An analytical review of vector- and pathogen-based transmission-blocking vaccine for malaria control.

Malaria is a vector borne disease, considered to be one of the most serious public health problems. The present review focused on the blocking of parasite development in mosquito vectors; one broad strategy for achieving this is Transmission Blocking Vaccines (TBV). The TBVs usually rely on immunization of vertebrate hosts with molecules derived from the vector or pathogen to reduce pathogen transmission from infected to uninfected hosts. Most of the studies on the TBVs are based on the antibodies targeted against the surface antigens of sexual stages of malaria parasite, but it is meagre to develop mosquito-based vaccine in this regard. Vector-based TBVs include surface proteins that are expressed by the mosquito midgut digestive enzymes which are induced upon blood-feeding, and receptors expressed on the epithelial line of the tissue. Many proteins are reported that can act as candidates for transmission-blocking vaccines. This review aims to summarize the vector midgut-based proteins identified till date, that can block the development and maturity of sexual stages of the parasite within mosquitoes as targets for transmission-blocking vaccine development. The TBVs intervention can block transmission of different malaria parasite species in various species of mosquitoes with future application perspective worldwide.

An Immunoinformatics Study to Predict Epitopes in the Envelope Protein of SARS-CoV-2.

COVID-19 is a new viral emergent disease caused by a novel strain of coronavirus. This virus has caused a huge problem in the world as millions of people are affected by this disease. We aimed at designing a peptide vaccine for COVID-19 particularly for the envelope protein using computational methods to predict epitopes inducing the immune system. The envelope protein sequence of SARS-CoV-2 has been retrieved from the NCBI database. The bioinformatics analysis was carried out by using the Immune Epitope Database (IEDB) to predict B- and T-cell epitopes. The predicted HTL and CTL epitopes were docked with HLA alleles and binding energies were evaluated. The allergenicity of predicted epitopes was analyzed, the conservancy analysis was performed, and the population coverage was determined throughout the world. Some overlapped CTL, HTL, and B-cell epitopes were suggested to become a universal candidate for peptide-based vaccine against COVID-19. This vaccine peptide could simultaneously elicit humoral and cell-mediated immune responses. We hope to confirm our findings by adding complementary steps of both in vitro and in vivo studies to support this new universal predicted candidate.

Plasmodium falciparum MSP3 Exists in a Complex on the Merozoite Surface and Generates Antibody Response during Natural Infection.

Plasmodium falciparum merozoite surface protein 3 (MSP3) is an abundantly expressed secreted merozoite surface protein and a leading malaria vaccine candidate antigen. However, it is unclear how MSP3 is retained on the surface of merozoites without a glycosylphosphatidylinositol (GPI) anchor or a transmembrane domain. In the present study, we identified an MSP3-associated network on the Plasmodium merozoite surface by immunoprecipitation of Plasmodium merozoite lysate using antibody to the N terminus of MSP3 (anti-MSP3N) followed by mass spectrometry analysis. The results suggested the association of MSP3 with other merozoite surface proteins: MSP1, MSP6, MSP7, RAP2, and SERA5. Protein-protein interaction studies by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) analysis showed that MSP3 complex consists of MSP1, MSP6, and MSP7 proteins. Immunological characterization of MSP3 revealed that MSP3N is strongly recognized by hyperimmune serum from African and Asian populations. Furthermore, we demonstrate that human antibodies, affinity purified against recombinant MSP3N (rMSP3N), promote opsonic phagocytosis of merozoites in cooperation with monocytes. At nonphysiological concentrations, anti-MSP3N antibodies inhibited the growth of P. falciparum in vitro Together, the data suggest that MSP3 and especially its N-terminal region containing known B/T cell epitopes are targets of naturally acquired immunity against malaria and also comprise an important candidate for a multisubunit malaria vaccine.

Molecular modeling and docking of recombinant HAP-phytase of a thermophilic mould Sporotrichum thermophile reveals insights into molecular catalysis and biochemical properties.

