Malaria transmission blocking activity of Anopheles stephensi alanyl aminopeptidase N antigen formulated with MPL, CpG, and QS21 adjuvants.

BACKGROUNDS: Malaria, a preventive and treatable disease, is still responsible for annual deaths reported in most tropical regions, principally in sub-Saharan Africa. Subunit recombinant transmission-blocking vaccines (TBVs) have been proposed as promising vaccines to succeed in malaria elimination and eradication. Here, a provisional study was designed to assess the immunogenicity and functional activity of alanyl aminopeptidase N (APN1) of Anopheles stephensi, as a TBV candidate, administered with MPL, CpG, and QS21 adjuvants in the murine model. METHODOLOGY/PRINCIPAL FINDINGS: The mouse groups were immunized with recombinant APN1 (rAPN1) alone or formulated with CpG, MPL, QS-21, or a combination of adjuvants (CMQ), and the elicited immune responses were evaluated after the third immunization. The standard membrane feeding assay (SMFA) measured the functional activity of antibodies against bacterial-expressed APN1 protein in adjuvanted vaccine groups on transmission of P. falciparum (NF54) to An. stephensi mosquitoes. Evaluation of mice vaccinated with rAPN1 formulated with distinct adjuvants manifested a significant increase in the high-avidity level of anti-APN1 IgG and IgG subclasses; however, rAPN1 induced the highest level of high-avidity anti-APN1 IgG1, IgG2a, and IgG2b antibodies in the immunized vaccine group 5 (APN1/CMQ). In addition, vaccine group 5 (receiving APN1/CMQ), had still the highest level of anti-APN1 IgG antibodies relative to other immunized groups after six months, on day 180. The SMFA data indicates a trend towards higher transmission-reducing activity in groups 2 and 5, which received the antigen formulated with CpG or a combination of three adjuvants. CONCLUSIONS/SIGNIFICANCE: The results have shown the capability of admixture to stimulate high-affinity and long-lasting antibodies against the target antigen to hinder Plasmodium parasite development in the mid-gut of An. stephensi. The attained results authenticated APN1/CMQ and APN1/CpG as a potent APN1-based TBV formulation which will be helpful in designing a vaccine in the future.

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

[This corrects the article DOI: 10.1371/journal.pone.0266067.].

Optimized Refolding Buffers Oriented Humoral Immune Responses Versus PfGCS1 Self-Assembled Peptide Nanoparticle.

Developing a novel class of vaccine is pivotal for eliminating and eradicating malaria. Preceding investigations demonstrated partial blocking activity in malaria transmission against recombinant vaccine PfHAP2-GCS1 and conserved region of the cd loop. The effectiveness of immune response varies with the size and shape of the self-assembly of peptide nanoparticles (SAPNs) displaying antigen, affected by different components in refolding buffers. Plasmodium falciparum Generative Cell Specific 1 (PfGCS1), a promising malaria transmission-blocking vaccine (TBV) candidate, was expressed, purified, and followed by a four-step refolding process to form nanoparticles (PfGCS1-SAPNs). The influence of buffer components on the size and shape of SAPNs was investigated by DLS and FESEM. Furthermore, the immunogenicity of nanostructures was assessed in different mouse groups. The results showed that PfGCS1-SAPN was immunogenic and its administration with Poly (I:C), stimulated humoral and cellular responses in the mouse model. In the immunized mice groups, the level of IgG antibodies against PfGCS1-SAPN was significantly increased in different time points (second and third boost) and heterogeneous boosters. The various IgG-subclasses profile shifted to Th1, Th2, or Th1/Th2 mix responses in mice immunized with PfGCS1-SAPN refolded in different buffers, indicating a prerequisite for further investigations to optimize vaccine formulation to enhance and modulate Th1/cellular responses. Such studies pave the way to improve biophysical features related to the nanoparticles' size, shape, and conformational epitopes of candidate antigens and T- and B-cells presented on the superficial structure to elicit robust immune responses.

