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It is obvious that there is a critical need for an efficient malaria vaccine to accelerate malaria eradication. Currently, recombinant subunit vaccination against malaria using proteins and peptides is gaining attention. However, one of the major drawbacks of this approach is the lack of an efficient and durable immune response. Therefore, subunit vaccines require adjuvants to make the vaccine sufficiently immunogenic. Considering the history of the RTS,S vaccine, it seems likely that no single adjuvant is capable of eliciting all the protective immune responses required in many malarial subunit vaccines and the use of combination adjuvants will be increasingly important as the science of malaria vaccines advances. In light of this, it appears that identifying the most effective mixture of adjuvants with minimal adverse effects offers tremendous opportunities in improving the efficacy of vaccines against malaria. Owing to the importance of a multi-adjuvanted approach in subunit malaria vaccine development, this review paper outlines some of the best known combination adjuvants used in malaria subunit vaccines, focusing on their proposed mechanisms of action, their immunological properties, and their notable results. The aim of the present review is to consolidate these findings to aid the application of these combination adjuvants in experimental malaria vaccines.

Background: Streptococcus mutans as an acid‑generator of biofilm, sugar as a caries‑conducive environment, and oral hygiene have been implicated as major etiological agents in dental caries. This study was designed to assess the association and impact of S. mutans, sugar consumption, and tooth brushing on decayed, missing, and filled teeth (DMFT) score in Iranian 20–30‑year‑old individuals and compare the effect of the three mentioned factors to find the most effective one. Materials and Methods: In this cross‑sectional study, 459 adults completed a Sugar Frequency Questionnaire and were examined for dental caries using DMFT index, sugar consumption level, and tooth brushing frequency per day. Saliva and plaque samples were collected, and the target population without Streptococcus sobrinus in their microbial oral community was selected using polymerase chain reaction technique. Data were analyzed by one‑way analysis of variance and multiple linear regression tests (α = 0.05). Results: Nearly 77.1% of the study population were harboring S. mutans. Mean DMFT of the population was 6.62. Mean comparison analysis showed that there is a strong relationship between S. mutans existence in mouth flora and DMFT scores (P < 0.0001). Multiple linear regression test showed higher percentage of S. mutans contribution (28.2%) in DMFT score changes than sugar consumption (3.6%) and tooth brushing (0.7%). Conclusions: This study provides a recent report from S. mutans frequency and DMFT score in Iranian adult population. It is also the first study that shows significantly higher impact of S. mutans in microbial population of mouth microflora on caries development than sugar consumption and oral hygiene. Accordingly, S. mutans screening program should be more highlighted in preventive strategies.

One of the main challenges in protein-based vaccines is the poor immunogenicity of antigens, which can be solved by the use of adjuvants. Advax is a novel microparticle polysaccharide adjuvant that in combination with antigens can induce both cellular and humoral immunity based on the intrinsic features of the antigen. It has been shown that poly(I:C) can be a suitable adjuvant for the PfMSP-142-based malaria vaccine. Advax is a suitable co-adjuvant for poly(I:C) to increase its half-life and reduce dose-dependent toxicity.To investigate whether advax alone or advax /poly(I:C) combination can enhance the immunogenicity with increased parasite inhibitory anti-PfMSP-142 antibodies in comparison to poly(I:C).Mice groups were inoculated with rPfMSP-142 alone or formulated in poly(I:C), poly(I:C)/advax, or advax. Then, humoral and cellular immune responses, the ratio of Th1/Th2 and growth inhibitory activity of induced antibodies were analyzed.Poly(I:C)/advax formulated PfMSP-142 induced higher levels of anti-PfMSP-142 IgG, IgG2a, and IgG2b antibodies relative to poly(I:C)-formulated PfMSP-142. The maximum ratio of IFN-?/IL-4 (50.13) and IgG2a/IgG1 (2.65), was induced in mice receivedadvax-formulated PfMSP-142. Besides, poly(I:C)/advax formulated PfMSP-142 induced a higher ratio of IFN-?/IL-4 (25.33) and IgG2a/IgG1 (1.89) when compared with poly(I:C) alone. Strong growth inhibitory activity was observed in antibodies induced in mice received poly(I:C)/advax-formulated PfMSP-142.These findings indicate that advax is a favorable adjuvant to be combined with poly(I:C), and this combination of adjuvants could induce Th1 immune responses and growth inhibitory antibodies against rPfMSP-142.

A vaccine targeting Plasmodium vivax signifies an additional necessary tool when considering the malaria elimination/eradication goal. In this study, in vivo immunological evaluation of two novel engineered proteins of P. vivax circumsporozoite (PvCS127 and PvCS712) with two different arrangements of the repeat sequences of VK210 and VK247 was assessed. The immunological properties of the Escherichia coli-expressed chimeric proteins were evaluated by the immunization of C57BL/6 mice administered in NLX, CpG-ODNs, and QS21, alone or in combination as adjuvants. A significant increase in anti-rPvCS127 and -rPvCS712 IgG antibodies was observed in all the vaccine groups after the first boost, and the predominant isotypes were high-avidity cytophilic antibodies, IgG2b, and IgG2c. The highest ratio of IgG2b/IgG1 (2.74) and IgG2c/IgG1 (2.1) levels was detected in mouse groups immunized with rPvCS712 + NLX-CpG-QS21. The lowest level of IFN-γ (mean: 441 and 588 pg/mL, respectively) was produced by the mouse group, which received both antigens without any adjuvant, while significant levels of IFN-γ were detected in the mouse groups immunized with rPvCS127- or rPvCS712-NLX-CpG-QS21 formulation (mean: 1200 and 3092 pg/mL, respectively). The current results indicated that in C57BL/6 mice, both recombinant antigens were efficient immunogens and could induce humoral and cellular immune responses and their combination with three Th1 potent adjuvants had an impact on the magnitude and the quality of humoral responses (specific antibody subclasses, titer, and high avidity). Although the overall response was marginally higher for rPvCS712 than rPvCS127, all immunized mice induced some immune responses against both proteins, and the present findings indicate that rPvCS127 and rPvCS712 meet the criteria to be potentially useful vaccine candidates against P. vivax malaria.

