Evaluation of a new fusion antigen, cd loop and HAP2-GCS1 domain (cd-HAP) of Plasmodium falciparum Generative Cell Specific 1 antigen formulated with various adjuvants, as a transmission blocking vaccine.

BACKGROUND: Malaria is a major global health challenge, and for the elimination and eradication of this disease, transmission-blocking vaccines (TBVs) are a priority. Plasmodium falciparum Generative Cell Specific 1 (PfGCS1), a promising TBV candidate, is essential for gamete fertilization. The HAP2-GCS1 domain of this antigen as well as its cd loop could induce antibodies that partially inhibit transmission of P. falciparum. METHODS: In the current study, a new synthetic fusion antigen containing cd loop and HAP2-GCS1 domain (cd-HAP) of PfGCS1 was evaluated as a transmission blocking vaccine candidate. Initially, the profile of naturally acquired IgG antibodies to the cd-HAP antigen was analysed in Iranian individuals infected with P. falciparum, to confirm that this new fusion protein has the appropriate structure containing common epitopes with the native form of PfGCS1. Then, the immunogenicity of cd-HAP was evaluated in BALB/c mice, using different adjuvant systems such as CpG, MPL, QS-21, and a combination of them (CMQ). Furthermore, the blocking efficacy of polyclonal antibodies induced against these formulations was also assessed by oocyst intensity and infection prevalence in the Standard Membrane Feeding Assay (SMFA). RESULTS: The naturally acquired antibodies (dominantly IgG1 and IgG3 subclasses) induced in P. falciparum-infected individuals could recognize the cd-HAP antigen which implies that the new fusion protein has a proper conformation that mimics the native structure of PfGCS1. Concerning the immunogenicity of cd-HAP antigen, the highest IgG levels and titers, by a Th1-type immune profile, and elevated antibody avidity were induced in mice immunized with the cd-HAP antigen formulated with a combination of adjuvants (P < 0.0001). Additionally, cytokine profiling of the immunized mice displayed that a high level of IFN-γ response, a Th1-type immune response, was produced by splenocytes from immunized mice that received cd-HAP antigen in combination with CMQ adjuvants (P < 0.0001). This formulation of cd-HAP antigen with CMQ adjuvants could reduce oocyst intensity and infection prevalence by 82%, evidenced by the SMFA and hold significant implications for future malaria vaccine development. CONCLUSION: Altogether, the results showed that cd-HAP antigen formulated with a combination of the adjuvants (CMQ), could be a promising formulation to develop a PfGCS1-based transmission-blocking vaccine.

Isolation and identification of microbiota of Culex quinquefasciatus for their application as paratransgenic tools in vector control.

Background and Objectives: Although the study on the bacteria residing in the mid-gut, salivary gland, and reproductive organs of insect vectors have drawn appeal to the host-pathogen interactions, we know comparatively less about microbiota that naturally exist in different mosquito organs within Iran. Materials and Methods: In the current investigation, PCR assay by using 16S rRNA gene amplification and DNA sequencing, in addition to the traditional culture-based approach utilized for the detection of cultivable bacterial assemblages in mid-gut and reproductive tracts of Culex quinquefasciatus. Results: The identified bacteria isolated from different tissues of 45 individuals were consisted of Achromobacter, Aeromonas, Arthrobacter, Asaia, Enterobacter, Gluconobacter, Klebsiella, Lysinibacillus, Micrococcus, Psuedomonas and Serratia. The results showed that Proteobacteria was the most prevalent phylum in both genders' mid-gut and reproductive tracts, and Asaia was the most common bacteria that originated in adult females and males' tissues. Conclusion: These outcomes recommend that the discovered microbiome may span through Cx. quinquefasciatus populations. This data can be utilized to interfere with the transmission of pathogens and design new strategies for the control of mosquito-borne diseases.

Molecular Detection of Plasmodium Infection among Anophelinae Mosquitoes and Differentiation of Biological Forms of Anopheles Stephensi Collected from Malarious Areas of Afghanistan and Iran.

