The Plasmodium falciparum circumsporozoite protein produced in Lactococcus lactis is pure and stable

The Plasmodium falciparum circumsporozoite protein (PfCSP) is a sporozoite surface protein whose role in sporozoite motility and cell invasion has made it the leading candidate for a pre-erythrocytic malaria vaccine. However, production of high yields of soluble recombinant PfCSP, including its extensive NANP and NVDP repeats, has proven problematic. Here, we report on the development and characterization of a secreted, soluble, and stable full-length PfCSP (containing 4 NVDP and 38 NANP repeats) produced in the Lactococcus lactis expression system. The recombinant full-length PfCSP, denoted PfCSP4/38, was produced initially with a histidine tag and purified by a simple two-step procedure. Importantly, the recombinant PfCSP4/38 retained a conformational epitope for antibodies as confirmed by both in vivo and in vitro characterizations. We characterized this complex protein by HPLC, light scattering, MS analysis, differential scanning fluorimetry, CD, SDS-PAGE, and immunoblotting with conformation-dependent and -independent mAbs, which confirmed it to be both pure and soluble. Moreover, we found that the recombinant protein is stable at both frozen and elevated-temperature storage conditions. When we used L. lactis–derived PfCSP4/38 to immunize mice, it elicited high levels of functional antibodies that had the capacity to modify sporozoite motility in vitro. We concluded that the reported yield, purity, results of biophysical analyses, and stability of PfCSP4/38 warrant further consideration of using the L. lactis system for the production of circumsporozoite proteins for preclinical and clinical applications in malaria vaccine development.

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Antibody Targets and Properties for Complement-Fixation Against the Circumsporozoite Protein in Malaria Immunity

Frontiers in Immunology ◽

10.3389/fimmu.2021.775659 ◽

2021 ◽

Vol 12 ◽

Author(s):

Liriye Kurtovic ◽

Damien R. Drew ◽

Arlene E. Dent ◽

James W. Kazura ◽

James G. Beeson

Keyword(s):

Plasmodium Falciparum ◽

Malaria Vaccine ◽

Vaccine Development ◽

Complement Fixation ◽

Terminal Region ◽

Antibody Responses ◽

Circumsporozoite Protein ◽

Full Length ◽

Acquired Immunity ◽

Vaccine Candidates

The Plasmodium falciparum circumsporozoite protein (CSP) forms the basis of leading subunit malaria vaccine candidates. However, the mechanisms and specific targets of immunity are poorly defined. Recent findings suggest that antibody-mediated complement-fixation and activation play an important role in immunity. Here, we investigated the regions of CSP targeted by functional complement-fixing antibodies and the antibody properties associated with this activity. We quantified IgG, IgM, and functional complement-fixing antibody responses to different regions of CSP among Kenyan adults naturally exposed to malaria (n=102) and using a series of rabbit vaccination studies. Individuals who acquired functional complement-fixing antibodies had higher IgG, IgM and IgG1 and IgG3 to CSP. Acquired complement-fixing antibodies targeted the N-terminal, central-repeat, and C-terminal regions of CSP, and positive responders had greater antibody breadth compared to those who were negative for complement-fixing antibodies (p<0.05). Using rabbit vaccinations as a model, we confirmed that IgG specific to the central-repeat and non-repeat regions of CSP could effectively fix complement. However, vaccination with near full length CSP in rabbits poorly induced antibodies to the N-terminal region compared to naturally-acquired immunity in humans. Poor induction of N-terminal antibodies was also observed in a vaccination study performed in mice. IgG and IgM to all three regions of CSP play a role in mediating complement-fixation, which has important implications for malaria vaccine development.

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The P. falciparum CSP repeat region contains three distinct epitopes required for protection by antibodies in vivo

PLoS Pathogens ◽

10.1371/journal.ppat.1010042 ◽

2021 ◽

Vol 17 (11) ◽

pp. e1010042

Author(s):

Yevel Flores-Garcia ◽

Lawrence T. Wang ◽

Minah Park ◽

Beejan Asady ◽

Azza H. Idris ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Monoclonal Antibodies ◽