Malaria, a mosquito-borne infection, is the most widespread parasitic disease. Despite numerous efforts to eradicate malaria, this disease is still a health concern worldwide. Owing to insecticide-resistant vectors and drug-resistant parasites, available controlling measures are insufficient to achieve a malaria-free world. Thus, there is an urgent need for new intervention tools such as efficient malaria vaccines. Subunit vaccines are the most promising malaria vaccines under development. However, one of the major drawbacks of subunit vaccines is the lack of efficient and durable immune responses including antigen-specific antibody, CD4+, and CD8+ T-cell responses, long-lived plasma cells, memory cells, and functional antibodies for parasite neutralization or inhibition of parasite invasion. These types of responses could be induced by whole organism vaccines, but eliciting these responses with subunit vaccines has been proven to be more challenging. Consequently, subunit vaccines require several policies to overcome these challenges. In this review, we address common approaches that can improve the efficacy of subunit vaccines against malaria.

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 (P0.0001, 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 (P0.05; 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.

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 ± 0.00203) and Iranian (0.00259 ± 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.

Background: The emergence of antimalarial drug resistance is one of the great problems of malaria control and elimination of worldwide. In order to overcome anti-malarial drug resistance, cytotoxicity and in vivo anti-Plasmodiumal activity of four different Iranian propolis were investigated. Materials and Methods:Four Iranian propolis samples were collected from four different regions of Iran and extracted by %70 ethanol and dichloromethane. The cytotoxicity of ethanolic and dichloromethane extract of propolis, using L969 fibroblast cell lines, were evaluated by MTT assay. The in vitro anti-Plasmodial activities of the propolis samples on BALB/c mice were assayed. Results:The cytotoxicity results showed that ethanol and dichloromethane extracts of four Iranian propolis samples at doses of 25, 50, 100 and 200 µg/ml were non-toxic (P

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.

Plasmodium falciparum cell-traversal protein for ookinetes and sporozoites (PfCelTOS) is an advanced vaccine candidate that has a crucial role in the traversal of the malaria parasite in both mosquito and mammalian hosts. As recombinant purified proteins are normally poor immunogens, they require to be admixed with an adjuvant(s); therefore, the objective of the present study was to evaluate the capacity of different vaccine adjuvants, monophosphoryl lipid A (MPL), CpG, and Quillaja saponaria Molina fraction 21 (QS-21), alone or in combination (MCQ [MPL/CpG/QS-21]), to enhance the immunogenicity of Escherichia coli-expressed PfCelTOS in BALB/c mice. This goal was achieved by the assessment of anti-PfCelTOS IgG antibodies (level, titer, IgG isotype profile, avidity, and persistence) and extracellular Th1 cytokines using an enzyme-linked immunosorbent assay (ELISA) on postimmunized BALB/c mouse sera and PfCelTOS-stimulated splenocytes, respectively. Also, an assessment of the transmission-reducing activity (TRA) of anti-PfCelTOS obtained from different vaccine groups was carried out in female Anopheles stephensi mosquitoes by using a standard membrane feeding assay (SMFA). In comparison to PfCelTOS alone, administration of PfCelTOS with three distinct potent Th1 adjuvants in vaccine mouse groups showed enhancement and improvement of PfCelTOS immunogenicity that generated more bias toward a Th1 response with significantly enhanced titers and avidity of the anti-PfCelTOS responses that could impair ookinete development in A. stephensi. However, immunization of mice with PfCelTOS with MCQ mixture adjuvants resulted in the highest levels of induction of antibody titers, avidity, and inhibitory antibodies in oocyst development (88%/26.7% reductions in intensity/prevalence) in A. stephensi. It could be suggested that adjuvant combinations with different mechanisms stimulate better functional antibody responses than adjuvants individually against challenging diseases such as malaria.

Background Plasmodium falciparum parasite is the most deadly species of human malaria, and the development of an effective vaccine that prevents P. falciparum infection and transmission is a key target for malarial elimination and eradication programmes. P. falciparum cell-traversal protein for ookinetes and sporozoites (PfCelTOS) is an advanced vaccine candidate. A comparative study was performed to characterize the immune responses in BALB/c mouse immunized with Escherichia coli-expressed recombinant PfCelTOS (rPfCelTOS) in toll-like receptor (TLR)-based adjuvants, CpG and Poly I:C alone or in combination (CpG + Poly I:C), followed by the assessment of transmission-reducing activity (TRA) of anti-rPfCelTOS antibodies obtained from different vaccine groups in Anopheles stephensi. Methods The aim of the current work was achieved by head-to-head comparison of the vaccine groups using conventional and avidity enzyme-linked immunosorbent assay (ELISA), immunofluorescence test (IFAT), and standard membrane feeding assay (SMFA). Results Comparing to rPfCelTOS alone, administration of rPfCelTOS with two distinct TLR-based adjuvants in vaccine mouse groups showed a significant increase in responses (antibody level, IgG subclass analysis, avidity, and Th1 cytokines) and was able to induce reasonable transmission-reducing activity. Also, comparable functional activity of anti-rPfCelTOS antibodies was found in group that received antigen in either CpG or Poly I:C (69.9%/20% and 73.5%/24.4%, respectively, reductions in intensity/prevalence). However, the vaccine group receiving rPfCelTOS in combination with CpG + Poly I:C showed a significant induction in antibody titers and inhibitory antibodies in oocysts development (78.3%/19.6% reductions in intensity/prevalence) in An. stephensi. Conclusions A key finding in this investigation is that rPfCelTOS administered alone in BALB/c mouse is poorly immunogenic, with relatively low IgG level, avidity, inhibitory antibodies, and mixed Th1/Th2 responses. However, immunological characteristic (IgG level, cytophilic IgG2a and IgG2b, avidity, and Th1 cytokines) and TRA of anti-rPfCelTOS significantly enhanced in the presence of co-administration of TLR-based adjuvants, confirming that targeting TLRs would be an effective means for the enhancement of inducing TRA against rPfCelTOS. Electronic supplementary material The online version of this article (10.1186/s12936-019-2773-3) contains supplementary material, which is available to authorized users.

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.

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.

