including 3 merozoite surface proteins(MSPl-42,MSP8 and MSPIO)were measured with sera from P.vwax-infected patients and healthv individuals using Ni-NTA chips.Results Ni-NTA surface based chips were successfully applied to detect immune responses to P.vivaz infection.Fifteen high immunoreactive recombinant P.vivax proteins were identified using Ni-NTA chips,including 10 MSPs,2 Cys6 lipid raft-associated proteins and 3 hypothetical proteins.MSPI-42.MSP8 and MSPIO recognized by IgG immune response to 100.0%(20/20),90.0%(18/20),and 70.0%(14/20)serum samples from P.vivax infected patients,respectively.Moreover,no crossreactivity to MSPs was found in serum samples from healthy individuals with area under the curve(AUC)values of 0.87-1.00.Conclusions In this report,Ni-NTA chips have been applied to investigate blood stage-specific immunogenic proteins from vivax malaria.The method may aid in determining the immunogenicity of candidate antigens and the functional identification of the large number of unknown and hypothetical proteins in P.vivax genome.     

Monoclonal and polyclonal antibodies both block and enhance transmission of human Plasmodium vivax malaria

Antibodies against gametes of the malarial parasite inhibit the development of the parasite in the mosquito and curtail the transmission of malaria. We now report that a monoclonal antibody against gametes of the human malaria pathogen Plasmodium vivax and antibodies induced during natural infections of P. vivax in humans which suppress infectivity of the parasites to the vector at high concentrations can, at lower concentrations, have the opposite effect and enhance the level of malaria infection in the mosquitoes. Infectivity enhancing effects of up to 12-fold were demonstrated when a transmission blocking monoclonal antibody and immune human sera were diluted, in some undiluted immune human sera, and in the sera of vivax malaria patients during convalescence after drug cure.     

Plasmodium vivax Isolates from Cambodia and Thailand Show High Genetic Complexity and Distinct Patterns of P. vivax Multidrug Resistance Gene 1 (pvmdr1) Polymorphisms

Plasmodium vivax accounts for an increasing fraction of malaria infections in Thailand and Cambodia. We compared P. vivax genetic complexity and antimalarial resistance patterns in the two countries. Use of a heteroduplex tracking assay targeting the merozoite surface protein 1 gene revealed that vivax infections in both countries are frequently polyclonal (84%), with parasites that are highly diverse (H_E = 0.86) but closely related (G_ST = 0.18). Following a history of different drug policies in Thailand and Cambodia, distinct patterns of antimalarial resistance have emerged: most Cambodian isolates harbor the P. vivax multidrug resistance gene 1 (pvmdr1) 976F mutation associated with chloroquine resistance (89% versus 8%, P < 0.001), whereas Thai isolates more often display increased pvmdr1 copy number (39% versus 4%, P < 0.001). Finally, genotyping of paired isolates from individuals suspected of suffering relapse supports a complex scheme of relapse whereby recurrence of multiple identical variants is sometimes accompanied by the appearance of novel variants.     

Genetic diversity in the C-terminal 42 kDa region of merozoite surface protein-1 of Plasmodium vivax (PvMSP-1(42)) among Indian isolates

Plasmodium vivax merozoite surface protein 1 (PvMSP-1) is a leading malaria vaccine candidate. This protein is processed to give rise to various sized fragments during merozoite maturation. Here, we describe the analysis of genetic diversity in the 42 kDa C-terminal part of this protein among 33 Indian P. vivax isolates. A total of 27 haplotypes with 72 mutations and 0.0212 ± 0.0005S.D. over all π nucleotide diversity were observed among the isolates. Twenty-six of 27 haplotypes reported here were new as they have not been reported so far from any other country. The difference between non-synonymous (dN) and synonymous (dS) mutations was found to be positive (0.0081 ± 0.0051) for the entire 42 kDa region. Further analysis revealed that 33 kDa (MSP-1₃₃) fragment of the MSP-1₄₂ was highly polymorphic with π nucleotide diversity 0.0290 ± 0.0007S.D. The dN-dS for this region of MSP-1 was also positive (0.0114 ± 0.0071S.E.). On the other hand, there was no non-synonymous mutation in the 19 kDa (MSP-1₁₉) fragment of the MSP-1₄₂ and thus it was highly conserved. In conclusion, MSP-1₃₃ fragment was highly polymorphic and appeared to be under diversifying selection whereas there was no selection at MSP-1₁₉ region among the isolates. Present study will be helpful for the development of PvMSP-1 based vaccine against P. vivax malaria.     

Primary structure of the merozoite surface antigen 1 of Plasmodium vivax reveals sequences conserved between different Plasmodium species.

Merozoite surface antigen 1 (MSA1) of several species of plasmodia has been shown to be a promising candidate for a vaccine directed against the asexual blood stages of malaria. We report the cloning and characterization of the MSA1 gene of the human malaria parasite Plasmodium vivax. This gene, which we call Pv200, encodes a polypeptide of 1726 amino acids and displays features described for MSA1 genes of other species, such as signal peptide and anchoring sequences, conserved cysteine residues, number of potential N-glycosylation sites, and repeats consisting here of 23 glutamine residues in a row. When the nucleotide and deduced amino acid sequences of the MSA1 of P. vivax are compared to those of another human malaria parasite, Plasmodium falciparum, and to those of the rodent parasite Plasmodium yoelii, 10 regions of high amino acid similarity are observed despite the very different dG + dC contents of the corresponding genes. All of the interspecies conserved regions reside within the conserved or semiconserved blocks delimited by the sequences of different alleles of the MSA1 gene of P. falciparum.