Transmission blocking immunity in Plasmodium vivax malaria: antibodies raised against a peptide block parasite development in the mosquito vector.

One approach towards the development of a vaccine against malaria is to immunize against the parasite sexual stages that mediate transmission of the parasite from man to mosquito. Antibodies against these stages, ingested with the blood meal, inhibit the parasite development in the mosquito vector, constituting "transmission blocking immunity." Most epitopes involved in transmission-blocking immunity depend on the tertiary conformational structure of surface antigens. However, one of the transmission-blocking monoclonal antibodies we have raised against Plasmodium vivax reacts with a linear epitope on both asexual stages and gametes. This monoclonal antibody (A12) is capable of totally blocking development of the parasite in the mosquito host when tested in membrane feeding assays with gametocytes from P. vivax-infected patients. Immune screening of a P. vivax lambda gt11 genomic expression library with A12 led to the isolation of a clone to which was mapped the six-amino acid epitope recognized by A12. Antisera raised in mice against a 12-mer synthetic peptide containing this epitope coupled to bovine serum albumin not only had high titers of antipeptide antibodies as measured by enzyme-linked immunosorbent assay, but in addition recognized the same 24- and 57-kD parasite components as A12 on Western blots and reacted with the parasite by immunofluorescence. When tested in membrane feeding assays, these antibodies have significant suppressive effects on parasite development in the mosquito.

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Optimization of Plasmodium vivax sporozoite production from Anopheles stephensi in South West India

Malaria Journal ◽

10.1186/s12936-021-03767-2 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Ajeet Kumar Mohanty ◽

Charles de Souza ◽

Deepika Harjai ◽

Prathamesh Ghavanalkar ◽

Mezia Fernandes ◽

...

Keyword(s):

South Asia ◽

Plasmodium Vivax ◽

Anopheles Stephensi ◽

Parasite Development ◽

Future Research ◽

High Quality ◽

Gametocyte Density ◽

Feeding Experiments ◽

South West ◽

Membrane Feeding

Abstract Background Efforts to study the biology of Plasmodium vivax liver stages, particularly the latent hypnozoites, have been hampered by the limited availability of P. vivax sporozoites. Anopheles stephensi is a major urban malaria vector in Goa and elsewhere in South Asia. Using P. vivax patient blood samples, a series of standard membrane-feeding experiments were performed with An. stephensi under the US NIH International Center of Excellence for Malaria Research (ICEMR) for Malaria Evolution in South Asia (MESA). The goal was to understand the dynamics of parasite development in mosquitoes as well as the production of P. vivax sporozoites. To obtain a robust supply of P. vivax sporozoites, mosquito-rearing and mosquito membrane-feeding techniques were optimized, which are described here. Methods Membrane-feeding experiments were conducted using both wild and laboratory-colonized An. stephensi mosquitoes and patient-derived P. vivax collected at the Goa Medical College and Hospital. Parasite development to midgut oocysts and salivary gland sporozoites was assessed on days 7 and 14 post-feeding, respectively. The optimal conditions for mosquito rearing and feeding were evaluated to produce high-quality mosquitoes and to yield a high sporozoite rate, respectively. Results Laboratory-colonized mosquitoes could be starved for a shorter time before successful blood feeding compared with wild-caught mosquitoes. Optimizing the mosquito-rearing methods significantly increased mosquito survival. For mosquito feeding, replacing patient plasma with naïve serum increased sporozoite production > two-fold. With these changes, the sporozoite infection rate was high (> 85%) and resulted in an average of ~ 22,000 sporozoites per mosquito. Some mosquitoes reached up to 73,000 sporozoites. Sporozoite production could not be predicted from gametocyte density but could be predicted by measuring oocyst infection and oocyst load. Conclusions Optimized conditions for the production of high-quality P. vivax sporozoite-infected An. stephensi were established at a field site in South West India. This report describes techniques for producing a ready resource of P. vivax sporozoites. The improved protocols can help in future research on the biology of P. vivax liver stages, including hypnozoites, in India, as well as the development of anti-relapse interventions for vivax malaria.

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Evaluation of two Plasmodium vivax sexual stage antigens as transmission-blocking vaccine candidates

Parasites & Vectors ◽

10.1186/s13071-021-04909-w ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Yongzhe Zhang ◽

Fei Liu ◽

Yan Zhao ◽

Fan Yang ◽

Jie Bai ◽

...

