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.

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 by sequencing 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.

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 signifi cantly 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-specifi c 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 vectordirected 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.

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.

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.

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.

Seasonal variations of culicine mosquitoes in district Rohtak.

Seasonal pattern in relation to culicine fauna of Rohtak district of Haryana--India studied for two successive years i.e. from October 1988 to September 1990 is presented. A total of seven culicine species in the descending order of their densities were Cx. quinquefasciatus > Cx. tritaeniorhynchus > Ae. aegypti > Cx. vishnui/Ma. uniformis > Cs. fuscanus > Ar. kuchingensis their were found. Seasonal pattern of abundance were classified on the basis of fluctuations in different seasons for different species. i) Unimodal monsoon - Ae. aegypti, Cx tritaeniorhynchus, Cx. fuscanus; ii) Bimodal spring and monsoon - Cx. vishnui, iii) Bimodal spring and winter-Cx. quinquefasciatus; iv) Unimodal summer-Ma. uniformis.