Potent Malaria Transmission-Blocking Antibody Responses Elicited by Plasmodium falciparum Pfs25 Expressed in Escherichia coli after Successful Protein Refolding

ABSTRACTProduction of Pfs25, aPlasmodium falciparumtransmission-blocking vaccine target antigen, in functional conformation with the potential to elicit effective immunogenicity still remains a major challenge. In the current study, codon-harmonized recombinant Pfs25 (CHrPfs25) was expressed inEscherichia coli, and purified protein after simple oxidative refolding steps retained reduction-sensitive conformational epitopes of transmission-blocking monoclonal antibodies. CHrPfs25 formulated in several adjuvants elicited strong immunogenicity in preclinical studies in mice. Antibodies elicited after immunization recognized native Pfs25 on the surface of live gametes ofP. falciparumand demonstrated complete malaria transmission-blocking activity. The transmission-blocking efficacy was 100% even after a 1:128 dilution of sera from immunized mice in the complete Freund's adjuvant and Montanide ISA51 groups and after a 1:16 dilution of sera from mice in the alum group. The blocking was mediated by antibodies; purified IgG at concentrations as low as 31.25 μg/ml exhibited 100% transmission blocking in membrane feeding assays employing two different species of mosquitoes,Anopheles gambiaeandAnopheles stephensi. This study provides the first evidence for successful expression of biologically functional rPfs25 inE. coli. The extremely potent malaria transmission-blocking activity of antibodies elicited by immunization with purified protein provides strong support for further evaluation ofE. coli-derived CHrPfs25 as a malaria transmission-blocking vaccine in human clinical trials.

Download Full-text

N-Terminal Prodomain of Pfs230 Synthesized Using a Cell-Free System Is Sufficient To Induce Complement-Dependent Malaria Transmission-Blocking Activity

Clinical and Vaccine Immunology ◽

10.1128/cvi.05104-11 ◽

2011 ◽

Vol 18 (8) ◽

pp. 1343-1350 ◽

Cited By ~ 49

Author(s):

Mayumi Tachibana ◽

Yimin Wu ◽

Hideyuki Iriko ◽

Olga Muratova ◽

Nicholas J. MacDonald ◽

...

Keyword(s):

Malaria Transmission ◽

Expression System ◽

Surface Protein ◽

Western Blot Assay ◽

Free System ◽

Transmission Blocking ◽

Content Type ◽

Membrane Feeding ◽

Blocking Activity ◽

Transmission Blocking Vaccine

ABSTRACTThe aim of a malaria transmission-blocking vaccine is to block the development of malaria parasites in the mosquito and thus prevent subsequent infection of the human host. Previous studies have demonstrated that the gametocyte/gamete surface protein Pfs230 can induce transmission-blocking immunity and have evaluatedEscherichia coli-produced Pfs230 as a transmission-blocking vaccine candidate. In this study, we used the wheat germ cell-free expression system to produce N-terminal fragments of Pfs230 and evaluated the transmission-blocking activity of antisera raised against the recombinant Pfs230 protein. The rabbit antisera reacted to the surface of cultured gametocytes and gametes of thePlasmodium falciparumNF54 line, recognized the 360-kDa form of parasite-produced Pfs230 by Western blot assay, and reduced the infectivity of NF54 parasites toAnopheles stefensimosquitoes in the presence of complement in a standard membrane feeding assay. Thus, our data demonstrate that the N-terminal pro domain of Pfs230 is sufficient to induce complement-dependent transmission-blocking activity againstP. falciparum.