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

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.

International audience; Despite considerable efforts toward vaccine development over decades, there is no available effective vaccine against Plasmodium vivax. Thrombospondin-related adhesive protein of P. vivax (PvTRAP) is essential for sporozoite motility and invasions into mosquito’s salivary gland and vertebrate’s hepatocyte; hence, it is a promising target for pre-erythrocytic vaccine. In the current investigation, the role of antibodies and cellular immune responses induced by purified recombinant PvTRAP (rPvTRAP) delivered in three adjuvants, naloxone (NLX), CpG oligodeoxynucleotides ODN1826 (CpG-ODN), and 3-O-deacylated monophosphoryl lipid A (MPL), alone and in combination was evaluated in immunized C57BL/6 mice. The highest level and the avidity of anti-PvTRAP IgG (mean OD490nm2.55), IgG2b (mean OD490nm 1.68), and IgG2c (mean OD490nm 1.466) were identified in the group received rPvTRA/NLX–MPL–CpG. This group also presented the highest IgG2c/ IgG1 (2.58) and IgG2b/IgG1 (2.95) ratio when compared to all other groups, and among the adjuvant groups, the lowest IgG2c/IgG1 (1.86) and IgG2b/IgG1 (2.25) ratio was observed in mice receiving rPvTRAP/NLX. Mice receiving rPvTRAP/ adjuvants induced significantly the higher levels of interferon gamma (IFN-γ), low level of detectable IL-10, and no detectable IL-4 production. The present result revealed that PvTRAP is immunogenic and its administration with CPG, MPL, and NLX in C57BL/6 mice induced Th1 immune response. Besides, the rPvTRAP delivery in the mixed formulation of those adjuvants had more potential to increase the level, avidity, and persistence of anti-TRAP antibodies. However, it warrants further assessment to test the blocking activity of the produced antibodies in immunized mice with different adjuvant formulations.

The binding domain of Plasmodium vivax Duffy binding protein (PvDBP-II) is a promising blood-stage vaccine candidate for vivax malaria. For the development of a successful vivax malaria vaccine based on DBP-II, the antigenic diversity and also naturally occurring functional antibodies to different PvDBP-II variant types in the various populations must be determined. However, similar to other blood-stage antigens, allelic variation within the PvDBP-II is a fundamental challenge for the development of a broadly efficient vaccine. The present study was performed to define whether the polymorphisms in PvDBP-II influence the nature of functional inhibitory activity of naturally acquired or induced anti-DBP-II antibodies in mice. In this investigation, five genetically distinct variants of PvDBP-II were transiently expressed on the COS-7 cell surface. Erythrocyte-binding inhibition assay (EBIA) was performed using human sera infected with corresponding and non-corresponding P. vivax variants as well as by the use of mice sera immunized with different expressed recombinant PvDBP-IIs. EBIA results showed that the inhibitory percentage varied between 50 and 63 % by using sera from infected individuals, and in case of mouse antisera, inhibition was in the range of 76-86 %. Interestingly, no significant difference was detected in red blood cell binding inhibition when different PvDBP-II variants on the COS-7 cell surfaces were incubated with heterologous and homologous sera infected with PvDBP-II variants. This suggests that the detected polymorphisms in all five forms of PvDBP-II may not affect functional activity of anti-DBP-II antibodies. In conclusion, our results revealed that there are functional cross-reactive antibody responses to heterologous PvDBP-II variants that might provide a broader inhibitory response against all, or at least the majority of strains compared to single allele of this protein that should be considered in development of PvDBP-II-based vaccine.

In Sudan, Plasmodium vivax accounts for approximately 5-10% of malaria cases. This study was carried out to determine the genetic diversity of P. vivax population from Sudan by analyzing the polymorphism of P. vivax csp (pvcsp) and pvmsp-3α genes. Blood samples (n=76) were taken from suspected malaria cases from 2012-2013 in three health centers of Eastern and Central Sudan. Parasite detection was performed by microscopy and molecular techniques, and genotyping of both genes was performed by PCR-RFLP followed by DNA sequence for only pvcsp gene (n=30). Based on microscopy analysis, 76 (%100) patients were infected with P. vivax, whereas nested-PCR results showed that 86.8% (n=66), 3.9% (n=3), and 3.9% (n=3) of tested samples had P. vivax as well as Plasmodium falciparum mono- and mixed infections, respectively. Four out of 76 samples had no results in molecular diagnosis. All sequenced samples were found to be of VK210 (100%) genotype with six distinct amino acid haplotypes, and 210A (66.7%) was the most prevalent haplotype. The Sudanese isolates displayed variations in the peptide repeat motifs (PRMs) ranging from 17 to 19 with GDRADGQPA (PRM1), GDRAAGQPA (PRM2) and DDRAAGQPA (PRM3). Also, 54 polymorphic sites with 56 mutations were found in repeat and post-repeat regions of the pvcsp and the overall nucleotide diversity (π) was 0.02149±0.00539. A negative value of dN-dS (-0.0344) was found that suggested a significant purifying selection of Sudanese pvcsp, (Z test, P

The circumsporozoite protein (CSP) of the malaria parasite Plasmodium vivax is a major pre-erythrocyte vaccine candidate. The protein has a central repeat region that belongs to one of repeat families (VK210, VK247, and the P. vivax-like). In the present study, computer modelling was employed to select chimeric proteins, comprising the conserved regions and different arrangements of the repeat elements (VK210 and VK247), whose structure is similar to that of the native counterparts. DNA encoding the selected chimeras (named CS127 and CS712) were synthetically constructed based on E. coli codons, then cloned and expressed. Mouse monoclonal antibodies (mAbs; anti-Pv-210-CDC and -Pv-247-CDC), recognized the chimeric antigens in ELISA, indicating correct conformation and accessibility of the B-cell epitopes. ELISA using IgG from plasma samples collected from 221 Iranian patients with acute P. vivax showed that only 49.32% of the samples reacted to both CS127 and CS712 proteins. The dominant subclass for the two chimeras was IgG1 (48% of the positive responders, OD492=0.777±0.420 for CS127; 48.41% of the positive responders, OD492=0.862±0.423 for CS712, with no statistically significant difference P>0.05; Wilcoxon signed ranks test). Binding assays showed that both chimeric proteins bound to immobilized heparan sulphate and HepG2 hepatocyte cells in a concentration-dependent manner, saturable at 80μg/mL. Additionally, anti-CS127 and -CS712 antibodies raised in mice recognized the native protein on the surface of P. vivax sporozoite with high intensity, confirming the presence of common epitopes between the recombinant forms and the native proteins. In summary, despite structural differences at the molecular level, the expression levels of both chimeras were satisfactory, and their conformational structure retained biological function, thus supporting their potential for use in the development of vivax-based vaccine.