Background: Updated information on the vectorial capacity of vectors is required in each malarious areas as well in Iran and its neighboring countries such as Afghanistan. The aims of this study were to investigate the potential infection of about 800 specimens collected from malarious areas of Afghanistan and Iran, and to differentiate biological forms of Anopheles stephensi. Method: Two molecular markers, 18S RNA gene subunit and AsteObp1 intron I, were used respectively for investigation Plasmodium infection and identifying the biological forms of An. stephensi. Results: Plasmodium infection was detected in 4 pools of Afghanistan specimens, including An. stephensi, collected from Nangarhar. Individually examination showed infection in 5 An. stephensi (infection rate: 1.25), to P. falciparum (2), P. vivax (2) and a mix infection. Out of five infected specimens, three were intermediate forms and two were mysorensis. No infection was found in specimens collected from Iran (Chabahar County), probably due to the active malaria control program in south-east of Iran. Conclusion: The key role of An. stephensi, as a known Asian malaria vector, was re-emphasized in Afghanistan by the results achieved here. The fauna of vectors and the pattern of biological forms of An. stephensi are similar in both countries that urge regional investigations to provide evidence-based and applied data for decision-maker in malaria control.

Optimization of the heterologous expression and purification of Plasmodium falciparum generative cell specific 1 in Escherichia coli.

Generative cell specific 1 (GCS1) or Hapless2 (Hap2) is a main transmission-blocking vaccine (TBV) candidate against malaria. Experience has shown that this protein is difficult to express in heterologous hosts. In a study, Plasmodium falciparum GCS1 (PfGCS1) could be expressed in fusion with Glutathione S Transferase (GST). Since the large fusions could influence the immunogenicity of the recombinant antigens, in the current study, we tried to express PfGCS1 protein without large fusion tags with an appropriate yield and purity in E. coli. To this end, pfgcs1 gene was codon-optimized and cloned in pET23a plasmid. The expression was evaluated in different E. coli hosts [E. coli BL21(DE3), E. coli BL21(DE3) pLysS, E. coli Rosetta(DE3), and E. coli Rosettagami(DE3)] and media cultures. In addition, the effect of post-induction times, inducer concentration, temperature, and supplementation of glucose and ethanol to culture media were evaluated. The obtained results revealed that rPfGCS1 protein was expressed in all examined E. coli hosts and media cultures with different yields, with the best yield in E. coli BL21(DE3), and E. coli Rosetta(DE3) hosts in TB medium, 16 h post-induction. The expression of rPfGCS1 was confirmed by western blotting using anti-His antibodies. Expression in low temperature at 20 °C and addition of glucose and ethanol to TB media could improve the expression of rPfGCS1. We could express and purify rPfGCS1 without a large fusion protein with an appropriate yield and purity in E. coli Rosetta(DE3). We will evaluate this antigen as TBV candidate against P. falciparum transmission in the future.

Untangling population structure and genetic diversity of reticulocyte binding protein 2b (PvRBP2b) erythrocytic stage vaccine candidate in worldwide Plasmodium vivax isolates.

BACKGROUNDS: Plasmodium vivax is the predominant Plasmodium species distributed extensively in the Americas and Asia-Pacific areas. Encoded protein by Plasmodium vivax Reticulocyte Binding Proteins (PvRBPs) family member are of critical prominence to parasite invasion and have been considered the significant targets in development of malaria vaccine for the blood stage. As high genetic polymorphism of parasites may impede the effectiveness of vaccine development, more research to unraveling genetic polymorphism of pvrbp2b from various geographical regions seems indispensable to map the exact pattern of field isolates. METHODOLOGY/PRINCIPAL FINDINGS: The aim of this study was to determine the sequences of Iranian pvrbp2b (nt: 502-1896) gene and then, to ascertain polymorphism of pvrbp2b gene, recombination, the level of genetic distances, evaluation of natural selection, and the prediction of B-cell epitopes of Iranian and global P. vivax isolates. Pvrbp2b partial gene was amplified and sequenced from 60 Iranian P. vivax isolates. Iranian pvrbp2b sequences as well as 95 published sequences from five countries were used to evaluate the genetic diversity and neutral evolution signature in worldwide scale. A total of 38 SNPs were identified among 60 Iranian pvrbp2b sequences (32 non-synonymous and 6 synonymous mutations), and 32 amino acid substitutions were observed in 29 positions as compared to Sal-1 sequence. Worldwide sequence analysis showed that 44 amino acid changes had occurred in 37 positions of which seven polymorphic sites had trimorphic mutations while the rest was dimorphic. The overall nucleotide diversity for Iranian isolates was 0.00431 ± 0.00091 while the level of nucleotide diversity was ranged from 0.00337 ± 0.00076 (Peru) to 0.00452 ± 0.00092 (Thailand) in global scale. CONCLUSIONS/SIGNIFICANCE: Of amino acid substitutions, 12 replacements were located in the B-cell epitopes in which nine polymorphic sites were positioned in N-terminal and three polymorphic sites in predicted B-cell epitopes of C-terminal, signifying both variable and conserved epitopes for vaccine designing. Using the achieved outcome of the current investigation interrogate questions to the selection of conserved regions of pvrbp2b and understanding polymorphism and immune system pressure to pave a way for developing a vaccine based on PvRBP2b candidate antigen.