Download Full-text

Murine Model for Assessment of Plasmodium falciparum Transmission-Blocking Vaccine Using Transgenic Plasmodium berghei Parasites Expressing the Target Antigen Pfs25

Infection and Immunity ◽

10.1128/iai.01409-07 ◽

2008 ◽

Vol 76 (5) ◽

pp. 2018-2024 ◽

Cited By ~ 24

Author(s):

Godfree Mlambo ◽

Jorge Maciel ◽

Nirbhay Kumar

Keyword(s):

Animal Model ◽

Plasmodium Falciparum ◽

Malaria Transmission ◽

Plasmodium Berghei ◽

Target Antigen ◽

Wild Type ◽

Transmission Blocking ◽

Transmission Blocking Vaccines ◽

Transmission Blocking Vaccine

ABSTRACT Currently, there is no animal model for Plasmodium falciparum challenge to evaluate malaria transmission-blocking vaccines based on the well-established Pfs25 target antigen. The biological activity of transmission-blocking antibodies is typically assessed using an assay known as the membrane feeding assay (MFA). It is an in vitro method that involves mixing antibodies with cultured P. falciparum gametocytes and feeding them to mosquitoes through an artificial membrane followed by assessment of infection in the mosquitoes. We genetically modified Plasmodium berghei to express Pfs25 and demonstrated that the transgenic parasites (TrPfs25Pb) are susceptible to anti-Pfs25 antibodies during mosquito-stage development. The asexual growth kinetics and mosquito infectivity of TrPfs25Pb were comparable to those of wild-type parasites, and TrPfs25Pb displayed Pfs25 on the surface of ookinetes. Immune sera from nonhuman primates immunized with a Pfs25-based vaccine when passively transferred to mice blocked transmission of TrPfs25Pb to Anopheles stephensi. Furthermore, mice immunized with Pfs25 DNA vaccine and challenged with TrPfs25Pb displayed reduced malaria transmission compared to mice immunized with wild-type plasmid. These studies describe development of an animal malaria model alternative to the in vitro MFA and show that the model can facilitate P. falciparum transmission-blocking vaccine evaluation based on the target antigen Pfs25. We believe that an animal model to test transmission-blocking vaccines would be superior to the MFA, since there may be additional immune factors that synergize the transmission-blocking activity of antibodies in vivo.

Download Full-text

DSM265 at 400 Milligrams Clears Asexual Stage Parasites but Not Mature Gametocytes from the Blood of Healthy Subjects Experimentally Infected with Plasmodium falciparum

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01837-18 ◽

2019 ◽

Vol 63 (4) ◽

Cited By ~ 10

Author(s):

Katharine A. Collins ◽

Thomas Rückle ◽

Suzanne Elliott ◽

Louise Marquart ◽

Emma Ballard ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Pharmacodynamic Modeling ◽

Dihydroorotate Dehydrogenase ◽

Transmission Blocking ◽

Content Type ◽

Blocking Activity ◽

Cutaneous Rash ◽

Phase 1B ◽

Study Support ◽

Previous Phase

ABSTRACT DSM265 is a novel antimalarial drug in clinical development that acts as a selective inhibitor of Plasmodium dihydroorotate dehydrogenase. In a previous phase 1b study, a single 150-mg dose of DSM265 showed partial efficacy against experimentally induced blood-stage Plasmodium falciparum malaria (IBSM). Pharmacokinetic/pharmacodynamic modeling predicted a human efficacious dose of 340 mg. The primary objectives of the current study were to determine the safety and efficacy of a single oral 400-mg dose of DSM265 against P. falciparum in the IBSM model. Eight healthy participants were inoculated intravenously with 2,800 parasites and treated with DSM265 7 days later. Unexpectedly, one participant did not develop parasitemia during the study. All other participants developed parasitemia, with the complete clearance of asexual parasites occurring following DSM265 treatment. All seven subjects also became gametocytemic. The secondary objectives were to investigate the gametocytocidal and transmission-blocking activity of a second 400-mg dose of DSM265, which was administered 23 days after inoculation. Gametocytes were not cleared by the second dose of DSM265, and transmission-blocking activity could not be determined due to low gametocyte densities. Three DSM265-related adverse events occurred, including a cutaneous rash in one subject on the day of the second DSM265 dose. The results obtained in this study support the prediction of the efficacious dose of DSM265 and provide further evidence that DSM265 is generally safe and well tolerated. In addition, this study confirms preclinical data indicating that DSM265 permits the development and maturation of gametocytes and does not clear mature circulating gametocytes. (This study has been registered at ClinicalTrials.gov under identifier NCT02573857.)