Malaria vaccine development has been confronted with various challenges such as poor immunogenicity of malaria vaccine candidate antigens, which is considered as the main challenge. However, this problem can be managed using appropriate formulations of antigens and adjuvants. Poly(I:C) is a potent Th1 inducer and a human compatible adjuvant capable of stimulating both B- and T-cell immunity. Plasmodium falciparum merozoite surface protein 142 (PfMSP-142) is a promising vaccine candidate for blood stage of malaria that has faced several difficulties in clinical trials, mainly due to improper adjuvants. Therefore, in the current study, poly(I:C), as a potent Th1 inducer adjuvant, was evaluated to improve the immunogenicity of recombinant PfMSP-142, when compared to CFA/IFA, as reference adjuvant. Poly(I:C) produced high level and titers of anti-PfMSP-142 IgG antibodies in which was comparable to CFA/IFA adjuvant. In addition, PfMSP-142 formulated with poly(I:C) elicited a higher ratio of IFN-γ/IL-4 (23.9) and IgG2a/IgG1 (3.77) with more persistent, higher avidity, and titer of IgG2a relative to CFA/IFA, indicating a potent Th1 immune response. Poly(I:C) could also help to induce anti-PfMSP-142 antibodies with higher growth-inhibitory activity than CFA/IFA. Altogether, the results of the current study demonstrated that poly(I:C) is a potent adjuvant that can be appropriate for being used in PfMSP-142-based vaccine formulations.

Eradication of malaria will depend on discovery of new intervention tools such as anti-malarial drugs. Due to the increasing interest in the application of propolis against significant clinical pathogenic agents, the aim of the present investigation was to evaluate the anti-plasmodial effect of Iranian propolis extracts against chloroquine (CQ)-sensitive Plasmodium falciparum 3D7 and Plasmodium berghei (ANKA strain).Crude samples of honeybee (Apis mellifera) propolis were collected from four provinces in northern (Kalaleh, Golestan), northeastern (Chenaran, Razavi Khorasan), central (Taleghan, Alborz) and western (Morad Beyg, Hamedan) areas of Iran with different types of flora. The dried propolis samples were extracted with three different solvents, including ethanol 70% (EtOH), ethyl acetate (EA) and dichloromethane (DCM).All extracts were shown to have in vitro anti-plasmodial activity with IC50 ranging from 16.263 to 80.012 µg/mL using parasite lactate dehydrogenase (pLDH) assay. The DCM extract of Morad Beyg propolis indicated the highest anti-plasmodial activity (IC50: 16.263 ± 2.910 μg/mL; P = 0.027, Kruskal-Wallis H-test). The samples were also evaluated in mice for their in vivo anti-plasmodial effect. The curative effect against established infection (Rane test) showed that both extracts at all doses (50, 100, and 200 mg/kgBW) produced anti-plasmodial activity against the parasite. Furthermore, using gas chromatography-mass spectrometry (GC-MS), the quantity of flavonoids in DCM and EtOH 70% extracts were found to be 7.42% and 3.10%, respectively.The potent anti-plasmodial activity of both EtOH 70% and DCM extracts of the propolis of Morad Beyg, Hamedan suggests further analyses of individual components to assess its utilization as anti-malarial drugs.

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.

Objective: Different studies have shown an association of TLR4 polymorphisms with susceptibility/resistance to malaria disease. In the current immunogenetic study, we assessed the TLR4 genotypes formed by the two common single nucleotide polymorphisms (SNPs) (Asp299Gly and Thr399Ile) in the co-segregate state in Baluchi Plasmodium falciparum infected and healthy populations from malaria hypoendemic areas of Iran. The study was performed to evaluate the distribution and correlation of TLR4 co-segregating genotypes in patients with mild malaria. Moreover, the frequency of these genotypes was compared with reported results from other populations in similar or contrasting malaria settings around the world. Materials and Methods: In this case control study, the presence of 2 SNPs in the TLR4 gene (Asp299Gly and Thr399Ile) were analyzed in 350 Baluchi patients with mild malaria and 350 unrelated healthy controls by using polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) techniques followed by sequencing analysis. Differences in the TLR4 co-segregate genotype frequencies among the studied group were determined by Fisher’s exact test. Results: Although the distribution of the two commonly co-segregating TLR4 genotypes presented a diverse and distinct pattern in the Baluchi population, no significant difference was detected between the cases and controls (p>0.05). A lower frequency of TLR4 Asp299Gly/Thr399Thr was observed in Baluchis with mild malaria compared to African populations (p< 0.05). Conclusion: Differences in the co-segregation patterns of TLR4 Asp299Gly/Thr399Ile genotypes in the Baluchi population compared to other malaria endemic populations may suggest different local evolutionary pressure on TLR4 polymorphisms by malaria in this region. The higher frequency of Asp299Gly/Thr399Ile genotypes among the Baluchi population compared with the African population (p< 0.05) which suffers from a larger number of severe cases might suggest that this genotype has a role in protecting against severe malaria. These findings are useful for further understanding the pathogenesis of severe malaria.