Keyword(s):

Amino Acids ◽

Plasmodium Vivax ◽

Recombinant Proteins ◽

Expression System ◽

Anopheles Dirus ◽

Transmission Blocking ◽

Yeast Expression System ◽

Membrane Feeding ◽

Reducing Activity ◽

Membrane Feeding Assay

Abstract Background Plasmodium vivax transmission-blocking vaccines (TBVs) are receiving increasing attention. Based on excellent transmission-blocking activities of the PbPH (PBANKA_0417200) and PbSOP26 (PBANKA_1457700) antigens in Plasmodium berghei, their orthologs in P. vivax, PVX_098655 (PvPH) and PVX_101120 (PvSOP26), were selected for the evaluation of their potential as TBVs. Methods Fragments of PvPH (amino acids 22–304) and PvSOP26 (amino acids 30–272) were expressed in the yeast expression system. The recombinant proteins were used to immunize mice to obtain antisera. The transmission-reducing activities of these antisera were evaluated using the direct membrane feeding assay (DMFA) using Anopheles dirus mosquitoes and P. vivax clinical isolates. Results The recombinant proteins PvPH and PvSOP26 induced robust antibody responses in mice. The DMFA showed that the anti-PvSOP26 sera significantly reduced oocyst densities by 92.0 and 84.1% in two parasite isolates, respectively, whereas the anti-PvPH sera did not show evident transmission-reducing activity. The variation in the DMFA results was unlikely due to the genetic polymorphisms of the two genes since their respective sequences were identical in the clinical P. vivax isolates. Conclusion PvSOP26 could be a promising TBV candidate for P. vivax, which warrants further evaluation. Graphical Abstract

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Elucidating functional epitopes within the N-terminal region of malaria transmission blocking vaccine antigen Pfs230

npj Vaccines ◽

10.1038/s41541-021-00423-3 ◽

2022 ◽

Vol 7 (1) ◽

Author(s):

Kazutoyo Miura ◽

Eizo Takashima ◽

Thao P. Pham ◽

Bingbing Deng ◽

Luwen Zhou ◽

...

Keyword(s):

Malaria Transmission ◽

Synthetic Peptide ◽

Vaccine Antigen ◽

Parasite Development ◽

Transmission Blocking ◽

Peptide Antigens ◽

Membrane Feeding ◽

Functional Antibodies ◽

Enrichment Techniques ◽

Transmission Blocking Vaccine

AbstractPfs230 is a leading malaria transmission blocking vaccine (TBV) candidate. Comprising 3135 amino acids (aa), the large size of Pfs230 necessitates the use of sub-fragments as vaccine immunogens. Therefore, determination of which regions induce functional antibody responses is essential. We previously reported that of 27 sub-fragments spanning the entire molecule, only five induced functional antibodies. A “functional” antibody is defined herein as one that inhibits Plasmodium falciparum parasite development in mosquitoes in a standard membrane-feeding assay (SMFA). These five sub-fragments were found within the aa 443–1274 range, and all contained aa 543–730. Here, we further pinpoint the location of epitopes within Pfs230 that are recognized by functional antibodies using antibody depletion and enrichment techniques. Functional epitopes were not found within the aa 918–1274 region. Within aa 443–917, further analysis showed the existence of functional epitopes not only within the aa 543–730 region but also outside of it. Affinity-purified antibodies using a synthetic peptide matching aa 543–588 showed activity in the SMFA. Immunization with a synthetic peptide comprising this segment, formulated either as a carrier-protein conjugate vaccine or with a liposomal vaccine adjuvant system, induced antibodies in mice that were functional in the SMFA. These findings provide key insights for Pfs230-based vaccine design and establish the feasibility for the use of synthetic peptide antigens for a malaria TBV.

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Evaluation of two Plasmodium vivax sexual-stage antigens as transmission-blocking vaccine candidates

10.21203/rs.3.rs-373830/v1 ◽

2021 ◽

Author(s):

Yongzhe Zhang ◽

Fei Liu ◽

Yan Zhao ◽

Fan Yang ◽

Jie Bai ◽

...