Haemoproteosis and avian malaria in Columbidae and Corvidae from Iran.

Avian malaria (Plasmodium) and related genera (Haemoproteus and Leucocytozoon) are diverse and widespread parasites. Despite the extent of knowledge on avian haemosporidian parasites, information about domestic and wild bird's blood parasites is overall insufficient in Iran. Prevalence of the haemosporidian parasites' and phylogenetic relationship of lineages are studied by using molecular and morphological results of 152 examined hosts belonging to 17 species. Molecular analysis for haemosporidian detections demonstrated overall prevalence 22.36%. Inspected hosts mostly belonging to Common Pigeons (Columba livia) parasitized by Haemoproteus spp., and Hooded Crows (Corvus cornix) and Carrion Crow (C. corone) were identified as hosting Plasmodium spp. Detected lineages COLIV03, COQUI05, LINN01, ROFI04 and SGS01 are identified as new reports from Iran. We detected no evidence of Leucocytozoon lineages, while the high prevalence of H. columbae was found in Common Pigeons. Such investigation on avian blood parasites contributes to providing new information on the prevalence, epidemiology and geographical distribution of haemosporidian parasites circulating in domestic, pets and wild birds.

Molecular Monitoring of Knockdown Resistance in Head Louse (Phthiraptera: Pediculidae) Populations in Iran.

Knockdown resistance (kdr) is a common mechanism of insecticide resistance in head lice to the conventionally used pyrethroid pediculosis and can be the result of various amino acid substitutions within the voltage-sensitive sodium channel (VSSC). In this study, 54 sequences from varied specimens were investigated to monitor well-known resistance mutations and probable new mutations. The Pediculus humanus capitis de Geer specimens were collected from 13 provinces in Iran. The specimens were stored in 70% ethanol until DNA extraction and PCR amplification of ~900-bp fragment of VSSC. The sequences were analyzed using different bioinformatics software for the detection of well-known kdr substitutions and additional mutations potentially associated with kdr resistance in head lice. There were six new and an old (haplotype I) kdr haplotypes within the Iranian head louse population. K794E, F815I, and N818D amino acid substitutions were reported for the first time. The P813H mutation was the most prevalent amino acid substitution in eight provinces. Among 53 sequences, 26 (49%) were homozygous susceptible, and 27 (51%) were heterozygotes. Thus, 51% of the head lice collected in Iran harbored only the P813H allele. The exact test for the Hardy-Weinberg (H-W) equilibrium showed that genotype frequencies differed significantly from the expectation in East-Azerbaijan and Tehran provinces. Moreover, these populations had an inbreeding coefficient (Fis) <0, indicating the excess of heterozygotes. This observation suggests that the populations of head lice from Iran are currently under active selective pressure. For the rest of the populations, H-W equilibrium and the expectations were significantly in harmony. The results of the current study highlight molecular techniques in the accurate detection of resistance genotypes before their establishment within the head louse population. Accurate detection of resistant genotypes seems to be helpful in decision-making on lice control programs and resistance monitoring and management.

Relationship between Wolbachia infection in Culex quinquefasciatus and its resistance to insecticide.

Many studies have been done on the various factors affecting resistance to insecticides. The relationship between Wolbachia bacteria and resistance to insecticides is one of the factors that has attracted a lot of attentions. Wolbachia are obligatory intracellular endosymbionts that naturally occur in a wide range of arthropods and nematodes, including the mosquito Culex quinquefasciatus. Initially, the presence of bacteria was proved by molecular assays. Then the resistance level of this species was evaluated in adults against DDT 4.0% and deltamethrin 0.05% using the standard WHO guideline. After elimination of Wolbachia by tetracycline and its proof by molecular assays, the susceptibility tests were conducted again on uninfected line. Finally, the two lines were compared in terms of responding to insecticides. The findings indicated that there is no significant correlation between susceptibility of two lines in response to DDT 4.0% while they represented a significant correlation for deltamethrin (P =0.00). We propose that Wolbachia bacteria increase the susceptibility to deltamethrin but they show neutral effect on DDT susceptibility in Cx. quinquefasciatus. However, more studies on other vectors and insecticides still need to be done.