In this study, the nature and extent of genetic diversity of Plasmodium vivax populations circulating in Afghanistan have been investigated by analyzing three genetic markers: csp, msp-1, and msp-3 alpha. Blood samples (n=202) were collected from patients presenting with vivax malaria from south-western (Herat) and south-eastern (Nangarhar) parts of Afghanistan, and analysed using nested-PCR/RFLP and sequencing methods. Genotyping pvmsp-1 revealed type 1, type 2 and recombinant type 3 allelic variants, with type 1 predominant in parasites in both study areas. The sequence analysis of 57 P. vivax isolates identified a total of 26 distinct alleles. Genotyping pvcsp gene showed that VK210 type (86.6%) is predominant in Afghanistan. Moreover, three major types of the pvmsp-3 alpha locus: type A, type B and type C were distinguished among Afghani isolates. The predominant fragments among Nangarhar and Herat parasites were type A (70.8% and 67.9%, respectively). PCR/RFLP products with Hha I and Alu I were detected 52 and 38 distinct variants among Nangarhar and Herat isolates, respectively. These results strongly indicate that the P. vivax populations in Afghanistan are highly diverse.

International audience; C-terminal region of merozoite surface protein-1 of Plasmodium falciparum (PfMSP-1) isolated from different parts of the world revealed sequence variability, however no data exist on sequence heterogeneity of this region from Iran. To address this question, DNA encoding the carboxyl (C)-terminal region of PfMSP-1 was amplified in 144 Iranian P. falciparum clinical isolates, using allele type-specific primers. In this study both MAD20 (88.2%) and K1 (7.6%) types were detected. Sequence analysis of 33 and 92 fragments corresponding to pfmsp-1(42) and pfmsp-1(19) revealed eight (15MAD1-15MAD7 and 15KCH) and five [A1 (E/TSR/L), A2 (Q/KNG/F), A3 (E/KNG/F), A4 (E/TSG/L), and A5 (Q/KNG/L)] distinct haplotypes, respectively. E/TSG/L variant type was the predominant haplotype, and reported only from Thailand and India, but E/KNG/L is widespread in Africa, Asia, and Latin America; but not found among Iranian isolates. In summary, result of this study indicates limited antigenic diversity, and thus support the potential utility of the C-terminal region of PfMSP-1 in designing polyvalent vaccine constructs.

Acquired antibody responses following natural infection provide valuable information for selection of candidate antigens for malaria vaccines. Apical membrane antigen-1 of Plasmodium vivax (PvAMA-1) has potential as a component of a subunit vaccine for vivax malaria. In addition, genetic diversity in this antigen is responsible for challenges in the development of an effective PvAMA-1 based vaccine. Therefore, the main aim of this study was to determine whether allelic polymorphisms in pvama-1 influence the recognition of naturally occurring antibodies. Also, the profile of IgG isotypes to two sequence types of PvAMA-1 antigen was evaluated among subjects exposed to P. vivax in areas of low and unstable transmission.For this purpose, the two variant forms of PvAMA-1 (PvAMA-1A and B) were expressed in Escherichia coli M15-pQE30 system using genomic DNA from Iranian individuals with patent P. vivax infection. Anti-AMA-1 response and isotype composition to two variant forms were measured in target P. vivax-infected individuals (n = 110, 2 to 65 years old) using Enzyme-linked immunosorbent assay.The results showed that 65.5% of the studied individuals had positive IgG responses to two PvAMA-1 variants, and the prevalence of responders did not differ significantly (P = 0.32). Also, a marked isotype switching to cytophilic (IgG1 /IgG3) antibodies was evident with increasing age, and adults responded more frequently to these antigens than did younger children.The presence of mature, protective isotype antibodies and equal immune responses to two genetically distinct variant forms of antigens in individuals from low transmission areas implicates that one of these forms could be used in a universal blood-stage vaccine based on PvAMA-1 antigen.

International audience; Plasmodium vivax apical membrane antigen-1 (PvAMA-1) is a major candidate antigen for human malaria vaccine. In the present study, polymorphism of pvama-1 among Iranian isolates was investigated to generate useful information on this vaccine candidate antigen, which is required for the rational design of a vaccine against P. vivax. Blood samples were collected from P. vivax-infected Iranian patients during 2009-2010. Of 99 collected isolates, 37 were analyzed for almost the entire pvama-1 gene using sequencing. The overall nucleotide diversity (π) was 0.00826±0.0004 and the majority of polymorphic sites were identified in domain I (DI) of the pvama-1 gene. Neutrality analysis using Tajima's D, Fu and Li's D* and F* and McDonald Kreitman tests showed a significant positive departure from neutral substitution patterns, indicating a possible balancing selection across the entire ectodomain and DI sequences of pvama-1 gene. However, no evidence was found for the balancing selection in DII and DIII regions of Iranian PvAMA-1. Also, 29 haplotypes with different frequencies were identified and the overall haplotype diversity was 0.982±0.012. Epitope mapping prediction of PvAMA-1 showed the potential B-cell epitopes across DI-DIII overlap with E145K, P210S, R249H, G253E, K352E, R438H and N445D mutations; however, no mutation has been found in intrinsically unstructured/disordered regions. The fixation index (Fst) estimation between Iran and the closest geographical sites such as India (0.0707) showed a slight geographical genetic differentiation; however, the Fst estimation between Iran and Thailand (0.1253) suggested a moderate geographical isolation. In summary, genetic investigation in pvama-1 among Iranian P. vivax isolates indicates that this antigen showed limited antigenic diversity and most of the detected mutations are located outside B-cell epitopes. Therefore, the present results have significant implications in understanding the nature of P. vivax population circulating in Iran as well as in providing useful information for malaria vaccine development based on this antigen.

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.

To date, there is no information on the genetic diversity of the circumsporozoite protein (CSP), a leading vaccine candidate, in Plasmodium vivax populations circulating in Iran. The gene for this protein, Pvcsp, was amplified from 374 P. vivax isolates collected in the temperate northern, and in the tropical southern endemic areas. PCR-RFLP analysis of the repeated central region revealed that the parasites collected in the northern area were almost exclusively of the VK210 type. Parasites collected in the south-eastern areas were of both VK210 and VK247 types. We detected VK210 parasite in 70.5% of the samples, VK247 parasites in 17.5% and mixed type infections in 12% of the isolates. Sequence analysis of 137 isolates obtained from both areas identified a total of 25 distinct genotypes. The degree of genetic diversity was generally higher for the tropical (21 genotypes) than the temperate (7 genotypes) P. vivax populations, a difference possibly reflecting the high cross-border exchanges between Afghanistan and Pakistan and southern Iran. Interestingly, all but two VK210 type isolates sequenced harboured a 36-bp post-repeat insert previously only observed in North Korea and China. This large-scale survey of parasite diversity in the Eastern Mediterranean Region provides a set of baseline data suitable for future molecular epidemiological studies of P. vivax.