A thermostable and protease-resistant HAP-phytase of Sporotrichum thermophile was over-expressed in Pichia pastoris X-33. Purified recombinant phytase displayed all its biochemical properties similar to wild type. Molecular modeling and docking of phytase with various substrates showed differential binding patterns with GoldScore values ranging from 40.61 to 79.78. Docking with different substrates revealed strong binding affinity with ATP and phytic acid, while the lowest with AMP and phosphoenol pyruvate. This was further confirmed using biochemical assays, as the recombinant enzyme displayed broad substrate specificity. Docking with inhibitors also showed differential binding with GoldScore values ranging from 22.94 (2,3-butanedione) to 85.72 (myo-inositol hexasulphate). Validation using biochemical analysis revealed that both 2,3-butanedione and phenyl glyoxal inhibited the phytase activity significantly. Furthermore, presence of inorganic phosphate in the reaction mixture also inhibited the phytase activity, as there was no activity at and beyond 0.8 mM. Docking of phytase with metavanadate showed binding at the same atom in the active-site where the substrate i.e. phytic acid binds. Vanadium incorporation resulted in the catalytic conversion of phytase into a peroxidase with concomitant inhibition of phytase activity. Peroxidase activity was high in acidic range and the product formation showed correlation with reaction time. Furthermore, molecular modeling and docking of recombinant HAP-phytase of a thermophilic mould S. thermophile reveals insights into molecular catalysis that is validated by the biochemical properties.

Population genetic structure of urban malaria vector Anopheles stephensi in India.

Malaria is a major public health problem in India because climatic condition and geography of India provide an ideal environment for development of malaria vector. Anopheles stephensi is a major urban malaria vector in India and its control has been hampered by insecticide resistance. In present study population genetic structure of A. stephensi is analyzed at macro geographic level using 13 microsatellite markers. Significantly high genetic differentiation was found in all studied populations with differentiation values (FST) ranging from 0.0398 to 0.1808. The geographic distance was found to be playing a major role in genetic differentiation between different populations. Overall three genetic pools were observed and population of central India was found to be coexisting in two genetic pools. High effective population size (Ne) was found in all the studied populations.

Population genetic structure of malaria vector Anopheles stephensi using mitochondrial cytochrome oxidase II gene in Indian populations.

The genetic differentiation in A. stephensi based on haplotype diversity using Restriction Fragment Length Polymorphism and bysequencing of CO II gene across different localities in India has been analyzed. The presence of only one DraI restriction site in CO II gene conferred to haplotype B indicating that the gene is very much conserved and the gene flow is not affected even by a major geographical distance barrier. The sequencing and analysisof various population parameters revealed seven haplotypes in all populations. The West Bengal population was found to be more genetically diverse than others. The geographic distance between populations was found to be contributing to the genetic differentiation. The sign of demographic expansion were found in three of the five populations. The local geographic barriers were found to be ineffective in prevention of gene flow.

Identification, characterization and analysis of expression of gene encoding carboxypeptidase A in Anopheles culicifacies A (Diptera: culicidae).

Carboxypeptidases are the digestive enzymes which cleave single amino acid residue from c-terminus of the protein. Digestive carboxypeptidase A gene regulatory elements in insects have shown their efficiency to drive midgut specific expression in transgenic mosquitoes. However no endogenous promoter has been reported for Indian malaria vector Anopheles culicifacies which is major vector in Indian subcontinent. Here we report cloning of carboxypeptidase A gene in the An. culicifacies A including its 5' upstream regions and named AcCP. In the upstream region of the gene an arthropod initiator sequence and two repeat sequences of the particular importance TTATC and GTTTT were also identified. The 1290 base pairs open reading frame encodes a protein of 48.5kDa. The coding region of the gene shares 82% and 72% similarity at nucleotide level with Anopheles gambiae and Ae. aegypti carboxypeptidase A gene, respectively. The peak expression of the gene was found to be at 3h after blood feeding and this is limited to midgut only. Based on the protein sequence, 3D structure of the AcCP was predicted and the active centre of the enzyme was predicted to consist of GLN 183, GLU 186, HIS 308 and Ser 309 amino acid residues. Comparison of the protein sequence among different genera revealed the conservation of zinc binding residues. Phylogenetically, AcCP was found most closely related to An. gambiae.

Population genetic structure of malaria vector Anopheles stephensi Liston (Diptera: Culicidae).

Malaria is a complex disease that afflicts human today. Malaria epidemiology is associated with drug resistance in parasite and differential distribution and insecticide resistance in vector. Efforts are being made to eradicate malaria but burden of malaria is still increasing. Vector control is essential for malaria prevention strategies. Knowledge of population genetic structure is pre-requisite for determining prevention strategies particularly using transgenic mosquitoes. Population genetic study can predict level of gene flow between different populations. Anopheles stephensi Liston is urban vector of malaria in Indo-Pakistan subcontinent. About 12% of malaria cases of malaria in India are contributed by A. stephensi. Studies conducted on population genetics of A. stephensi using various markers in different parts of the world are discussed in this communication.

