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.

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.

Advax, as a Co-adjuvant, in Combination with Poly(I:C) Elicits Enhanced Th1 Immune Responses and Parasite Growth-Inhibitory Antibodies Against Plasmodium Falciparum Merozoite Surface Protein-1 (PfMSP-142) in BALB/c Mice.

BACKGROUND: 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. OBJECTIVES: 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). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

How can we develop an effective subunit vaccine to achieve successful malaria eradication?

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.

A review of combination adjuvants for malaria vaccines: a promising approach for vaccine development.

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.

Developing a Vaccine to Block West Nile Virus Transmission: In Silico Studies, Molecular Characterization, Expression, and Blocking Activity of Culex pipiens mosGCTL-1.

BACKGROUND: Mosquito galactose-specific C-type lectins (mosGCTLs), such as mosGCTL-1, act as ligands to facilitate the invasion of flaviviruses like West Nile virus (WNV). WNV interacts with the mosGCTL-1 of Aedes aegypti (Culicidae) and facilitates the invasion of this virus. Nevertheless, there is no data about the role of mosGCTL-1 as a transmission-blocking vaccine candidate in Culex pipiens, the most abundant Culicinae mosquito in temperate regions. METHODS: Adult female Cx. pipiens mosquitoes were experimentally infected with a WNV infectious blood meal, and the effect of rabbit anti-rmosGCTL-1 antibodies on virus replication was evaluated. Additionally, in silico studies such as the prediction of protein structure, homology modeling, and molecular interactions were carried out. RESULTS: We showed a 30% blocking activity of Cx. pipiens mosGCTL-1 polyclonal antibodies (compared to the 10% in the control group) with a decrease in infection rates in mosquitoes at day 5 post-infection, suggesting that there may be other proteins in the midgut of Cx. pipiens that could act as cooperative-receptors for WNV. In addition, docking results revealed that WNV binds with high affinity, to the Culex mosquito lectin receptors. CONCLUSIONS: Our results do not support the idea that mosGCTL-1 of Cx. pipiens primarily interacts with WNV to promote viral infection, suggesting that other mosGCTLs may act as primary infection factors in Cx. pipiens.

Evolutionary analyses of the major variant surface antigen-encoding genes reveal population structure of Plasmodium falciparum within and between continents.

Malaria remains a major public health problem in many countries. Unlike influenza and HIV, where diversity in immunodominant surface antigens is understood geographically to inform disease surveillance, relatively little is known about the global population structure of PfEMP1, the major variant surface antigen of the malaria parasite Plasmodium falciparum. The complexity of the var multigene family that encodes PfEMP1 and that diversifies by recombination, has so far precluded its use in malaria surveillance. Recent studies have demonstrated that cost-effective deep sequencing of the region of var genes encoding the PfEMP1 DBLα domain and subsequent classification of within host sequences at 96% identity to define unique DBLα types, can reveal structure and strain dynamics within countries. However, to date there has not been a comprehensive comparison of these DBLα types between countries. By leveraging a bioinformatic approach (jumping hidden Markov model) designed specifically for the analysis of recombination within var genes and applying it to a dataset of DBLα types from 10 countries, we are able to describe population structure of DBLα types at the global scale. The sensitivity of the approach allows for the comparison of the global dataset to ape samples of Plasmodium Laverania species. Our analyses show that the evolution of the parasite population emerging out of Africa underlies current patterns of DBLα type diversity. Most importantly, we can distinguish geographic population structure within Africa between Gabon and Ghana in West Africa and Uganda in East Africa. Our evolutionary findings have translational implications in the context of globalization. Firstly, DBLα type diversity can provide a simple diagnostic framework for geographic surveillance of the rapidly evolving transmission dynamics of P. falciparum. It can also inform efforts to understand the presence or absence of global, regional and local population immunity to major surface antigen variants. Additionally, we identify a number of highly conserved DBLα types that are present globally that may be of biological significance and warrant further characterization.

Serological evidence of West Nile virus infection among birds and horses in some geographical locations of Iran.