To obtain the genetic structure of Plasmodium vivax populations in the northern and southern malaria-endemic areas in Iran, which differ in endemicity, sequence diversity in the variable block 5 and the C-terminal part of P. vivax merozoite surface protein 1 (Pvmsp 1) was analyzed. The variable block 5 fragment from 52 northern and 94 southern isolates was amplified and sequenced. Type 1, type 2, and recombinant type 3 allelic variants were found in both northern and southern isolates, with type 1 predominant in parasites from the north and type 2 in those from the south. A total of 7 and 27 distinct variants were detected among northern and southern isolates, respectively. A single variant predominated (71%) in the northern isolates, whereas variants were evenly distributed among southern isolates, with only two exceeding 10%. Thus, parasites from the southern malaria-endemic area were more polymorphic than those circulating in the northern area, where malaria is a re-emerging disease. Sequence alignments showed that although some variants were found only in northern or southern isolates, some were common to both and had also been observed in parasites from Azerbaijan, Turkey, Thailand, Bangladesh, and China. The Pvmsp 1 fragment corresponding to the C-terminal region was also amplified and the sequences derived from 20 northern and 50 southern isolates were identical. This high degree of conservation reinforces the potential of this polypeptide fragment for inclusion in synthetic vaccines being developed against P. vivax.

International audience; Plasmodium falciparum remains globally an important cause of mortality and morbidity and despite decades of research, no effective vaccine is available against this deadly parasite. The 19-kDa C-terminal fragment of P. falciparum merozoite surface protein 1 (PfMSP-1(19)) is a target for protective immunity against malaria and the major concern in development of vaccine based on this antigen is the presence of polymorphisms. This investigation was designed to evaluate naturally acquired antibodies and antigen-binding avidity of IgG antibodies to three variant forms of PfMSP-1(19) antigen (E/TSG/L, E/KNG/F and Q/KNG/L) in malaria individuals who are living in hypoendemic areas in Iran (n=92, 4-75 years old). The three variant forms of PfMSP-1(19) were expressed in Escherichia coli and IgG isotype composition and avidity of naturally acquired antibodies to the 19-kDa antigen were measured by ELISA assay. Results showed that almost 72% of the studied individuals had positive antibody responses to three PfMSP-1(19) variants and the prevalence of responders did not differ significantly (P>0.05). High-avidity IgG (62.7%, 65.7% and 47.76%) and IgG1 (64.2%, 50.75%, and 50.75%) were found in positive sera for E/TSG/L, E/KNG/F and Q/KNG/L variants, respectively. Moreover, the prevalence and titers of IgG1 antibody responses to the three variants increased with age (P

International audience; Plasmodium vivax remains an important cause of morbidity outside Africa, and no effective vaccine is available against this parasite. The P. vivax Duffy binding protein (PvDBP) is essential during merozoite invasion into erythrocytes, and it is a target for protective immunity against malaria. This investigation was designed to evaluate naturally acquired antibodies to two variant forms of PvDBP-II antigen (DBP-I and -VI) in malaria individuals (N = 85; median = 22 years) who were living in hypoendemic areas in Iran. The two PvDBP-II variants were expressed in Escherichia coli, and immunoglobulin G (IgG) isotype composition and avidity of naturally acquired antibodies to these antigens were measured using enzyme-linked immunosorbent assay (ELISA). Results showed that almost 32% of the studied individuals had positive antibody responses to the two PvDBP-II variants, and the prevalence of responders did not differ significantly (P > 0.05; χ(2) test). The IgG-positive samples exhibited 37.03% and 40.8% high-avidity antibodies for PvDBP-I and PvDBP-VI variants, respectively. Furthermore, high-avidity IgG1 antibody was found in 39.1% of positive sera for each examined variant antigen. The avidity of antibodies for both PvDBP variant antigens and the prevalence of responders with high- and intermediate-avidity IgG, IgG1, and IgG3 antibodies were similar in patients (P > 0.05; χ(2) test). Moreover, the prevalence of IgG antibody responses to the two variants significantly increased with exposure and host age. To sum up, the results provided additional data in our understanding of blood-stage immunity to PvDBP, supporting the rational development of an effective blood-stage vaccine based on this antigen.

Background Plasmodium falciparum apical membrane antigen-1 (PfAMA-1) and the 19-kDa C-terminal region of merozoite surface protein-1 (PfMSP-119) are candidate malaria vaccine antigens expressed on merozoites and sporozoites. This investigation was performed to evaluate simultaneously the naturally-acquired antibodies to PfAMA-1 and PfMSP-119 and to compare IgG subclass profiles to both antigens in naturally exposed individuals living in malaria hypoendemic areas in Iran to determine which antigen has better ability to detect sero-positive individuals infected with P. falciparum. Methods In this investigation, 101 individuals from the malaria-endemic areas in Iran were examined. PfAMA-1 and PfMSP-119 were expressed in Escherichia coli, and IgG isotype composition of naturally acquired antibodies to the antigens (as single or in combination) was measured by ELISA assay. Results The result showed that 87.1% and 84.2% of the studied individuals had positive anti-PfAMA-1 and -PfMSP-119 IgG antibody responses, respectively, and the prevalence of responders did not differ significantly (P > 0.05). Moreover, IgG1 and IgG3 were predominant over IgG2 and IgG4 antibodies and the prevalence of IgG and its subclasses to two tested antigens had no significant correlation with age and exposure (P > 0.05). The present data confirmed that when recombinant PfAMA-1 and recombinant PfMSP-119 antigens were combined in ELISA at equal ratios of 200 ng (100 ng each antigen/well) and 400 ng (200 ng each antigen/well), 86.1% and 87.1% of positives sera were detected among the examined samples, respectively. Conclusions The two tested recombinant antigens are immunogenic molecules, and individuals in low transmission areas in Iran could develop and maintain equal immune responses to PfAMA-1 and PfMSP-119. Therefore, these results could support the design of a universal PfAMA-1- and PfMSP-119-based vaccine. Also, both recombinant antigens could be used in combination as reliable serology markers to perform immuno-epidemiological studies in malaria-endemic areas of Iran during elimination strategy. The present information could be of use in control and elimination programmes in Iran and other similar malaria settings.