Effect of anti-mosquito midgut antibodies on development of malaria parasite, Plasmodium vivax and fecundity in vector mosquito Anopheles culicifacies (Diptera: culicidae).

The effect of anti-mosquito-midgut antibodies on the development of the malaria parasite, P. vivax was studied by feeding the vector mosquito, An. culicifacies with infected blood supplemented with serum from immunized rabbits. In order to get antisera, rabbits were immunized with midgut proteins of three siblings species of Anopheles culicifacies, reported to exhibit differential vectorial capacity. The mosquitoes that ingested anti-midgut antibodies along with infectious parasites had significantly fewer oocysts compared to the control group of mosquitoes. The immunized rabbits generated high titer of antibodies. Their cross reactivity amongst various tissues of the same species and with other sibling species was also determined. Immunogenic polypeptides expressed in the midgut of glucose or blood fed An. culicifacies sibling species were identified by Western blotting. One immunogenic polypeptide of 62 kDa was exclusively present in the midgut of species A. Similarly, three polypeptides of 97, 94 and 58 kDa and one polypeptide of 23 kDa were present exclusively in species B and C respectively. Immunoelectron microscopy revealed the localization of these antigens on baso-lateral membrane and microvilli. The effects of anti-mosquito midgut antibodies on fecundity, longevity, mortality and engorgement of mosquitoes were studied. Fecundity was also reduced significantly. These observations open an avenue for research toward the development of a vector-based malaria parasite transmission-blocking vaccine.

Monoclonal antibodies AC-43 and AC-29 disrupt Plasmodium vivax development in the Indian malaria vector Anopheles culicifacies (Diptera: Culicidae).

A repertoire of monoclonal antibodies (mAbs) was generated against the midgut proteins of Anopheles culicifacies mosquitoes. The mAbs AC-43 and AC-29 significantly inhibited Plasmodium vivax development inside the mosquito midgut. The number of oocysts that developed was reduced by 78.6% when mosquitoes ingested a combination of these two mAbs along with the blood meal. AC-43 mAb binds to the epitope common in 97, 80 and 43 kDa polypeptides from the midgut protein extract, as indicated by western blot analysis. Similarly, the mAb AC-29 recognized 52, 44, 40 and 29 kDa polypeptides. These female midgut-specific polypeptides are shared between An. culicifacies and An. stephensi, two major vectors of malaria in India. Deglycosylation assays revealed that O-linked carbohydrates are the major components in epitopes corresponding to AC-43 and AC-29. Gold particle labelling revealed that both these mAbs preferentially bind to glycoproteins at the apical microvilli and the microvillus-associated network present inside transverse sections of the gut epithelium. These regions are particularly known to have receptors for ookinetes, which enable them to cross this epithelial barrier and provide them with certain necessary chemicals or components for further development into oocysts. Therefore, these glycoproteins appear to be potential candidates for a vector-directed transmission-blocking vaccine (TBV).

Antibodies raised against hemolymph of Anopheles culicifacies reduce the fecundity and malaria parasite development.

BACKGROUND & OBJECTIVES: Several studies have been made to study the effect of antisera raised against different tissues (hemolymh, ovary, midgut and salivary glands) on the fecundity and malaria parasite development in the different species of mosquitoes but there are no reports on the antisera raised against the hemolymph of Anopheles culicifacies, the principal malaria vector in India accounting for 65% of malaria cases. Hence, an attempt was made to study the same and evaluate its impact on malaria parasite development. METHODS: Polyclonal and multifactorial antibodies were produced in rabbits against heterogenous mixture of hemolymph proteins. Antibodies against hemolymph proteins were screened for their potential to influence reproductive performance of mosquitoes. Antibody titer in rabbit serum was determined by ELISA and putative candidate antigens were identified in the hemolymph of An. culicifacies by western blotting. Cross reactivity amongst various tissues vis-a-vis hemolymph protein was also identified. In addition, a significant reduction in oocyst development was also observed in An. culicifacies mosquitoes that ingested antihemolymph antibodies along with Plasmodium vivax. RESULTS: The maximum reduction in fecundity (57%) was observed during fourth week, after the last booster and number of oocyts per infected mosquito reduced by 73.35% in the group of mosquitoes that ingested antihemolymph antibodies along with the infected blood meal respectively. However, the ingestion of antibodies against hemolymph proteins did not have significant influence on hatchability. Antisera raised against hemolymph proteins of An. culicifacies recognized 11 polypeptides by western blotting. INTERPRETATION & CONCLUSION: During the present study, 11 putative candidate antigens were identified in the hemolymph of An. culicifacies, against which antibodies produced significantly reduced the fecundity by 57%. In addition, a significant reduction in oocyst development was also observed in An. culicifacies that ingested antihemolymph antibodies along with P.vivax.