Recent expansion of arboviruses such as West Nile (WNV), Usutu (USUV), and tick-borne encephalitis (TBEV) over their natural range of distribution needs strengthening their surveillance. As common viral vertebrate hosts, birds and horses deserve special attention with routine serological surveillance. Here, we estimated the seroprevalence of WNV, USUV and TBEV in 160 migrating/resident birds and 60 horses sampled in Mazandaran, Golestan, North Khorasan, Kordestan provinces and Golestan province of Iran respectively. ELISA results showed that of 220 collected samples, 32 samples (14.54%), including 22 birds and 10 horses, were positive. Microsphere immunoassay results showed that 16.7% (10/60) of horse blood samples collected in Golestan province were seropositive against WNV (7; 11.7%), Flavivirus (2; 3.3%) and seropositive for USUV or WNV (1; 1.7%). Furthermore, micro virus neutralization tests revealed that four of seven ELISA-positive bird blood samples were seropositive against WNV: two Egyptian vultures, and one long-legged buzzard collected in Golestan province as well as a golden eagle collected in North Khorasan province. No evidence of seropositivity with TBEV was observed in collected samples. We showed that WNV, responsible for neuroinvasive infection in vertebrates, is circulating among birds and horses in Iran, recommending a sustained surveillance of viral infections in animals, and anticipating future infections in humans.

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.

Detection of arboviruses in mosquitoes: Evidence of circulation of chikungunya virus in Iran.

Mosquitoes are vectors of viruses affecting animal and human health. In Iran, the prevalence of mosquito-borne viruses remains poorly investigated. Once infected, mosquito females remain infected for all their life making virus detections possible at early steps before infections are reported in vertebrate hosts. In this study, we used a recently developed high-throughput chip based on the BioMark Dynamic arrays system capable of detecting 37 arboviruses in a single experiment. A total of 1,212 mosquitoes collected in Mazandaran, North-Khorasan, and Fars provinces of Iran were analyzed. Eighteen species were identified, belonging to five genera; the most prevalent species were Anopheles maculipennis s.l. (42.41%), Culex pipiens (19.39%), An. superpictus (11.72%), and Cx. tritaeniorhynchus (10.64%). We detected chikungunya virus (CHIKV) of the Asian genotype in six mosquito pools collected in North Khorasan and Mazandaran provinces. To our knowledge, this is the first report of mosquitoes infected with CHIKV in Iran. Our high-throughput screening method can be proposed as a novel epidemiological surveillance tool to identify circulating arboviruses and to support preparedness to an epidemic in animals and humans.

New Insights Into Culturable and Unculturable Bacteria Across the Life History of Medicinal Maggots Lucilia sericata (Meigen) (Diptera: Calliphoridae).

Because of the nutritional ecology of dung- and carrion-feeding, bacteria are the integral part of Lucilia sericata life cycle. Nevertheless, the disinfected larvae of the blowfly are applied to treat human chronic wounds in a biosurgery named maggot debridement therapy (MDT). To realize the effects of location/diet on the gut bacteria, to infer the role of bacteria in the blowfly ecology plus in the MDT process, and to disclose bacteria circulating horizontally in and vertically between generations, bacterial communities associated with L. sericata specimens from various sources were investigated using culture-based and culture-independent methods. In total, 265 bacteria, including 20 families, 28 genera, and 40 species, were identified in many sources of the L. sericata. Culture-dependent method identified a number of 144 bacterial isolates, including 21 species, in flies reared in an insectary; specimens were collected from the field, and third-instar larvae retrieved from chronic wounds of patients. Metagenetic approach exposed the occurrences of 121 operational taxonomic units comprising of 32 bacterial species from immature and adult stages of L. sericata. Gammaproteobacteria was distinguished as the dominant class of bacteria by both methods. Bacteria came into the life cycle of L. sericata over the foods and transovarially infected eggs. Enterococcus faecalis, Myroides phaeus, Proteus species, Providencia vermicola, and Serratia marcescens were exchanged among individuals via transstadial transmission. Factors, including diets, feeding status, identification tool, gut compartment, and life stage, governed the bacteria species. Herein, we reemphasized that L. sericata is thoroughly connected to the bacteria both in numerous gut compartments and in different life stages. Among all, transstadially transmitted bacteria are underlined, indicating the lack of antagonistic effect of the larval excretions/secretions on these resident bacteria. While the culture-dependent method generated useful data on the viable aerobic gut bacteria, metagenomic method enabled us to identify bacteria directly from the tissues without any need for cultivation and to facilitate the identification of anaerobic and unculturable bacteria. These findings are planned to pave the way for further research to determine the role of each bacterial species/strain in the insect ecology, as well as in antimicrobial, antibiofilm, anti-inflammatory, and wound healing activities.