Region II of Duffy binding protein (PvDBP-II) is one of the most promising blood-stage vaccine candidate antigens against Plasmodium vivax and having knowledge of the nature and genetic polymorphism of PvDBP-II among global P. vivax isolates is important for developing a DBP-based vaccine. By using PCR and sequencing, the present molecular population genetic approach was carried out to investigate sequence diversity and natural selection of dbp-II gene in 63 P. vivax isolates collected from unstable and low transmission malaria-endemic areas of Iran during 2008–2012. Also, phylogenetic analysis, the diversifying natural selection, and recombination across the pvdbp-II gene, including regions containing B-cell epitopes were analyzed using the DnaSP and MEGA4 programs. Twenty two single nucleotide polymorphisms (SNPs, including 20 non-synonymous and 2 synonymous) were identified in PvDBP-II, resulting in 16 different PvDBP-II haplotypes among the Iranian P. vivax isolates. High binding inhibitory B-cell epitope (H3) overlapping with intrinsically unstructured/disordered region (aa: 384–392) appeared to be highly polymorphic (D384G/E385K/ K386N/Q/R390H), and positive selective pressure acted on this region. Most of the polymorphic amino acids, which are located on the surface of the protein, are under selective pressure that implies increased recombination events and exposure to the human immune system. In summary, PvDBP-II gene displays genetic polymorphism among Iranian P. vivax isolates and it is under selective pressure. Mutations, recombination, and positive selection seem to play a role in the resulting genetic diversity, and phylogenetic analysis of DNA sequences demonstrates that Iranian isolates represent a sample of the global population. These results are useful for understanding the nature of the P. vivax population in Iran and also for development of PvDBP-II-based malaria vaccine.

Among vaccines, those that have an impact on transmission are in priority for malaria elimination and eradication. One of the new identified transmission-blocking vaccine (TBV) candidate antigens is Generative Cell Specific 1 (GCS1) located on the male gametocytes of Plasmodium species. Since the antigenic diversity could hamper vaccine development, it is essential to determine the gene diversity of gcs1 in global malaria-endemic areas in order to develop efficient TBVs. Therefore, in this study, nucleotide diversity and selection in the Plasmodium falciparum GCS1 (PfGCS1) antigen were analyzed in 36 Iranian clinical isolates by using PCR sequencing in order to provide useful information on this TBV candidate antigen. For this purpose, successful sequence analysis was carried out in 36 isolates. The results showed three single-nucleotide polymorphisms including one synonymous (G1475A) and two non-synonymous (A697G and G1479A) mutations leading to 3 distinct haplotypes with different frequencies: GCS1-A (N184/D445, 16.7%), GCS1-B (S184/D445, 63.9%), and GCS1-C (N184/N445, 19.4%). The overall nucleotide diversity (π) for all 36 sequences of Iranian pfgcs1 was 0.00066 ± 0.00012, and the dN–dS value (−0.00028) was negative, suggesting the possible action of purifying selection in this gene. Epitope mapping prediction of PfGCS1 antigen showed that most of the potential linear and conformational B-cell epitopes are located in conserved regions. However, N184S and D445N mutations were also involved in linear and conformational B-cell epitopes, respectively that should be considered in vaccine design. In conclusion, the present study showed a very low genetic diversity of pfgcs1 gene among Iranian isolates. Considering PfGCS1 as a conserved TBV candidate, our data provides valuable information to develop a PfGCS1-based TBV.

International audience; One of the main host genetic factors involved in inflammation, immune responses and pathogenesis of malaria is FcγRIIa (cd32) gene. A single point mutation at position 131 replace an arginine (R) with a histidine (H) that can affect the affinity of the receptor for human IgG subclasses. This investigation was designed to explore the polymorphisms at FcγRIIa gene in association with both anti-malarial total IgG antibody and IgG subclass profiles to C-terminal region of Plasmodium falciparum merozoite surface protein 1 (PfMSP-1(19)). In this study, 166 infected patients with P. falciparum who are living in a malaria endemic area of Iran were studied using PCR-RFLP and ELISA methods. The results showed that the frequency of FcγRIIa-R/R131, -R/H131 and -H/H131 genotypes was 9.6%, 42.8% and 47.6%, respectively. Level of total IgG to recombinant PfMSP-1(19) antigen showed that there was no difference among the FcγRIIa-R/R131, -R/H131 and -H/H131 groups. With regards to the IgG subclasses, the anti-malarial IgG1 antibodies predominated. Also, there was a significant difference between the frequency of positive responders for anti-PfMSP-1(19) IgG and IgG1 antibodies in P. falciparum-infected individuals with FcγRIIa-R/R131, -R/H131 or -H/H131 genotypes (P

International audience; The Plasmodium falciparum apical membrane antigen1 (AMA1) is a leading malaria vaccine candidate antigen. In the present investigation, for the first time, the almost full length of the ama1 gene covering domain I (DI), DII and DIII was PCR amplified and sequenced in 21 P. falciparum isolates collected from the southeastern parts of Iran. The result showed the low genetic diversity of Iranian PfAMA1 with 11 PfAMA1 haplotypes in which nine out of 11 haplotypes are novel and have been reported for the first time. The Iranian P. falciparum population indicated a moderate level of genetic differentiation. The difference among the rates of non-synonymous and synonymous mutations, Tajima's D and McDonald-Kreitman tests suggested that the diversity at DI is due to positive natural selection. In addition, recombination contributes to the diversity of Iranian PfAMA1 and this is supported by the decline of the linkage disequilibrium index R(2) with increasing the nucleotide distance. The highly polymorphic residues (positions: 187, 197, 200, 230 and 243) were polymorphic; however, most of the SNPs in non-polymorphic residues were conserved except the residue at position 395. Nevertheless, no mutation was found in the DII loop of the Iranian PfAMA1, indicating that it is subjected to purifying selection. In conclusion, the low genetic diversity in PfAMA1 among Iranian isolates supports and provides valuable information for the development of a PfAMA1-based malaria vaccine.