Identification of all members of the anopheles culicifacies complex using allele-specific polymerase chain reaction assays.

Anopheles culicifacies, a complex of five isomorphic sibling species, is a major vector of malaria in India and neighboring countries. The five species are provisionally designated as species A, B, C, D, and E. Polytene chromosome examination has been the only method available that differentiates four members of this complex in areas where species E is not prevalent. However, this technique requires the mosquitoes to be in the half-gravid stage and thus limits its application to only about one fourth to one third of the total adult collection and excludes immature stages completely. For species E, both polytene chromosome examination and mitotic chromosome examination of F1 males are required. A polymerase chain reaction (PCR) assay based on the D3 domain (D3-PCR) of 28S rDNA and a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay involving ITS2 of rDNA are available for the discrimination of the members of the An. culicifacies complex. However, both these can only differentiate species A and D from species B, C, and E. We report here two allele-specific PCR assays (AD-PCR and BCE-PCR) using sequence differences in the mitochondrial cytochrome oxidase II (CO II) subunit. The AD-PCR assay distinguishes species A and D, whereas the BCE-PCR assay distinguishes species B, C, and E. Thus, with a combination of two PCR assays, namely the D3-PCR/ITS2-RsaI assay, followed by either the AD-PCR or the BCE-PCR assay, it is possible to identify individual specimens of any of the species of this complex. This assay system is the first, and the best available at present to distinguish all sibling species and especially to discriminate non-vector, species B from all the vector species, A, C, D, and E, of the An. culicifacies complex. Until another DNA-based method involving fewer steps is developed, this assay system can be used in all malaria epidemiologic studies where An. culicifacies is prevalent.

PCR-RFLP of mitochondrial cytochrome oxidase subunit II and ITS2 of ribosomal DNA: markers for the identification of members of the Anopheles culicifacies complex (Diptera: Culicidae).

Anopheles culicifacies Giles is a complex of five sibling species, provisionally designated as species A, B, C, D and E. Species A, C, D and E are vectors of malaria in India. Species A, B, C and D can be identified by polytene chromosome examination except in areas where species B and E are sympatric. Species B and E share the same configuration of the polytene chromosomes but can be differentiated by examining the mitotic chromosomes of F(1) progeny from field collection. Further, polytene chromosome examination method requires the mosquitoes to be at the semigravid stage, which limits on use of this method to a very small proportion of the population. The present study investigated whether the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method can be used to differentiate the members of this complex. Complete ITS2 region along with part of the 5.8S and 28S rDNA sequences (512 bp) and the mitochondrial cytochrome oxidase II (530 bp) were amplified and digested with different restriction endonucleases. The Alu I digest of the COII amplicon and Rsa I digest of the ITS2 amplicon could distinguish two categories: species A and D forming one category and species B, C and E forming another. Further, Dde I digestion of the COII amplicon could distinguish species E from species B and C within the latter category. The PCR-RFLP techniques developed in this study can be applied to areas where species A and B and species B and E are sympatric.

Midgut antibodies reduce the reproductive capacity of Anopheles stephensi (Diptera: Culicidae).

Rabbits immunized with polypeptides of midgut of glucose fed A. stephensi resulted in high titer of antibodies (10(4)-10(6)) as detected by ELISA. Effect of antisera on fecundity, hatchability and engorgement was investigated. Fecundity was reduced drastically (62.4%). Eight polypeptides were recognized by the antisera raised against midgut tissues viz., 92, 85, 55, 52, 45, 38, 29 and 13 kDa. Cross reactivity of these antibodies with different tissues of A. stephensi as well as different species of Anopheles was also analyzed. The results indicated that anti-mosquito midgut antibodies had the potential to disrupt the reproductive physiology of mosquitoes in view of the present study, there is a need for further investigation with target antigens.

Immune haemolymph proteins in response to bacterial infection and identification of a putative bacteria binding protein in malaria vector Anopheles stephensi.

Induction of haemolymph proteins in mosquito A. stephensi due to wounding or bacterial infection (E. coli) was analyzed using SDS-PAGE. Wounding response of pupa revealed subsequent induction of two polypeptides (21 and 74 kDa). Two other polypeptides (44 and 57 kDa) were induced commonly in both pupa and adult female haemolymph upon bacterial infection. In vitro binding assay revealed identification of 44 kDa, a putative bacterial binding protein, a more relevant protein for further elucidation of molecular mechanism involved in host parasite interactions.