Download Full-text

Engineering a Virus-Like Particle as an Antigenic Platform for a Pfs47-Targeted Malaria Transmission-Blocking Vaccine

Scientific Reports ◽

10.1038/s41598-019-53208-z ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Lampouguin Yenkoidiok-Douti ◽

Adeline E. Williams ◽

Gaspar E. Canepa ◽

Alvaro Molina-Cruz ◽

Carolina Barillas-Mury

Keyword(s):

Plasmodium Falciparum ◽

Amino Acid ◽

Malaria Transmission ◽

Transmission Blocking ◽

High Affinity ◽

Virus Like Particle ◽

Antibody Titers ◽

Reducing Activity ◽

Sexual Stages ◽

Transmission Blocking Vaccine

AbstractWe recently characterized Pfs47, a protein expressed on the surface of sexual stages and ookinetes of Plasmodium falciparum, as a malaria transmission-blocking vaccine (TBV) target. Mice immunization induced antibodies that conferred strong transmission-reducing activity (TRA) at a concentration of 200 μg/mL. Here, we sought to optimize the Pfs47 vaccine to elicit higher titers of high-affinity antibodies, capable of inducing strong TRA at a lower concentration. We report the development and evaluation of a Pfs47-based virus-like particle (VLP) vaccine generated by conjugating our 58 amino acid Pfs47 antigen to Acinetobacter phage AP205-VLP using the SpyCatcher:SpyTag adaptor system. AP205-Pfs47 complexes (VLP-P47) formed particles of ~22 nm diameter that reacted with polyclonal anti-Pfs47 antibodies, indicating that the antigen was accessible on the surface of the particle. Mice immunized with VLP-P47 followed by a boost with Pfs47 monomer induced significantly higher antibody titers, with higher binding affinity to Pfs47, than mice that received two immunizations with either VLP-P47 (VLP-P47/VLP-P47) or the Pfs47 monomer (P47/P47). Purified IgG from VLP-P47/P47 mice had strong TRA (83–98%) at concentrations as low as 5 μg/mL. These results indicate that conjugating the Pfs47 antigen to AP205-VLP significantly enhanced antigenicity and confirm the potential of Pfs47 as a TBV candidate.

Download Full-text

Plasmodium falciparum: Immunogenicity of Alum-Adsorbed Clinical-Grade TBV25–28, a Yeast-Secreted Malaria Transmission-Blocking Vaccine Candidate

Experimental Parasitology ◽

10.1006/expr.2000.4580 ◽

2001 ◽

Vol 97 (2) ◽

pp. 61-69 ◽

Cited By ~ 21

Author(s):

Mary Margaret G Gozar ◽

Olga Muratova ◽

David B Keister ◽

Charlotte R Kensil ◽

Virginia L Price ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Malaria Transmission ◽

Vaccine Candidate ◽

Clinical Grade ◽

Transmission Blocking ◽

Transmission Blocking Vaccine

Download Full-text

Alga-Produced Malaria Transmission-Blocking Vaccine Candidate Pfs25 Formulated with a Human Use-Compatible Potent Adjuvant Induces High-Affinity Antibodies That Block Plasmodium falciparum Infection of Mosquitoes

Infection and Immunity ◽

10.1128/iai.02980-14 ◽

2015 ◽

Vol 83 (5) ◽

pp. 1799-1808 ◽

Cited By ~ 20

Author(s):

Kailash P. Patra ◽

Fengwu Li ◽

Darrick Carter ◽

James A. Gregory ◽

Sheyenne Baga ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Malaria Vaccine ◽