Detection of haemosporidian parasites in wild and domestic birds in northern and central provinces of Iran: Introduction of new lineages and hosts.

Haemosporidian parasites characterize multi-host and multi-parasite structures which are prevalent among wild bird populations. Here, determination of host records, estimation of the prevalence and diversity of haemosporidian lineages were performed in wild and domestic birds in 11 provinces in Iran. To our knowledge, for the first time in this region, molecular characterization of haemosporidians in migratory water birds, raptors, and domestic birds was carried out: blood or tissue samples were collected from 246 birds belonging to 36 species, 12 families, and 11 orders. The prevalence of Plasmodium, Haemoproteus, and Leucocytozoon were documented as 1.21%, 3.65%, and 0.4%, respectively. Of 36 birds' species inspected in this investigation, 13 individuals of 9 species were parasitized by blood parasites. To our knowledge, five lineages including hANACRE03, hAYTFER01, hAYTFER02, hAQUCYR01, and hSTAL06 were found as un-described lineages, while six known lineages of hLK03, pLK05, lTUSW04, pSW5, hMILANS02, and hHAECOL1 were recorded in hosts within novel geographical regions. Such results are required to fill the gaps in understanding the geographical distribution patterns of wildlife related vector-borne parasites in migratory birds as potential carriers, raptors with high vulnerability, and domestic birds as pet or with economic value.

Measuring of IgG2c isotype instead of IgG2a in immunized C57BL/6 mice with Plasmodium vivax TRAP as a subunit vaccine candidate in order to correct interpretation of Th1 versus Th2 immune response.

Evaluation of the murine isotype antibodies is essential in subunit vaccine development because inbred mouse strains with diverse genetic backgrounds respond different to recombinant proteins. In this regard, the main goal of this study was to measuring and comparing the profile of IgG isotype responses in C57BL/6 mice. For this purpose, the extracellular region of plasmodium vivax thrombospondin-related adhesive protein (PvTRAP) gene was expressed in Escherichia coli Rosetta (DE3)-pET23a. Then, the recombinant PvTRAP alone or emulsified with Freund's complete adjuvant were applied for immunization of the C57BL/6 mice. The role of antibodies and cellular immune responses induced by recombinant PvTRAP were evaluated. The results showed the level of anti-rPvTRAP IgG2c was significantly higher than IgG2a in the groups that received rPvTRAP alone (mean OD490 = 0.798 ± 0.12 and 0.39 ± 0.1, respectively) and emulsified with CFA/IFA (mean OD490 = 1.48 ± 0.07 and 0.605 ± 0.13, respectively; P < 0.05, independent sample t-test). Additionally, the immunized mice with rPvTRAP and rPvTRAP + CFA/IFA had an intermediate-avidity IgG2a antibody but high-avidity IgG2c antibody as well as the mean of serum antibody titers results exhibited that in both rPvTRAP and rPvTRAP + CFA/IFA mouse groups, IgG2a end-point titer (1:3200 and 1:25,600, respectively) was noteworthy lower than IgG2c (1:25,600 and 1:102,400, respectively). Moreover, the results revealed the eliciting significant levels of IFN-γ (P < 0.05, independent sample t-test) and no detectable level of IL-4 in the mouse groups received rPvTRAP alone and emulsified with CFA/IFA as compared to the mouse control groups. In general, our results showed that for correctly interpreting of Th1 immune responses in C57BL/6 mouse strain it is critical to measure IgG2c instead of IgG2a along with IFN-γ.

Molecular characteristics of odorant-binding protein 1 in Anopheles maculipennis.

BACKGROUND: Anopheles maculipennis complex, the historic vector of malaria, causes serious medical problems worldwide and exhibits different behaviours. Studying the odorant-binding proteins (OBPs), which influence the chemosensory system and behavioural responses, is essential to understanding the population structure and developing effective control measures against this vector. The present study was designed to identify and analyse the obp1 gene in An. maculipennis. METHODS: Adults of An. maculipennis sensu stricto were collected in Zanjan Province, northwest of Iran, and gDNAs of female mosquitoes were extracted. Fragments of An. maculipennis obp1 (Amacobp1) gene were amplified using degenerate and specific primers, and some of amplicons were selected for sequencing. RESULTS: Analysis of amplified products identified that the sequence of Amacobp1 gene was 1341 bp long. This gene contains three exons (5', internal, and 3'of 160, 256, and 18 bp, respectively) and encodes 144 amino acids. The sizes of introns I and II in deduced gene are 268 and 358 nucleotides, respectively. The amino acid sequence in the C-terminal of AmacOBP1 is similar to that of major malaria vector Anopheles species. However, its N-terminal has a specific signal peptide with 19 amino acids. This peptide is conserved in different studied populations, and its sequence of amino acids shows the most variation among anopheline species. CONCLUSIONS: Degenerate primers in this study are suggested for studying obp1 gene in Anopheles species. Amacobp1 gene is proposed as a molecular marker for the detection of intraspecific ecotypes and diagnosis of different species within Maculipennis Group. Moreover, the N-terminal of AmacOBP1 peptide is recommended as a molecular marker to identify the Amacobp1 expression patterns in different chemosensory organs for assessing the molecular mechanisms and developing novel behavioural disturbance agents to control An. maculipennis.