Malaria Infection ◽

Vaccine Development ◽

Circumsporozoite Protein ◽

Repeat Region ◽

Human Mabs ◽

Necessary And Sufficient

Rare and potent monoclonal antibodies (mAbs) against the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) on infective sporozoites (SPZ) preferentially bind the PfCSP junctional tetrapeptide NPDP or NVDP minor repeats while cross-reacting with NANP major repeats in vitro. The extent to which each of these epitopes is required for protection in vivo is unknown. Here, we assessed whether junction-, minor repeat- and major repeat-preferring human mAbs (CIS43, L9 and 317 respectively) bound and protected against in vivo challenge with transgenic P. berghei (Pb) SPZ expressing either PfCSP with the junction and minor repeats knocked out (KO), or PbCSP with the junction and minor repeats knocked in (KI). In vivo protection studies showed that the junction and minor repeats are necessary and sufficient for CIS43 and L9 to neutralize KO and KI SPZ, respectively. In contrast, 317 required major repeats for in vivo protection. These data establish that human mAbs can prevent malaria infection by targeting three different protective epitopes (NPDP, NVDP, NANP) in the PfCSP repeat region. This report will inform vaccine development and the use of mAbs to passively prevent malaria.

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Vaccination of Monkeys with Recombinant Plasmodium falciparum Apical Membrane Antigen 1 Confers Protection against Blood-Stage Malaria

Infection and Immunity ◽

10.1128/iai.70.12.6961-6967.2002 ◽

2002 ◽

Vol 70 (12) ◽

pp. 6961-6967 ◽

Cited By ~ 128

Author(s):

Anthony W. Stowers ◽

Michael C. Kennedy ◽

Brian P. Keegan ◽

Allan Saul ◽

Carole A. Long ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Malaria Vaccine ◽

Vaccine Development ◽

Apical Membrane ◽

Surface Protein ◽

Merozoite Surface Protein ◽

Lethal Challenge ◽

Apical Membrane Antigen 1 ◽

Apical Membrane Antigen

ABSTRACT A major challenge facing malaria vaccine development programs is identifying efficacious combinations of antigens. To date, merozoite surface protein 1 (MSP1) is regarded as the leading asexual vaccine candidate. Apical membrane antigen 1 (AMA1) has been identified as another leading candidate for an asexual malaria vaccine, but without any direct in vivo evidence that a recombinant form of Plasmodium falciparum AMA1 would have efficacy. We evaluated the efficacy of a form of P. falciparum AMA1, produced in Pichia pastoris, by vaccinating Aotus vociferans monkeys and then challenging them with P. falciparum parasites. Significant protection from this otherwise lethal challenge with P. falciparum was observed. Five of six animals had delayed patency; two of these remained subpatent for the course of the infection, and two controlled parasite growth at <0.75% of red blood cells parasitized. The protection induced by AMA1 was superior to that obtained with a form of MSP1 used in the same trial. The protection induced by a combination vaccine of AMA1 and MSP1 was not superior to the protection obtained with AMA1 alone, although the immunity generated appeared to operate against both vaccine components.

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In vitro and in vivo inhibition of malaria parasite infection by monoclonal antibodies against Plasmodium falciparum circumsporozoite protein (CSP)

Scientific Reports ◽

10.1038/s41598-021-84622-x ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Merricka C. Livingstone ◽

Alexis A. Bitzer ◽

Alish Giri ◽

Kun Luo ◽

Rajeshwer S. Sankhala ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Monoclonal Antibodies ◽

Disease Burden ◽

Vaccine Candidate ◽

Specific Binding ◽

Circumsporozoite Protein ◽

Target Epitope ◽

Selection Of

AbstractPlasmodium falciparum malaria contributes to a significant global disease burden. Circumsporozoite protein (CSP), the most abundant sporozoite stage antigen, is a prime vaccine candidate. Inhibitory monoclonal antibodies (mAbs) against CSP map to either a short junctional sequence or the central (NPNA)n repeat region. We compared in vitro and in vivo activities of six CSP-specific mAbs derived from human recipients of a recombinant CSP vaccine RTS,S/AS01 (mAbs 317 and 311); an irradiated whole sporozoite vaccine PfSPZ (mAbs CIS43 and MGG4); or individuals exposed to malaria (mAbs 580 and 663). RTS,S mAb 317 that specifically binds the (NPNA)n epitope, had the highest affinity and it elicited the best sterile protection in mice. The most potent inhibitor of sporozoite invasion in vitro was mAb CIS43 which shows dual-specific binding to the junctional sequence and (NPNA)n. In vivo mouse protection was associated with the mAb reactivity to the NANPx6 peptide, the in vitro inhibition of sporozoite invasion activity, and kinetic parameters measured using intact mAbs or their Fab fragments. Buried surface area between mAb and its target epitope was also associated with in vivo protection. Association and disconnects between in vitro and in vivo readouts has important implications for the design and down-selection of the next generation of CSP based interventions.