Keyword(s):

Amino Acids ◽

Plasmodium Vivax ◽

Recombinant Proteins ◽

Expression System ◽

Transmission Blocking ◽

Yeast Expression System ◽

Membrane Feeding ◽

Reducing Activity ◽

Membrane Feeding Assay ◽

Transmission Blocking Vaccines

Abstract Background: Plasmodium vivax transmission-blocking vaccines (TBVs) have received high attention. PVX_098655 (PvPH) and PVX_101120 (PvSOP26) were predicted to be potential TBV antigens based on the studies of their orthologs in Plasmodium berghei. Methods: Fragments of PvPH (amino acids 22–304) and PvSOP26 (amino acids 30–272) were expressed in the yeast expression system. The recombinant proteins were used to immunize mice to obtain the antisera. The transmission-reducing activities of these antisera were evaluated using the standard membrane feeding assay (SMFA) using Anopheles dirus mosquitoes and P. vivax clinical isolates. Results: The recombinant proteins PvPH and PvSOP26 induced robust antibody responses in mice. With SMFA, the anti-PvSOP26 sera significantly reduced oocyst densities by 92.0% and 84.1% in two parasite isolates, while the anti-PvPH sera did not show evident transmission-reducing activity. Both PvPH and PvSOP26 showed limited gene polymorphisms in the clinical P. vivax isolates. Conclusion: PvSOP26 could be a promising TBV candidate for P. vivax.

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Induction of Plasmodium falciparum Transmission-Blocking Antibodies in Nonhuman Primates by a Combination of DNA and Protein Immunizations

Infection and Immunity ◽

10.1128/iai.72.1.253-259.2004 ◽

2004 ◽

Vol 72 (1) ◽

pp. 253-259 ◽

Cited By ~ 43

Author(s):

Cevayir Coban ◽

Mario T. Philipp ◽

Jeanette E. Purcell ◽

David B. Keister ◽

Mobolaji Okulate ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Antibody Response ◽

Dna Vaccine ◽

Nonhuman Primates ◽

Rhesus Macaques ◽

Surface Protein ◽

Mosquito Vector ◽

Transmission Blocking ◽

Membrane Feeding ◽

Antibody Titers

ABSTRACT Malaria transmission-blocking vaccination can effectively reduce and/or eliminate transmission of parasites from the human host to the mosquito vector. The immunity achieved by inducing an antibody response to surface antigens of male and female gametes and parasite stages in the mosquito. Our laboratory has developed DNA vaccine constructs, based on Pfs25 (a Plasmodium falciparum surface protein of 25 kDa), that induce a transmission-blocking immune response in mice (C. A. Lobo, R. Dhar, and N. Kumar, Infect. Immun. 67:1688-1693, 1999). To evaluate the safety, immunogenicity, and efficacy of the Pfs25 DNA vaccine in nonhuman primates, we immunized rhesus macaques (Macaca mulatta) with a DNA vaccine plasmid encoding Pfs25 or a Pfg27-Pfs25 hybrid or with the plasmid (empty plasmid) alone. Immunization with four doses of these DNA vaccine constructs elicited antibody titers that were high but nonetheless unable to reduce the parasite's infectivity in membrane feeding assays. Further boosting of the antibody response with recombinant Pfs25 formulated in Montanide ISA-720 increased antibody titers (30-fold) and significantly blocked transmission of P. falciparum gametocytes to Anopheles mosquitoes (∼90% reduction in oocyst numbers in the midgut). Our data show that a DNA prime-protein boost regimen holds promise for achieving transmission-blocking immunity in areas where malaria is endemic and could be effective in eradicating malaria in isolated areas where the level of malaria endemicity is low.

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Functional Conservation of P48/45 Proteins in the Transmission Stages of Plasmodium vivax (Human Malaria Parasite) and P. berghei (Murine Malaria Parasite)

mBio ◽

10.1128/mbio.01627-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 5

Author(s):

Yi Cao ◽

Robert J. Hart ◽

Geetha P. Bansal ◽

Nirbhay Kumar

Keyword(s):