Population genetic structure analysis of thrombospondin-related adhesive protein (TRAP) as a vaccine candidate antigen in worldwide Plasmodium falciparum isolates.

Antigenic diversity is a major concern in malaria vaccine development that requires to be considered in developing a malaria vaccine. Plasmodium falciparum thrombospondin-related adhesive protein (PfTRAP) is a leading malaria vaccine candidate antigen. In the current study, we investigated the level of genetic diversity and natural selection of pftrap sequences in P. falciparum isolates from Iran (n = 47). The gene diversity of Iranian pftrap sequences was also compared to available global pftrap sequences deposited in the GenBank or PlasmoDB databases (n = 220). Comparison of Iranian PfTRAP sequences with T9/96 reference sequence showed the presence of 35 amino acid changes in 32 positions and a limited variation in repeat sequences, leading to 13 distinct haplotypes. The overall nucleotide diversity (π) for the ectodomain of Iranian pftrap sequences was 0.00444 ± 0.00043, with the highest diversity in Domain IV. Alignment comparison of global PfTRAP sequences with T9/96 reference sequence indicated 96 amino acid replacements as well as extensive variable repeat sequences (9-23 repeats), which led to 192 haplotypes. Among the global isolates, the lowest nucleotide diversity was detected in French Guianan (0.00428 ± 0.00163) and Iranian (0.00444 ± 0.00043) pftrap sequences, and the most variation was observed in domains II and IV in all populations. The dN-dS value displayed the evidence of positive selection due to recombination and immune system pressure. The Fst analysis revealed a gene flow between African populations; however, genetic differentiation observed between Iranian and other populations probably was due to gene flow barriers. Both conserved and variable epitopes were predicted in B- and T-cell epitopes of PfTRAP antigen. The obtained results from this study could be helpful for developing a PfTRAP-based malaria vaccine.

Identification, Molecular Characterization, and In Silico Structural Analysis of Carboxypeptidase B2 of Anopheles stephensi.

Malaria is a vector-borne infectious disease that is considered a priority of the World Health Organization due to its enormous impacts on global health. Plasmodium spp. (Haemosporida: Plasmodiidae), Anopheles spp. (Diptera: Culicidae), and a suitable host are the key elements for malaria transmission. To disrupt the parasitic life cycle of malaria or prevent its transmission, these three key elements should be targeted by effective control strategies. Development of vaccines that interrupt malaria transmission is one of the solutions that has been recommended to the countries that aim to eliminate malaria. With respect to the important role of Anopheles stephensi in malaria transmission and involvement of Anopheles carboxypeptidase B1 in sexual parasite development, we characterized the second member of cpb gene family (cpbAs2) of An. Stephensi to provide some basic information and evaluate significance of cpbAs2's role in complementing sexual plasmodium development role of cpbAs1. The cpbAs2 mRNA sequence was characterized by 3' and 5' RACE and the structural features of its coded protein were studied by in silico modeling. The coding sequence and gene structure of cpbAs2 were determined empirically and compared with the in silico predictions from the An. stephensi genome sequencing project. Furthermore, homology modeling revealed that its structure is very similar to the structurally important domains of procarboxypeptidase B2 in humans. This study provides basic molecular and structural information about another member of the cpb gene family of An. stephensi. The reported results are informative and necessary for evaluation of the role of this gene in sexual parasite development by future studies.

Heterogeneity in the acquisition of naturally acquired antibodies to cell-traversal protein for ookinetes and sporozoites (CelTOS) and thrombospondin-related adhesion protein (TRAP) of Plasmodium falciparum in naturally infected patients from unstable malaria areas in Iran.