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

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

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

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

CRISPR/Cas advancements for genome editing, diagnosis, therapeutics, and vaccine development for Plasmodium parasites, and genetic engineering of Anopheles mosquito vector.

Malaria as vector-borne disease remains important health concern with over 200 million cases globally. Novel antimalarial medicines and more effective vaccines must be developed to eliminate and eradicate malaria. Appraisal of preceding genome editing approaches confirmed the CRISPR/Cas nuclease system as a novel proficient genome editing system and a tool for species-specific diagnosis, and drug resistance researches for Plasmodium species, and gene drive to control Anopheles population. CRISPR/Cas technology, as a handy tool for genome editing can be justified for the production of transgenic malaria parasites like Plasmodium transgenic lines expressing Cas9, chimeric Plasmodium transgenic lines, knockdown and knockout transgenic parasites, and transgenic parasites expressing alternative alleles, and also mutant strains of Anopheles such as only male mosquito populations, generation of wingless mosquitoes, and creation of knock-out/ knock-in mutants. Though, the incorporation of traditional methods and novel molecular techniques could noticeably enhance the quality of results. The striking development of a CRISPR/Cas-based diagnostic kit that can specifically diagnose the Plasmodium species or drug resistance markers is highly required in malaria settings with affordable cost and high-speed detection. Furthermore, the advancement of genome modifications by CRISPR/Cas technologies resolves contemporary restrictions to culturing, maintaining, and analyzing these parasites, and the aptitude to investigate parasite genome functions opens up new vistas in the better understanding of pathogenesis.

Prevalence of co-infection and genetic diversity of avian haemosporidian parasites in two rehabilitation facilities in Iran: implications for the conservation of captive raptors.

BACKGROUND: Various haemosporidian parasites infect raptors, especially captive hosts who may be more exposed. Diagnosis of threatening factors such as infectious diseases indirectly has a significant role in protecting endangered or threatened species that may boost the mortality or extinction resulting from declined reproduction. Few investigations have been performed in captive hosts to detect the prevalence of haemosporidian parasites and define genetic diversity in west Asia. For the first time, the current study was designed to determine the prevalence and genetic diversity of haemosporidian parasites in captive raptors by molecular methods in two rehabilitation facilities in North and North-east Iran and to define phylogenetic relationships of detected lineages circulating in raptors. RESULTS: Molecular characterization of the haemosporidian parasite was accomplished by PCR-based method and DNA sequencing in 62 captive raptors. The overall prevalence was ~ 36% with higher infection of Haemoproteus spp. than Leucocytozoon spp. Plasmodium infection was not detected in any host. Results showed that 22 individuals (of 10 species) were infected with unique lineages. Genus Haemoproteus was detected in 26.66% of examined individuals (of eight species) and Leucocytozoon was found in 10% of individuals (of four species). The molecular analysis could detect ten lineages (nine Haemoproteus spp. and one Leucocytozoon spp.) which were categorizes as new and six lineages which have been previously detected in the other investigations. CONCLUSIONS: The Bayesian phylogenetic analysis derived from obtained data in the present study and published lineages in previous investigations indicated the probable host specificity of Haemoproteus and Leucocytozoon parasites in several sub-clades at hosts' order and genus level. As monitoring the parasite loads of captive birds when admitted reduce the risk of infecting hosts in captivity at those locations, we designed this study to determine infection prevalence and genetic diversity of blood parasites in raptors examined in Iran. These results allow mapping of haemosporidian distribution and shed light on the depth of their diversity in Iran to protect species by identification of risk in rehabilitation facilities.

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

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

Haemoproteosis and avian malaria in Columbidae and Corvidae from Iran.

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

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.

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

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

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.

Correction: Molecular detection and genetic diversity of avian haemosporidian parasites in Iran.

[This corrects the article DOI: 10.1371/journal.pone.0206638.].

Molecular detection and genetic diversity of avian haemosporidian parasites in Iran.