Plasmodium Vivax ◽

Malaria Transmission ◽

Malaria Parasite ◽

Mosquito Vector ◽

Test Model ◽

Transmission Blocking ◽

Content Type ◽

Functional Conservation ◽

Transgenic Parasites

ABSTRACT Sexual-stage proteins have a distinct function in the mosquito vector during transmission and also represent targets for the development of malaria transmission-blocking vaccine. P48/45, a leading vaccine candidate, is critical for male gamete fertility and shows >50% similarity across various species of Plasmodium. We evaluated functional conservation of P48/45 in Plasmodium vivax and P. berghei with the motivation to establish transgenic P. berghei strains expressing P. vivax P48/45 (Pvs48/45) in an in vivo assay to evaluate the transmission-blocking activity of antibodies elicited by Pvs48/45. Homologous recombination was employed to target P. berghei s48/45 (pbs48/45) for knockout (KO) or for its replacement by two different forms of P. vivax s48/45 (pvs48/45) (the full-length gene and a chimeric gene consisting of pbs48/45 5′ signal and 3′ anchor sequences flanking pvs48/45). Expression of Pvs48/45 in transgenic parasites and lack of expression of any P48/45 in KO parasites were confirmed by reverse transcription-PCR (RT-PCR) and Western blotting. Both transgenic and knockout parasites revealed asexual growth kinetics in mice comparable to that seen with wild-type parasites. When employed in mosquito infection experiments, both transgenic parasite strains remained transmission competent and developed into infectious sporozoites, whereas the knockout parasites were incapable of establishing mosquito-stage infection. These results indicate the functional conservation of P48/45 protein during transmission, and the transgenic parasites generated in this study represent a valuable tool to evaluate the protective efficacy of transmission-blocking antibodies elicited by Pvs48/45-based vaccines using an in vivo mouse animal assay instead of ex vivo membrane feeding assays (MFA) relying on access to P. vivax gametocytes from infected patients. IMPORTANCE Malaria transmission depends upon successful sexual differentiation and maturation of parasites in the vertebrate host and further development in the mosquito midgut. Stage-specific proteins in the sexual stages have been shown to play a critical role in development and successful transmission through the anopheline mosquito vector. Studies presented in the current manuscript evaluated functional conservation of one such protein, P48/45, in two diverse species (P. berghei and P. vivax). Replacement of endogenous pbs48/45 in P. berghei with pvs48/45 (P. vivax homologue) did not affect the viability of the parasites, and the transgenic parasites expressing Pvs48/45 remained transmission competent. These studies establish not only the functional conservation of P48/45 in P. berghei and P. vivax but also offer an opportunity to develop an in vivo test model for Pvs48/45-based P. vivax transmission-blocking vaccines, currently under development.

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Functional Comparison of Plasmodium falciparum Transmission-Blocking Vaccine Candidates by the Standard Membrane-Feeding Assay

Infection and Immunity ◽

10.1128/iai.01056-13 ◽

2013 ◽

Vol 81 (12) ◽

pp. 4377-4382 ◽

Cited By ~ 72

Author(s):

Kazutoyo Miura ◽

Eizo Takashima ◽

Bingbing Deng ◽

Gregory Tullo ◽

Ababacar Diouf ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Recombinant Proteins ◽

The Other ◽

Enzyme Linked Immunosorbent Assay ◽

Dependent Manner ◽

Feeding Assay ◽

Transmission Blocking ◽

Content Type ◽

Membrane Feeding ◽

Membrane Feeding Assay

ABSTRACTRecently, there has been a renewed interest in the development of transmission-blocking vaccines (TBV) againstPlasmodium falciparummalaria. While several candidate TBVs have been reported, studies directly comparing them in functional assays are limited. To this end, recombinant proteins of TBV candidates Pfs25, Pfs230, and PfHAP2 were expressed in the wheat germ cell-free expression system. Outbred CD-1 mice were immunized twice with the antigens. Two weeks after the second immunization, IgG levels were measured by enzyme-linked immunosorbent assay (ELISA), and IgG functionality was assessed by the standard membrane-feeding assay (SMFA) using culturedP. falciparumNF54 gametocytes andAnopheles stephensimosquitoes. All three recombinant proteins elicited similar levels of antigen-specific IgG judged by ELISA. When IgGs purified from pools of immune serum were tested at 0.75 mg/ml in the SMFA, all three IgGs showed 97 to 100% inhibition in oocyst intensity compared to control IgG. In two additional independent SMFA evaluations, anti-Pfs25, anti-Pfs230, and anti-PfHAP2 IgGs inhibited oocyst intensity in a dose-dependent manner. When all three data sets were analyzed, anti-Pfs25 antibody showed significantly higher inhibition than the other two antibodies (P< 0.001 for both), while there was no significant difference between the other two (P= 0.15). A proportion of plasma samples collected from adults living in an area of malaria endemicity in Mali recognized Pfs230 and PfHAP2. This is the first study showing that the HAP2 protein ofP. falciparumcan induce transmission-blocking antibody. The current study supports the possibility of using this system for a comparative study with multiple TBV candidates.