Currently, there is no subunit malaria vaccine capable of providing long-lasting protection, and a vaccine based on a single-antigen has shown moderate to unsatisfactory efficacies in clinical trials. As in malaria elimination and eradication strategies, the primary objective is reduction in disease and death due to P. falciparum, in the present investigation, for the first time, we attempted to determine and compare the naturally acquired immune responses to two well-recognized sporozoite antigens, cell-traversal protein for ookinetes and sporozoites (CelTOS) and thrombospondin-related adhesion protein (TRAP), in P. falciparum-infected individuals (n = 204) in low malaria transmission settings of Iran using ELISA. Besides, the profile of IgG isotype responses, the avidity of IgG, IgG1, and IgG3, and the association of anti-PfCelTOS and -PfTRAP antibodies with host age were evaluated. Positive antibody responses to PfCelTOS and PfTRAP antigens were detected in 16.2% and 31.9% of Iranian P. falciparum-infected individuals, respectively, indicating significantly lower immune response to PfCelTOS than PfTRAP (P <0.0001, McNemar's test). Also, among the positive samples for anti-PfCelTOS (n = 33) and -PfTRAP (n = 65) total IgG, the cytophilic IgG1 and IgG3 antibodies were predominant. A significant proportion of the examined positive responders had high- and intermediate-avidity for IgG (93.9%, 87.7%), IgG1 (96.3%, 87.7%), and IgG3 (76%, 78.7%) antibodies to both PfCelTOS and PfTRAP antigens, respectively, with no correlation with age (P >0.05; Spearman's correlation test). In conclusion, the present data suggests the acquisition of heterogenic immune responses to both antigens in the same patients naturally infected with P. falciparum from settings of low malaria transmission intensity in Iran in which their role in protection to malaria needs further study.

Analysis of genetic diversity and population structure of gene encoding cell-traversal protein for ookinetes and sporozoites (CelTOS) vaccine candidate antigen in global Plasmodium falciparum populations.

Plasmodium falciparum cell-traversal protein for ookinetes and sporozoites (PfCelTOS) has been reported as one of the most attractive malaria vaccine candidate antigens. To design a broadly effective malaria vaccine based on this antigen, it is crucial to have adequate information on genetic diversity in global PfCelTOS. Therefore, the extent of sequence diversity at the full-length of the pfceltos was assessed among both natural P. falciparum isolates collected from Iran (n = 93) and from available global pfceltos sequence data retrieved from PlasmoDB database (n = 159). Also, recombination, natural selection, the degree of genetic differentiation as well as the predicted immunodominant regions in PfCelTOS were analyzed. In total, 40 SNPs (including 1 synonymous and 39 non-synonymous) were detected in 34 positions, as compared to 3D7 sequence, which led to 66 distinct haplotypes with different frequencies. Among those haplotypes, 34 (51.5%, excluded from further analysis) were singleton haplotype and mostly detected among Senegalese parasite isolates. PfCelt-1 was found as predominant haplotype (32.6% total frequency) that was only detected in Iranian P. falciparum isolates. Nucleotide diversity was low in French Guiana (0.00236 ±â€¯0.00203) and Iranian (0.00259 ±â€¯0.00048) P. falciparum isolates in comparison with African populations. Evidence for positive selection by host immunity and intragenic recombination were detected that are two key factors responsible for gene evolution and genetic diversity of pfceltos gene. The results of Fst analysis and haplotype network revealed that PfCelTOS antigen displayed evident genetic structure between geographical parasite populations. In conclusion, the present analysis demonstrates that there is a limited antigenic diversity and geographic variation in global PfCelTOS, and this finding may be associated with the critical function of this antigen in cell traversal of the parasite in sporozoite and ookinete. Besides, most of the predicted B- and T-cell epitopes were located in the conserved region of the gene, but most of the amino acid replacements were located at the C-terminal region of PfCelTOS. The obtained results in this investigation could provide knowledge for better design of PfCelTOS-based malaria vaccine.

Limited genetic diversity in the global Plasmodium vivax Cell traversal protein of Ookinetes and Sporozoites (CelTOS) sequences; implications for PvCelTOS-based vaccine development.