Vaccine Development ◽

Vaccine Candidate ◽

Water Emulsion ◽

Transmission Blocking ◽

Content Type ◽

Oil In Water ◽

Oil In Water Emulsion ◽

Transmission Blocking Vaccine

A vaccine to prevent the transmission of malaria parasites from infected humans to mosquitoes is an important component for the elimination of malaria in the 21st century, yet it remains neglected as a priority of malaria vaccine development. The lead candidate forPlasmodium falciparumtransmission-blocking vaccine development, Pfs25, is a sexual stage surface protein that has been produced for vaccine testing in a variety of heterologous expression systems. Any realistic malaria vaccine will need to optimize proper folding balanced against cost of production, yield, and potentially reactogenic contaminants. HereChlamydomonas reinhardtiimicroalga-produced recombinant Pfs25 protein was formulated with four different human-compatible adjuvants (alum, Toll-like receptor 4 [TLR-4] agonist glucopyranosal lipid A [GLA] plus alum, squalene–oil-in-water emulsion, and GLA plus squalene–oil-in-water emulsion) and compared for their ability to induce malaria transmission-blocking antibodies. Alga-produced recombinant Pfs25 plus GLA plus squalene–oil-in-water adjuvant induced the highest titer and avidity in IgG antibodies, measured using alga-produced recombinant Pfs25 as the enzyme-linked immunosorbent assay (ELISA) antigen. These antibodies specifically reacted with the surface ofP. falciparummacrogametes and zygotes and effectively prevented parasites from developing within the mosquito vector in standard membrane feeding assays. Alga-produced Pfs25 in combination with a human-compatible adjuvant composed of a TLR-4 agonist in a squalene–oil-in-water emulsion is an attractive new vaccine candidate that merits head-to-head comparison with other modalities of vaccine production and administration.

Download Full-text

A Potent Malaria Transmission Blocking Vaccine Based on Codon Harmonized Full Length Pfs48/45 Expressed in Escherichia coli

PLoS ONE ◽

10.1371/journal.pone.0006352 ◽

2009 ◽

Vol 4 (7) ◽

pp. e6352 ◽

Cited By ~ 86

Author(s):

Debabani Roy Chowdhury ◽

Evelina Angov ◽

Thomas Kariuki ◽

Nirbhay Kumar

Keyword(s):

Escherichia Coli ◽

Malaria Transmission ◽

Full Length ◽

Transmission Blocking ◽

Transmission Blocking Vaccine

Download Full-text

Display of malaria transmission-blocking antigens on chimeric duck hepatitis B virus-derived virus-like particles produced inHansenula polymorpha

10.1101/595538 ◽

2019 ◽

Cited By ~ 1

Author(s):

David Wetzel ◽

Jo-Anne Chan ◽

Manfred Suckow ◽

Andreas Barbian ◽

Michael Weniger ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Malaria Transmission ◽

Transmission Blocking ◽

Duck Hepatitis B Virus ◽

Vaccine Candidates ◽

Duck Hepatitis ◽

B Virus ◽

Transmission Blocking Vaccine

1.AbstractBackgroundMalaria caused byPlasmodium falciparumis one of the major threats to human health globally. Despite huge efforts in malaria control and eradication, highly effective vaccines are urgently needed, including vaccines that can block malaria transmission. Chimeric virus-like particles (VLP) have emerged as a promising strategy to develop new malaria vaccine candidates.MethodsWe developed yeast cell lines and processes for the expression of malaria transmission-blocking vaccine candidates Pfs25 and Pfs230 as VLP and VLP were analyzed for purity, size, protein incorporation rate and expression of malaria antigens.ResultsIn this study, a novel platform for the display ofPlasmodium falciparumantigens on chimeric VLP is presented. Leading transmission-blocking vaccine candidates Pfs25 and Pfs230 were genetically fused to the small surface protein (dS) of the duck hepatitis B virus (DHBV). The resulting fusion proteins were co-expressed in recombinantHansenula polymorpha(syn.Pichia angusta, Ogataea polymorpha) strains along with the wild-type dS as the VLP scaffold protein. Through this strategy, chimeric VLP containing Pfs25 or the Pfs230-derived fragments Pfs230c or Pfs230D1M were purified. Up to 100 mg chimeric VLP were isolated from 100 g dry cell weight with a maximum protein purity of 90 % on the protein level. Expression of the Pfs230D1M construct was more efficient than Pfs230c and enabled VLP with higher purity. VLP showed reactivity with transmission-blocking antibodies and supported the surface display of the malaria antigens on the native VLP.ConclusionThe incorporation of leadingPlasmodium falciparumtransmission-blocking antigens into the dS-based VLP scaffold is a promising novel strategy for their display on nano-scaled particles. Competitive processes for efficient production and purification were established in this study.

Download Full-text