BACKGROUND: The mobility of birds across or between continents exposes them to numerous vectors that have the potential to transmit pathogens and spread them into new regions. A combination of rich species diversity of birds along with the small amount of molecular studies in Iran makes observing the blood parasite distribution in wild avian populations indispensable for further estimation and administration of blood parasites. METHODOLOGY/PRINCIPAL FINDINGS: In order to evaluate the infection rate and molecular context of avian blood parasites, bird samples were collected (passerine = 316 and non-passerine = 14) in eight provinces of northern Iran between June to September 2015 and 2016. All bird samples were examined for haematoprotozoan infections by morphological screening using light microscope and mtDNA cytb gene amplification. A total of 115 birds were positive for blood parasites by molecular approach (34.84% overall infection). The infection rate of Haemoproteus, Plasmodium, and Leucocytozoon were 33.03%, 1.21%, and 0.6%, respectively. Sequences analysis has detected 43 lineages in Iranian birds' hosts. Lineages were attributed to three genera Haemoproteus (n = 37), Plasmodium (n = 4), and Leucocytozoon (n = 2), of which 23 lineages fully matched previously recorded sequences in GenBank and MalAvi data reciprocities. Five lineages of ACDUM1, ACDUM2, PARUS1, PYERY01, and SISKIN1 were detected in multiple hosts' species from dissimilar families. In Bayesian tree, all sequences were clustered in three main monophyletic clades as Haemoproteus, Plasmodium, and Leucocytozoon genera. CONCLUSIONS/SIGNIFICANCE: As the first study outlining the molecular detection of hematozoa of passerines from Iran, the current study has recorded 20 new lineages for three genera of Haemoproteus, Plasmodium, and Leucocytozoon. Additional investigations into these taxa in the avifauna for the other parts of Iran may provide extra information on blood parasites, hosts relationships and distribution patterns.

Occurrence of Haemoproteus spp. (Haemosporida: Haemoproteidae) in New Host Records of Passerine Birds from the East of Iran.

BACKGROUND: Avian haemosporidians are able to parasitize numerous bird species all over the world. The extensive range of blood parasites infection rate is between 50% and 100% or less percentage. Haemoparasites with major effects on physiology, ecology, health, population dynamics, sexual selection and production success of avian hosts may promote species extinction. METHODS: To evaluate haemosporidians infection rate in Iranian birds, 136 individuals were examined by microscopic observation of stained blood smears under light microscope. These samples belonged to 10 different families of Songbirds from the east of Iran from April to August 2014-2016. RESULTS: Fifty-one passerine birds were detected as harboring Haemoproteus spp. Furthermore, we recorded Haemoproteus spp. infection of Granativora bruniceps, Oenanthe pleschanka for the first time in the world and eight more species for Iran. CONCLUSION: Age and sampling localities do not influence the infection rate of Haemoproteus spp. from the eastern provinces of Iran. The relative high infection of avian haematozoa revealed this region might provide suitable sites for future studies on these parasites and the relationship with their hosts and vectors.

New Host Records for Haemoproteus Spp. (Apicomplexa: Haemosporidiasina) in Passeriformes from North-West of Iran.

BACKGROUND: The intracellular protozoan parasites of the genus Haemoproteus occur in different avian hosts all over the world. Various genus of blood sucking insects' families such as Hippoboscidae and Ceratopogonidae could transmit Haemoproteus in avian hosts. There are very limited number of studies on wild infected birds with blood parasites in Iran, so the aim of this study was to determine the frequency of Haemoproteus spp. infection in passerine birds from northwest of Iran. METHODS: Passerines were collected from four different localities in Zanjan Province, northwest Iran during June to August 2014. RESULTS: Of 86 passerines, we found Haemoproteus infection in 19 (22.09%) individuals. In general, 15 bird species were observed for haemosporidians, of which 53% were infected. CONCLUSION: Three species of passerines: Petronia petronia, Sitta tephronota and Acrocephalus melanopogon are new host records for Haemoproteus infection in the world. Results acquired by this study support widespread distribution of Haemoproteus in passerines and illustrated the prevalence of Haemoproteus species in wild birds of northwest of Iran. Conclusively, our study specified that more investigations are needed to reach exact prevalence rate in different families of birds in Iran.