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Evaluation and modeling of direct membrane-feeding assay with Plasmodium vivax to support development of transmission blocking vaccines

Scientific Reports ◽

10.1038/s41598-020-69513-x ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Kazutoyo Miura ◽

Bruce J. Swihart ◽

Michael P. Fay ◽

Chalermpon Kumpitak ◽

Kirakorn Kiattibutr ◽

...

Keyword(s):

Plasmodium Vivax ◽

Feeding Assay ◽

Transmission Blocking ◽

Membrane Feeding ◽

Membrane Feeding Assay ◽

Transmission Blocking Vaccines

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The effect of transmission-blocking antibody ingested in primary and secondary bloodfeeds, upon the development ofPlasmodium bergheiin the mosquito vector

Parasitology ◽

10.1017/s0031182000079191 ◽

1993 ◽

Vol 107 (3) ◽

pp. 225-231 ◽

Cited By ~ 23

Author(s):

G. Ranawaka ◽

R. Alejo-Blanco ◽

R. E. Sinden

Keyword(s):

Plasmodium Berghei ◽

Anopheles Stephensi ◽

Similar Efficacy ◽

Parasite Development ◽

Mosquito Vector ◽

Blocking Antibody ◽

Transmission Blocking ◽

Mean Intensity ◽

Monoclonal Immunoglobulins ◽

Transmission Enhancement

SummaryThe effects of purified monoclonal immunoglobulins from control, or transmission-blocking anti-Pbs21 antibodies, upon the infection ofAnopheles stephensiby ookinetes ofPlasmodium bergheiare compared. Anti-Phs21 antibody reduced mean intensity and prevalence of infection by 94·7 and 58·7% respectively if added to the infectious bloodfeed at a concentration of 100 μg/ml. Fab fragments were of similar efficacy. No transmission enhancement was detected with declining antibody concentrations. Addition to subsequent (second) feeds reduced mean oocyst intensity but not prevalence. The reduction in blockade declined from 41% at day 2, to 4% at day 8. Second bloodfeeds, containing control globulin taken 4 or 6 days (but not 2 days) after infection, increased sporozoite burden in the salivary glands. At all times anti-Pbs21 reduced sporozoite number in the thorax compared to time-matched controls, but again highest gland intensities were obtained when the second bloodfeed was given on day 4. We conclude that second bloodfeeds containing transmission-blocking antibody simultaneously serve two opposing roles, (1) inhibition of parasite development and (2) the supply of nutrients which permit more sporozoites to be produced by each oocyst.

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Expression, Structure, and Location of Epitopes of the Major Surface Glycoprotein of Pneumocystis carinii f. sp. carinii

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.5.1.50-57.1998 ◽

1998 ◽

Vol 5 (1) ◽

pp. 50-57 ◽

Cited By ~ 20

Author(s):

Michael J. Linke ◽

Susan M. Sunkin ◽

Ryan P. Andrews ◽

James R. Stringer ◽

Peter D. Walzer

Keyword(s):

Amino Acids ◽

Pneumocystis Carinii ◽

Amino Acid Sequences ◽

Surface Glycoprotein ◽

Enzyme Linked Immunosorbent Assay ◽

Expression Library ◽

Genomic Expression ◽

Expression Screening ◽

Major Surface Glycoprotein ◽

Major Surface

ABSTRACT The major surface glycoprotein (MSG) of Pneumocystis carinii f. sp. carinii consists of a heterogeneous family of proteins that are encoded by approximately 100 unique genes. A genomic expression library was screened with a panel of MSG-specific monoclonal antibodies (MAbs) to identify conserved and rare epitopes. All of the antibodies reacted with epitopes that are encoded within the 5′ end of MSG. The results from the expression screening identified antibodies that recognize highly conserved, moderately conserved, and rare epitopes. Four MAbs (MAbs RA-F1, RA-E7, RA-G10, and RB-E3) reacted with a maltose binding protein–MSG-B fusion protein (MBPMSG-B41–1065) by immunoblotting and enzyme-linked immunosorbent assay. Three of the MAbs (MAbs RA-F1, RA-G10, and RA-E7) reacted with the same continuous epitope that was localized to amino acids 278 to 290 of MSG-B. Comparison of the sequence of the RA-F1-, RA-G10-, and RA-E7-reactive epitope to the deduced amino acid sequences of multiple MSGs demonstrated that it is highly conserved. The reactivity of RB-E3 with MSG-B was shown to be dependent on amino acids 184 to 192, which may comprise a portion of a discontinuous epitope.

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