High Transmission Potential of West Nile Virus Lineage 1 for Cx. pipiens s.l. of Iran.

: Vector competence is an important parameter in evaluating whether a species plays a role in transmission of an arbovirus. Although the protocols are similar, interpretation of results is unique given the specific interactions that exist between a mosquito population and a viral genotype. Here, we assessed the infection (IR), dissemination (DR), and transmission (TR) rates of Cx. pipiens s.l., collected from Iran, for West Nile virus (WNV) lineage 1a. We showed that Cx. pipiens s.l. mosquitoes in Iran were susceptible to WNV with IR up to 89.7%, 93.6%, and 83.9% at 7, 14, and 21 days post-infection (dpi) respectively. In addition, DR and TR reached respectively 92.3% and 75.0% at 21 dpi, and the number of viral particles delivered with saliva reached up to 1.33 × 105 particles. Therefore, an unexpected high risk of WNV dissemination in the region where Cx. pipiens s.l. mosquitoes are well established should be considered carefully and surveillance measures implemented accordingly.

Human Plasmodium vivax diversity, population structure and evolutionary origin.

More than 200 million malaria clinical cases are reported each year due to Plasmodium vivax, the most widespread Plasmodium species in the world. This species has been neglected and understudied for a long time, due to its lower mortality in comparison with Plasmodium falciparum. A renewed interest has emerged in the past decade with the discovery of antimalarial drug resistance and of severe and even fatal human cases. Nonetheless, today there are still significant gaps in our understanding of the population genetics and evolutionary history of P. vivax, particularly because of a lack of genetic data from Africa. To address these gaps, we genotyped 14 microsatellite loci in 834 samples obtained from 28 locations in 20 countries from around the world. We discuss the worldwide population genetic structure and diversity and the evolutionary origin of P. vivax in the world and its introduction into the Americas. This study demonstrates the importance of conducting genome-wide analyses of P. vivax in order to unravel its complex evolutionary history.

Dynamics of prevalence and distribution pattern of avian Plasmodium species and its vectors in diverse zoogeographical areas - A review.

Avian Plasmodium is of special interest to health care scientists and veterinarians due to the potency of causing avian malaria in non-adapted birds and their evolutionary phylogenetic relationship with human malaria species. This article aimed to provide a comprehensive list of the common avian Plasmodium parasites in the birds and mosquitoes, to specify the common Plasmodium species and lineages in the selected regions of West of Asia, East of Europe, and North of Africa/Middle East, and to determine the contribution of generalist and host-specific Plasmodium species and lineages. The final list of published infected birds includes 146 species, among which Passer domesticus was the most prevalent in the studied areas. The species of Acrocephalus arundinaceus and Sylvia atricapilla were reported as common infected hosts in the examined regions of three continents. The highest numbers of common species of infected birds between continent pairs were from Asia and Europe, and no common record was found from Europe and Africa. The species of Milvus migrans and Upupa epops were recorded as common species from Asia and Africa. The lineage of GRW11 and species of P. relictum were the most prevalent parasites among all the infection records in birds. The most prevalent genus of vectors of avian malaria belonged to Culex and species of Cx. pipiens. The lineage SGS1 with the highest number of occurrence has been found in various vectors comprising Cx. pipiens, Cx. modestus, Cx. theileri, Cx. sasai, Cx. perexiguus, Lutzia vorax, and Culicoides alazanicus. A total of 31 Plasmodium species and 59 Plasmodium lineages were recorded from these regions. SGS1, GRW04, and GRW11, and P. relictum and P. vaughani are specified as common generalist avian malaria parasites from these three geographic areas. The presence of avian Plasmodium parasites in distant geographic areas and various hosts may be explained by the movement of the infected birds through the migration routes. Although most recorded lineages were from Asia, investigating the distribution of lineages in some of the countries has not been done. Thus, the most important outcome of this review is the determination of the distribution pattern of parasite and vector species that shed light on gaps requiring further studies on the monitoring of avian Plasmodium and common vectors extension. This task could be achieved through scientific field and laboratory networking, performing active surveillance and designing regional/continental control programs of birds' malaria and other zoonotic diseases.

Identification, molecular characterization and expression of aminopeptidase N-1 (APN-1) from Anopheles stephensi in SF9 cell line as a candidate molecule for developing a vaccine that interrupt malaria transmission.