Cell traversal protein of Ookinetes and Sporozoites (CelTOS) is a new malaria vaccine candidate antigen. Since one of the main challenges in malaria vaccine development is the extensive antigenic diversity of this parasite, local and global gene diversity analysis is of particular importance. Therefore, in this study, the genetic diversity of pvceltos gene was investigated among Iranian P. vivax isolates (n=46) and compared with available worldwide pvceltos sequences. One synonymous (C109A) and three amino acid replacements (V118L, K178T, and G179R) were observed in Iranian pvceltos sequences in compare with Sal-1 sequence leading to five haplotypes including PvCelt-A (GSVKGL, 13%), PvCelt-B (GSLKGL, 50%), PvCelt-C (GSLTGL, 17.4%), PvCelt-D (GSVTGL, 13%) and PvCelt-E (GSLTRL, 6.5%). However, amino acid replacements were observed in six positions (G10S, S40N, V118L/M, K178T, G179R/D and L181R) in PvCelTOS antigen of global isolates leading to 11 distinct haplotypes. PvCelt-A and PvCelt-B haplotypes were the most common haplotypes in the world. The overall nucleotide diversity for Iranian isolates was 0.00169, while, the level of nucleotide diversity was ranged from 0.00252 for Thailand to 0.00022 for Peru populations in the world. The analysis of SNPs in relation with the predicted immunodominant regions revealed that only K178T and G179R SNPs are located in putative B-cell epitopes. All replacements were located in CD4+ and/or CD8+ T-cell epitopes. However, the majority of epitopes are located in conserved regions. Knowing whether these changes may alter the affinity of the epitopes for antibodies and/or MHC molecules remains to be investigated in experimental studies. In conclusion, the present study showed a very limited genetic diversity in pvceltos gene among the global clinical isolates that can be regarded as a potential candidate antigen to apply for vivax-based malaria vaccine development.

Naturally acquired immune responses to thrombospondin-related adhesion protein (TRAP) of Plasmodium vivax in patients from areas of unstable malaria transmission.

A key tool for the control, elimination, and eradication of Plasmodium vivax is the development of an effective vaccine. The thrombospondin-related adhesion protein (TRAP) is one of the major sporozoite antigens that plays an important role in the invasion of mosquito salivary glands and hepatocytes by sporozoites. The main goal of this study was to evaluate the naturally acquired antibodies to the P. vivax TRAP (PvTRAP) in patients from malaria-endemic areas of Iran (n=116), Afghanistan (n=50), and Pakistan (n=50). The PvTRAP gene was expressed in Escherichia coli Rosetta (DE3)-pET23a and used as antigen in enzyme-linked immunosorbent assay (ELISA). The profile of immunoglobulin G (IgG) isotype and the avidity of IgG, IgG1, and IgG3 to PvTRAP, as well as the association between anti-PvTRAP isotype responses and host age were evaluated. Only 42.24% of Iranian, 38% of Afghani, and 44% of Pakistani patients infected with P. vivax had positive anti-PvTRAP IgG, and the prevalence of responders in the three countries did not differ significantly (P>0.05). Moreover, the prevalence of IgG1 and IgG3 antibody responses to PvTRAP showed no significant correlation with age (P>0.05). Individuals exposed to vivax malaria in the unstable malaria transmission areas are able to produce antibodies to the TRAP antigen at all ages in response to P. vivax infections. Finally, the presence of mature IgG1 and IgG3 antibodies with high to intermediate avidity against PvTRAP antigen (>60%) provide more information to understand the interactions between the host and P. vivax parasite. In summary, the present study provides data that support the rational development of an effective pre-erythrocytic stage vaccine based on PvTRAP antigen.

Worldwide population genetic analysis and natural selection in the Plasmodium vivax Generative Cell Specific 1 (PvGCS1) as a transmission-blocking vaccine candidate.

GENERATIVE CELL SPECIFIC 1 (GCS1) is one of the Transmission Blocking Vaccine (TBV) candidate antigens, which is expressed on the surface of male gametocytes and gametes of Plasmodium species. Since antigenic diversity could inhibit the successful development of a malaria vaccine, it is crucial to determine the diversity of gcs1 gene in global malaria-endemic areas. Therefore, gene diversity and selection of gcs1 gene were analyzed in Iranian Plasmodium vivax isolates (n=52) and compared with the corresponding sequences from worldwide clinical P. vivax isolates available in PlasmoDB database. Totally 12 SNPs were detected in the pvgcs1 sequences as compared to Sal-1 sequence. Five out of 12 SNPs including three synonymous (T797C, G1559A, and G1667T) and two amino acid replacements (Y133S and Q634P) were detected in Iranian pvgcs1 sequences. According to four amino acid replacements (Y133S, N575S, Q634P and D637N) observed in all world PvGCS1 sequences, totally 5 PvGCS1 haplotypes were detected in the world, that three of them observed in Iranian isolates including the PvGCS-A (133S/634Q, 92.3%), PvGCS-B (133Y/634Q, 5.8%), and PvGCS-C (133S/634P, 1.9%). The overall nucleotide diversity (π) for all 52 sequences of Iranian pvgcs1 gene was 0.00018±0.00006, and the value of dN-dS (-0.00031) were negative, however, it was not statistically significant. In comparison with global isolates, Iranian and PNG pvgcs1 sequences had the lowest nucleotide and haplotype diversity, while the highest nucleotide and haplotype diversity was observed in China population. Moreover, epitope prediction in this antigen showed that all B-cell epitopes were located in conserved regions. However, Q634P (in one Iranian isolate) and D637N (observed in Thailand, China, Vietnam and North Korea) mutations are involved in predicted IURs. The obtained results in this study could be used in development of PvGCS1 based malaria vaccine.