Background In malaria endemic areas, continuous exposure to Plasmodium parasites leads to asymptomatic carriers that provide a fundamental reservoir of parasites, contributing to the persistence of malaria transmission. Therefore, in the present investigation, the presence and prevalence of malaria asymptomatic cases were determined to evaluate the reservoir of infection in two malaria endemic areas with a previous history of malaria transmission in the south of Iran, Bashagard and Ghale-Ganj districts of Hormozgan and Kerman provinces, respectively, where malaria transmission has been drastically reduced in the recent years. Methods The population samples (n=500 from each of the studied areas) were randomly collected from non-febrile, long-term residing, aged two to over 60years, during 20092010. Three identical surveys were carried out in both study areas and in each phase all the consent participants were interviewed and clinically examined. In all, three surveys to detect hidden parasite reservoirs (both Plasmodium falciparum and Plasmodium vivax), thick and thin blood smears and a highly sensitive nested-PCR were applied. In addition, the sero-prevalence survey for detecting malaria exposure was done by using a serological marker. Results In this study, P. vivax and P. falciparum parasites were not detected by light microscopy and nested-PCR assay in all three surveys of samples. Antibody responses against P. vivax and P. falciparum were detected in 1 % and 0.2 % of the total examined individuals, respectively, in Bashagard district. Regarding to Ghale-Ganj district, about 0.9% of the individuals had IgG -specific antibody to P. vivax at the first and second surveys, but at the third survey 0.45% of the participants had positive antibody to P. vivax parasite. IgG -specific antibody to P. falciparum was detected in 0.2% of the participants at the first and follow-up surveys. The overall regional differences were not statistically significant (P>0.05). Conclusion Taken together, the lack of asymptomatic carrier with the evidence of extremely low sero-positive to both P. vivax and P. falciparum among examined individuals supported the limited recent transmission in the studied areas and, therefore, these parts of Iran have potential to eliminate the disease in the next few years. However, continued follow up and action are still needed in both studied areas and also in their neighbouring province, Sistan and Baluchistan, which has the highest reported cases of malaria in Iran and also, has the largest border line with Afghanistan and Pakistan, with no elimination activities. This data will provide useful information for managing elimination activities in Iran.

Background Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and their activation leads to the induction of effector genes involving inflammatory cytokines that may have contribute to controlling parasite growth and disease pathogenesis. The current immunogenetic study was designed to analyse the key components of innate immunity, TLRs and TIRAP (Toll-interleukin-1 receptor domain-containing adaptor protein), also known as MAL (MYD88 adaptor-like), in Iranian patients with mild malaria. Methods The tlr-4 (D299G and T399I), tlr-9 (T-1486C and T-1237C) and tirap (S180L) genes were assessed in 640 Baluchi individuals (320 Plasmodium falciparum-infected and 320 non-infected, median age of 28 years) from malaria-endemic regions using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. Results Common tlr-4 SNPs and promoter SNPs of tlr-9 were distributed among P. falciparum-infected and non-infected groups (P > 0.05) that showed no association of these variants with mild clinical manifestation. The comparison of the tirap S180L genotypes between patients with mild malaria and those healthy individuals showed that the frequency of heterozygosity was significantly higher in infected than non-infected individuals (33.8 vs. 25.6; OR, 1.479; 95% CI, 1.051-2.081; P = 0.024). The result also revealed a significant association of tirap S180L (P < 0.05) with development of mild malaria, which is common in Baluchi populations, who are living in malaria hypoendemic region of Iran but not in African populations (0%-6%). Conclusion These data point towards the need for addressing the exact role of TLRs in contributing to human genetic factors in malaria susceptibility/resistance/severity within different malaria settings in the world.

Background The C-terminal region of Plasmodium falciparum merozoite surface protein-1 (PfMSP-119) is a leading malaria vaccine candidate antigen. However, the existence of different variants of this antigen can limit efficacy of the vaccine development based on this protein. Therefore, in this study, the main objective was to define the frequency of PfMSP-119 haplotypes in malaria hypoendemic region of Iran and also to analyse cross-reactive and/or variant-specific antibody responses to four PfMSP-119 variant forms. Methods The PfMSP-119 was genotyped in 50 infected subjects with P. falciparum collected during 2006-2008. Four GST-PfMSP-119 variants (E/TSR/L, E/TSG/L, E/KNG/F and Q/KNG/L) were produced in Escherichia coli and naturally occurring IgG antibody to these proteins was evaluated in malaria patients' sera (n = 50) using ELISA. To determine the cross-reactivity of antibodies against each PfMSP-119 variant in P. falciparum-infected human sera, an antibody depletion assay was performed in eleven corresponding patients' sera. Results Sequence data of the PfMSP-119 revealed five variant forms in which the haplotypes Q/KNG/L and Q/KNG/F were predominant types and the second most frequent haplotype was E/KNG/F. In addition, the prevalence of IgG antibodies to all four PfMSP-119 variant forms was equal and high (84%) among the studied patients' sera. Immunodepletion results showed that in Iranian malaria patients, Q/KNG/L variant could induce not only cross-reactive antibody responses to other PfMSP-119 variants, but also could induce some specific antibodies that are not able to recognize the E/TSG/L or E/TSR/L variant forms. Conclusion The present findings demonstrated the presence of non-variant specific antibodies to PfMSP-119 in Iranian falciparum malaria patients. This data suggests that polymorphism in PfMSP-119 is less important and one variant of this antigen, particularly Q/KNG/L, may be sufficient to be included in PfMSP-119-based vaccine.

The C-terminal region of Plasmodium vivax merozoite surface protein 1 (PvMSP-1 19 ) 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-1 19 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-1 19 and IgG1 (81.9%) was the predominant subclass. In addition, to determine the persistence of specific IgG, IgG1 and IgG3 antibodies to PvMSP-1 19 , 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-1 19 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-1 19 . These results could help to understand the interactions between the host and P. vivax parasite in development of MSP-1 19 -based vaccine.