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Bacterially Expressed Full-Length Recombinant Plasmodium falciparum RH5 Protein Binds Erythrocytes and Elicits Potent Strain-Transcending Parasite-Neutralizing Antibodies

Infection and Immunity ◽

10.1128/iai.00970-13 ◽

2013 ◽

Vol 82 (1) ◽

pp. 152-164 ◽

Cited By ~ 54

Author(s):

K. Sony Reddy ◽

Alok K. Pandey ◽

Hina Singh ◽

Tajali Sahar ◽

Amlabu Emmanuel ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Neutralizing Antibodies ◽

Malaria Vaccine ◽

Expression System ◽

Full Length ◽

Blood Stage ◽

Parasite Protein ◽

Content Type ◽

Erythrocyte Binding ◽

Blood Stage Malaria

ABSTRACTPlasmodium falciparumreticulocyte binding-like homologous protein 5 (PfRH5) is an essential merozoite ligand that binds with its erythrocyte receptor, basigin. PfRH5 is an attractive malaria vaccine candidate, as it is expressed by a wide number ofP. falciparumstrains, cannot be genetically disrupted, and exhibits limited sequence polymorphisms. Viral vector-induced PfRH5 antibodies potently inhibited erythrocyte invasion. However, it has been a challenge to generate full-length recombinant PfRH5 in a bacterial-cell-based expression system. In this study, we have produced full-length recombinant PfRH5 inEscherichia colithat exhibits specific erythrocyte binding similar to that of the native PfRH5 parasite protein and also, importantly, elicits potent invasion-inhibitory antibodies against a number ofP. falciparumstrains. Antibasigin antibodies blocked the erythrocyte binding of both native and recombinant PfRH5, further confirming that they bind with basigin. We have thus successfully produced full-length PfRH5 as a functionally active erythrocyte binding recombinant protein with a conformational integrity that mimics that of the native parasite protein and elicits potent strain-transcending parasite-neutralizing antibodies.P. falciparumhas the capability to develop immune escape mechanisms, and thus, blood-stage malaria vaccines that target multiple antigens or pathways may prove to be highly efficacious. In this regard, antibody combinations targeting PfRH5 and other key merozoite antigens produced potent additive inhibition against multiple worldwideP. falciparumstrains. PfRH5 was immunogenic when immunized with other antigens, eliciting potent invasion-inhibitory antibody responses with no immune interference. Our results strongly support the development of PfRH5 as a component of a combination blood-stage malaria vaccine.

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Plasmodium falciparum Merozoite Surface Protein 1 (MSP-1)-MSP-3 Chimeric Protein: Immunogenicity Determined with Human-Compatible Adjuvants and Induction of Protective Immune Response

Infection and Immunity ◽

10.1128/iai.00427-09 ◽

2009 ◽

Vol 78 (2) ◽

pp. 872-883 ◽

Cited By ~ 19

Author(s):

Suman Mazumdar ◽

Paushali Mukherjee ◽

Syed Shams Yazdani ◽

S. K. Jain ◽

Asif Mohmmed ◽

...

Keyword(s):

Immune Response ◽

Plasmodium Falciparum ◽

Fusion Protein ◽

Malaria Vaccine ◽

Chimeric Protein ◽

Surface Protein ◽

Merozoite Surface Protein ◽

Protective Immune Response ◽

Merozoite Surface Protein 1

ABSTRACT A chimeric gene, MSP-Fu24 , was constructed by genetically coupling immunodominant, conserved regions of the two leading malaria vaccine candidates, Plasmodium falciparum merozoite surface protein 1 (C-terminal 19-kDa region [PfMSP-119]) and merozoite surface protein 3 (11-kDa conserved region [PfMSP-311]). The recombinant MSP-Fu24 protein was produced in Escherichia coli cells and purified to homogeneity by a two-step purification process with a yield of ∼30 mg/liter. Analyses of conformational properties of MSP-Fu24 using PfMSP-119-specific monoclonal antibody showed that the conformational epitopes of PfMSP-119 that may be critical for the generation of the antiparasitic immune response remained intact in the fusion protein. Recombinant MSP-Fu24 was highly immunogenic in mice and in rabbits when formulated with two different human-compatible adjuvants and induced an immune response against both PfMSP-119 and PfMSP-311. Purified anti-MSP-Fu24 antibodies showed invasion inhibition of P. falciparum 3D7 and FCR parasites, and this effect was found to be dependent on antibodies specific for the PfMSP-119 component. The protective potential of MSP-Fu24 was demonstrated by in vitro parasite growth inhibition using an antibody-dependent cell inhibition (ADCI) assay with anti-MSP-Fu24 antibodies. Overall, the antiparasitic activity was mediated by a combination of growth-inhibitory antibodies generated by both the PfMSP-119 and PfMSP-311 components of the MSP-Fu24 protein. The antiparasitic activities elicited by anti-MSP-Fu24 antibodies were comparable to those elicited by antibodies generated with immunization with a physical mixture of two component antigens, PfMSP-119 and PfMSP-311. The fusion protein induces a protective immune response with human-compatible adjuvants and may form a part of a multicomponent malaria vaccine.