Population genetics structure of Plasmodium vivax circumsporozoite protein during the elimination process in low and unstable malaria transmission areas, southeast of Iran.

In Iran, the prevalence of Plasmodium falciparum and Plasmodium vivax has dropped after a national malaria elimination program was launched. To estimate the likelihood of success and to measure the outcome of malaria intervention tools during elimination programs (2008-2012), the population genetic surveys of Iranian P. vivax isolates (n=60) were carried out using the CSP genetic marker. The results were compared with a similar work that was carried out during a control phase (2000-2003) in the same study areas. Based on PCR-RFLP analysis, 49 (81.67%) of 60 studied samples were VK210 and 11 (18.33%) were VK247 with no mixed genotypes. However, 10.97% of P. vivax isolates of control phase harbored the mixed genotypes. Sequencing analysis of 50 pvcsp gene showed 14 distinct haplotypes, of which 11 and 3 were VK210 and VK247 types, respectively. However, during the control phase, 19 distinct subtypes (11 VK210 and 8 VK247) were reported. Also, 7 of 11 VK210 and the VK247F subtypes were new, and 3 out of 7 new VK210 and VK247F were isolated from the patients with Pakistani nationality. The lower nucleotide diversity per site (π=0.02017±0.00436 and π=0.04525±0.00255) and haplotype diversity (Hd=0.513±0.093 and Hd=0.691±0.128) as well as lower In/Del haplotype [Hd(i)=0.243 and 0] and nucleotide diversity [π(i)=0.00078 and 0] were recorded for VK210 and VK247of the elimination samples, respectively. In conclusion, the comparison of PRMs and RATs in CRR along with the polymorphism analysis of the sequence lengths, SNPs, and In/Del polymorphisms in all analyzed samples showed lower genetic diversity for PvCSP in the elimination samples. Also, although there is a turnover of P. vivax parasite genotypes in the study areas, reduction in genetic diversity and transmission was detected due to scaling-up of the intervention tools during an elimination program in Iran. This notable challenge of the elimination program must be taken into account and controlled by active surveillance for limiting both reintroductions of new allelic forms as well as the spread of drug-resistant parasite to prevent any disease outbreaks.

A comparative study on worldwide genetic diversity and population structure analysis of Plasmodium vivax thrombospondin-related adhesive protein (PvTRAP) and its implications for the vivax vaccine design.

Plasmodium vivax thrombospondin-related anonymous protein (PvTRAP) is a promising malaria vaccine candidate; however, it exhibits sequence heterogeneity. Therefore, to design a broadly protective vivax vaccine, it is essential to have adequate information on signatures of selection and geospatial genetic diversity of global PvTRAP. For this purpose, 50 Iranian pvtrap were sequenced and compared with related available global sequences in GenBank. The nucleotide sequence analysis of Iranian pvtrap in comparison with the Sal-1 sequence showed the occurrence of 15 SNPs, and all sites were dimorphic. In total, 12 amino acid substitutions were detected and 2 of which were novel, resulting in 10 haplotypes that 8 of them were not reported in any other geographic regions. In comparison with global population, haplotype and nucleotide diversities were lowest in South Korean populations while higher levels of diversities were observed in Thai and Brazilian P. vivax populations. All 12 amino acid replacements in ectodomain of Iranian PvTRAP were distributed in predicted either B- or T-cells epitope as well as intrinsically unstructured/disordered regions (IURs). The present results revealed that observing the relatively low-level diversity in PvTRAP protein might actually be selected by immune response. In summary, the present analysis in parallel to the limited available published data has shown that genetic diversity in the global pvtrap exhibits low-level diversity and geographic variation. These results are of practical significance for the strategic development and deployment of control measures in particular for development of PvTRAP-based malaria vaccine.