Blocking of malaria parasite development in mosquito and fecundity reduction by midgut antibodies in Anopheles stephensi (Diptera: Culicidae).

Rabbits were immunized three times with extracts of Anopheles stephensi midgut. Immunized rabbits showed a high titer of antibodies when characterized by ELISA. We investigated the effect of anti-mosquito midgut antibodies on mosquito fecundity, longevity, mortality, engorgement, and the development of the malaria parasite in mosquitoes. Fecundity was reduced significantly (38%) and similarly hatchability by about 43.5%. There was no statistically significant effect on mortality, longevity, and engorgement. When the mosquito blood meal contained anti-midgut antibodies, fewer oocysts of Plasmodium vivax developed in the mosquito midgut and the proportion of mosquitoes becoming infected was significantly reduced. We also found that the midgut antibodies inhibit the development and/or translocation of the sporozoites. Antisera raised against midgut of A. stephensi recognized eight polypeptides (110, 92, 70, 45, 38, 29, 15, 13 kDa) by Western blotting. Cross-reactive antigens/epitopes present in other tissues of A. stephensi were also examined both by Western blotting and in vivo ELISA. Together, these observations open an avenue for research toward the development of a vector-based malaria parasite transmission blocking vaccine and/or anti-mosquito vaccine.

Alterations in polypeptides pattern in malaria vector Anopheles stephensi, fed upon immunized blood causing fecundity reduction.

Changes in polypeptides pattern of haemolymph, midgut, ovary and salivary glands of female mosquito A. stephensi were studied when fed upon anti-mosquito haemolymph antibodies. The expression of almost all polypeptides was reduced in haemolymph and ovary of the immune fed mosquitoes as compared to control. However, there was no significant difference in case of midgut and salivary glands. Seven polypeptides 100, 90, 84, 80, 62, 19 and 12.5 kDa were absent in haemolymph and five 92, 90, 80, 60 and 55 kDa were absent in ovaries. Changes in the polypeptide pattern have been correlated with the fecundity reduction due to immunized blood feeding.

Ovary specific immune response during Plasmodium yoelii yoelii infection in malaria vector Anopheles stephensi (Diptera:Insecta).

Innate immune related polypeptides expression during three gonotrophic cycles in the ovaries of major disease vector mosquito A. stephensi has been analyzed following infection by malaria parasite, P. yoelii yoelii. Seventeen polypeptides were induced in the ovaries of various stages due to parasitic infection. Most of proteins were induced systemically during early stages of infection suggesting the possibility of immune related signalling process. The reduction in the quantity of protein contents in infected mosquitoes has been ascribed to the repression of seven polypeptides and in turn correlated with the fecundity reduction. The mechanism of these responses and their significance for malaria transmission and fecundity reduction are discussed.

Anti-mosquito ovary antibodies reduce the fecundity of Anopheles stephensi (Diptera:Insecta).

Rabbit antibodies to five antigens (AJ** 29, 35, 43, 64, and 80 kDa) derived from the ovaries of Anopheles stephensi tended to reduce the number of eggs produced. Ingestion of anti-mosquito ovary antibodies did not show a detectable effect on the mortality of mosquitoes. Antisera raised against An. stephensi ovaries showed cross-reactivity in other tissues and in the ovaries of other Anopheles spp. by Western blotting. The results indicate that anti-mosquito ovary antibodies have the potential to disrupt the reproductive physiology of mosquitoes, and indicate the need for further studies with target antigens.

Midgut specific immune response of vector mosquito Anopheles stephensi to malaria parasite Plasmodium.

Innate immune-related polypeptides expression in midgut in the ageing vector mosquito A. stephensi following infection by malaria parasite, Plasmodium yoelii yoelii has been studied. Twenty polypeptides were induced by an infected blood meal during various stages of adult life. A 24 kDa polypeptide was induced generally in most of the stages. Maximum parasite induced polypeptides i.e. 22, 33, 111, 122, 127, 140, 143 and 146 kDa were found in 5 days of post blood feeding (PBF) which coincides with the presence of oocysts on the midgut. However, in addition, three polypeptides in 11 days PBF and 8 polypeptides in 20 days PBF were also induced due to parasite infection in aged mosquitoes. Quantitatively, the amount of soluble proteins in the midgut in oocyst-sporozoite-positive mosquitoes was always less as compared to their normal counterparts. The parasite evidently elicits defined immune responses by inducing specific polypeptides in the midgut of the mosquito.