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Immunoglobulin G Subclass-Specific Responses against Plasmodium falciparum Merozoite Antigens Are Associated with Control of Parasitemia and Protection from Symptomatic Illness

Infection and Immunity ◽

10.1128/iai.01129-08 ◽

2009 ◽

Vol 77 (3) ◽

pp. 1165-1174 ◽

Cited By ~ 165

Author(s):

Danielle I. Stanisic ◽

Jack S. Richards ◽

Fiona J. McCallum ◽

Pascal Michon ◽

Christopher L. King ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Immunoglobulin G ◽

Vaccine Development ◽

Surface Protein ◽

Merozoite Surface Protein ◽

Igg Subclass ◽

Apical Membrane Antigen 1 ◽

Falciparum Merozoite ◽

Merozoite Antigens

ABSTRACT Substantial evidence indicates that antibodies to Plasmodium falciparum merozoite antigens play a role in protection from malaria, although the precise targets and mechanisms mediating immunity remain unclear. Different malaria antigens induce distinct immunoglobulin G (IgG) subclass responses, but the importance of different responses in protective immunity from malaria is not known and the factors determining subclass responses in vivo are poorly understood. We examined IgG and IgG subclass responses to the merozoite antigens MSP1-19 (the 19-kDa C-terminal region of merozoite surface protein 1), MSP2 (merozoite surface protein 2), and AMA-1 (apical membrane antigen 1), including different polymorphic variants of these antigens, in a longitudinal cohort of children in Papua New Guinea. IgG1 and IgG3 were the predominant subclasses of antibodies to each antigen, and all antibody responses increased in association with age and exposure without evidence of increasing polarization toward one subclass. The profiles of IgG subclasses differed somewhat for different alleles of MSP2 but not for different variants of AMA-1. Individuals did not appear to have a propensity to make a specific subclass response irrespective of the antigen. Instead, data suggest that subclass responses to each antigen are generated independently among individuals and that antigen properties, rather than host factors, are the major determinants of IgG subclass responses. High levels of AMA-1-specific IgG3 and MSP1-19-specific IgG1 were strongly predictive of a reduced risk of symptomatic malaria and high-density P. falciparum infections. However, no antibody response was significantly associated with protection from parasitization per se. Our findings have major implications for understanding human immunity and for malaria vaccine development and evaluation.

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Transgenic Parasites Stably Expressing Full-Length Plasmodium falciparum Circumsporozoite Protein as a Model for Vaccine Down-Selection in Mice Using Sterile Protection as an Endpoint

Clinical and Vaccine Immunology ◽

10.1128/cvi.00066-13 ◽

2013 ◽

Vol 20 (6) ◽

pp. 803-810 ◽

Cited By ~ 31

Author(s):

Michael D. Porter ◽

Jennifer Nicki ◽

Christopher D. Pool ◽

Margot DeBot ◽

Ratish M. Illam ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Malaria Vaccine ◽

Circumsporozoite Protein ◽

Full Length ◽

Content Type ◽

Transmission Cycle ◽

Degree Of Protection ◽

Antibody Titers ◽

Malaria Vaccine Candidate ◽

Transgenic Parasites

ABSTRACTCircumsporozoite protein (CSP) ofPlasmodium falciparumis a protective human malaria vaccine candidate. There is an urgent need for models that can rapidly down-select novel CSP-based vaccine candidates. In the present study, the mouse-mosquito transmission cycle of a transgenicPlasmodium bergheimalaria parasite stably expressing a functional full-lengthP. falciparumCSP was optimized to consistently produce infective sporozoites for protection studies. A minimal sporozoite challenge dose was established, and protection was defined as the absence of blood-stage parasites 14 days after intravenous challenge. The specificity of protection was confirmed by vaccinating mice with multiple CSP constructs of differing lengths and compositions. Constructs that induced high NANP repeat-specific antibody titers in enzyme-linked immunosorbent assays were protective, and the degree of protection was dependent on the antigen dose. There was a positive correlation between antibody avidity and protection. The antibodies in the protected mice recognized the native CSP on the parasites and showed sporozoite invasion inhibitory activity. Passive transfer of anti-CSP antibodies into naive mice also induced protection. Thus, we have demonstrated the utility of a mouse efficacy model to down-select human CSP-based vaccine formulations.

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