BACKGROUND: According to the World Health Organization reports, billions of people around the world are at risk for malaria disease and it is important to consider the preventive strategies for protecting the people that are living in high risk areas. One of the main reasons of disease survival is diversity of vectors and parasites in different malaria regions that have their specific features, behaviour and biology. Therefore, specific regional strategies are necessary for successful control of malaria. One of the tools that needs to be developed for elimination and prevention of reintroduction of malaria is a vaccine that interrupt malaria transmission (VIMTs). VIMT is a broad concept that should be adjusted to the biological characteristics of the disease in each region. One type of VIMT is a vector-based vaccine that affects the sexual stage of Plasmodium life cycle. According to recent studies, the aminopeptidase N-1 of Anopheles gambiae (AgAPN-1) is as a potent vector-based VIMT with considerable inhibition activity against the sexual stage of Plasmodium parasite. METHODS: Systems for rapid amplification of cDNA ends (3'-RACE) and genome walking methods were used for sequence determination of apn-1 gene from Anopheles stephensi and distinct bioinformatics software were used for structural analysis. AsAPN-1 was expressed in Spodoptera frugiperda (Sf9) insect cell line using the baculovirus expression system. Recombinant AsAPN-1 was purified under the hybrid condition and its biological activity was assayed. RESULTS: Asapn-1 gene and its coded protein from An. stephensi were characterized for the first time in this study. Subsequently, the structural features and immunological properties of its coded protein were evaluated by in silico approaches. Enzymatic activity of the recombinant AsAPN-1, which was expressed in Sf9 insect cell line, was equal to 6 unit/µl. CONCLUSIONS: Results of this study revealed that AsAPN-1 is very similar to its counterpart in An. gambiae. In silico evaluation and fundamental data which are necessary for its evaluation as a VIMT-based vaccine in the next steps were acquired in this study and those could be useful for research groups that study on malaria vaccine for countries that An. stephensi is the main malaria vector there.

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.

Gene copy number and function of the APL1 immune factor changed during Anopheles evolution.

BACKGROUND: The recent reference genome assembly and annotation of the Asian malaria vector Anopheles stephensi detected only one gene encoding the leucine-rich repeat immune factor APL1, while in the Anopheles gambiae and sibling Anopheles coluzzii, APL1 factors are encoded by a family of three paralogs. The phylogeny and biological function of the unique APL1 gene in An. stephensi have not yet been specifically examined. METHODS: The APL1 locus was manually annotated to confirm the computationally predicted single APL1 gene in An. stephensi. APL1 evolution within Anopheles was explored by phylogenomic analysis. The single or paralogous APL1 genes were silenced in An. stephensi and An. coluzzii, respectively, followed by mosquito survival analysis, experimental infection with Plasmodium and expression analysis. RESULTS: APL1 is present as a single ancestral gene in most Anopheles including An. stephensi but has expanded to three paralogs in an African lineage that includes only the Anopheles gambiae species complex and Anopheles christyi. Silencing of the unique APL1 copy in An. stephensi results in significant mosquito mortality. Elevated mortality of APL1-depleted An. stephensi is rescued by antibiotic treatment, suggesting that pathology due to bacteria is the cause of mortality, and indicating that the unique APL1 gene is essential for host survival. Successful Plasmodium development in An. stephensi depends upon APL1 activity for protection from high host mortality due to bacteria. In contrast, silencing of all three APL1 paralogs in An. coluzzii does not result in elevated mortality, either with or without Plasmodium infection. Expression of the single An. stephensi APL1 gene is regulated by both the Imd and Toll immune pathways, while the two signaling pathways regulate different APL1 paralogs in the expanded APL1 locus. CONCLUSIONS: APL1 underwent loss and gain of functions concomitant with expansion from a single ancestral gene to three paralogs in one lineage of African Anopheles. We infer that activity of the unique APL1 gene promotes longevity in An. stephensi by conferring protection from or tolerance to an effect of bacterial pathology. The evolution of an expanded APL1 gene family could be a factor contributing to the exceptional levels of malaria transmission mediated by human-feeding members of the An. gambiae species complex in Africa.

Immunological evaluation of two novel engineered Plasmodium vivax circumsporozoite proteins formulated with different human-compatible vaccine adjuvants in C57BL/6 mice.

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.