scholarly journals Multistage Multiantigen Heterologous Prime Boost Vaccine forPlasmodium knowlesi Malaria Provides Partial Protection in Rhesus Macaques

2001 ◽  
Vol 69 (9) ◽  
pp. 5565-5572 ◽  
Author(s):  
William O. Rogers ◽  
J. Kevin Baird ◽  
Anita Kumar ◽  
John A. Tine ◽  
Walter Weiss ◽  
...  
Keyword(s):  
Dna Vaccine ◽  
Vaccine Development ◽  
Plasmodium Knowlesi ◽  
Rhesus Macaques ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Antibody Responses ◽  
Primate Model ◽  
Boost Vaccine

ABSTRACT A nonhuman primate model for malaria vaccine development allowing reliable, stringent sporozoite challenge and evaluation of both cellular and antibody responses is needed. We therefore constructed a multicomponent, multistage DNA vaccine for the simian malaria species Plasmodium knowlesi including two preerythrocytic-stage antigens, the circumsporozoite protein (PkCSP) and sporozoite surface protein 2 (PkSSP2), and two blood stage antigens, apical merozoite antigen 1 (PkAMA1) and merozoite surface protein 1 (PkMSP1p42), as well as recombinant canarypox viruses encoding the four antigens (ALVAC-4). The DNA vaccine plasmids expressed the corresponding antigens in vitro and induced antiparasite antibodies in mice. Groups of four rhesus monkeys received three doses of a mixture of the four DNA vaccine plasmids and a plasmid encoding rhesus granulocyte-monocyte colony-stimulating factor, followed by boosting with a single dose of ALVAC-4. Three groups received the priming DNA doses by different routes, either by intramuscular needle injection, by intramuscular injection with a needleless injection device, the Biojector, or by a combination of intramuscular and intradermal routes by Biojector. Animals immunized by any route developed antibody responses against sporozoites and infected erythrocytes and against a recombinant PkCSP protein, as well as gamma interferon-secreting T-cell responses against peptides from PkCSP. Following challenge with 100 P. knowlesi sporozoites, 1 of 12 experimental monkeys was completely protected and the mean parasitemia in the remaining monkeys was significantly lower than that in 4 control monkeys. This model will be important in preclinical vaccine development.

scholarly journals Strain-Transcending Fc-Dependent Killing ofPlasmodium falciparumby Merozoite Surface Protein 2 Allele-Specific Human Antibodies

2010 ◽  
Vol 79 (3) ◽  
pp. 1143-1152 ◽  
Author(s):  
Janine Stubbs ◽  
Sope Olugbile ◽  
Balam Saidou ◽  
Jacques Simpore ◽  
Giampietro Corradin ◽  
...  
Keyword(s):  
Polyclonal Antibodies ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Antibody Responses ◽  
Blood Stage ◽  
Human Antibodies ◽  
Candidate Peptide ◽  
Allele Specific ◽  
High Level

ABSTRACTIt is widely accepted that antibody responses against the human parasitic pathogenPlasmodium falciparumprotect the host from the rigors of severe malaria and death. However, there is a continuing need for the development ofin vitrocorrelate assays of immune protection. To this end, the capacity of human monoclonal and polyclonal antibodies in eliciting phagocytosis and parasite growth inhibition via Fcγ receptor-dependent mechanisms was explored. In examining the extent to which sequence diversity in merozoite surface protein 2 (MSP2) results in the evasion of antibody responses, an unexpectedly high level of heterologous function was measured for allele-specific human antibodies. The dependence on Fcγ receptors for opsonic phagocytosis and monocyte-mediated antibody-dependent parasite inhibition was demonstrated by the mutation of the Fc domain of monoclonal antibodies against both MSP2 and a novel vaccine candidate, peptide 27 from the genePFF0165c. The described flow cytometry-based functional assays are expected to be useful for assessing immunity in naturally infected and vaccinated individuals and for prioritizing among blood-stage antigens for inclusion in blood-stage vaccines.

scholarly journals Codon Optimization of Gene Fragments EncodingPlasmodium falciparum Merzoite Proteins Enhances DNA Vaccine Protein Expression and Immunogenicity in Mice

2001 ◽  
Vol 69 (12) ◽  
pp. 7250-7253 ◽  
Author(s):  
David L. Narum ◽  
Sanjai Kumar ◽  
William O. Rogers ◽  
Steven R. Fuhrmann ◽  
Hong Liang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Codon Usage ◽  
Dna Vaccine ◽  
Dna Vaccines ◽  
Homo Sapiens ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Erythrocyte Binding ◽  
Mouse Cells

ABSTRACT In contrast to conventional vaccines, DNA and other subunit vaccines exclusively utilize host cell molecules for transcription and translation of proteins. The adenine plus thymine content of Plasmodium falciparum gene sequences (∼80%) is much greater than that of Homo sapiens(∼59%); consequently, codon usage is markedly different. We hypothesized that modifying codon usage of P. falciparumgenes encoded by DNA vaccines from that used by the parasite to those resembling mammalian codon usage would lead to increased P. falciparum protein expression in vitro in mouse cells and increased antibody responses in DNA-vaccinated mice. We synthesized gene fragments encoding the receptor-binding domain of the 175-kDaP. falciparum erythrocyte-binding protein (EBA-175 region II) and the 42-kDa C-terminal processed fragment of the P. falciparum merozoite surface protein 1 (MSP-142) using the most frequently occurring codon in mammals to code for each amino acid, and inserted the synthetic genes in DNA vaccine plasmids. In in vitro transient-expression assays, plasmids containing codon-optimized synthetic gene fragments (pS plasmids) showed greater than fourfold increased protein expression in mouse cells compared to those containing native gene fragments (pN plasmids). In mice immunized with 0.5, 5.0, or 50 μg of the DNA plasmids, the dose of DNA required to induce equivalent antibody titers was 10- to 100-fold lower for pS than for pN plasmids. These data demonstrate that optimizing codon usage in DNA vaccines can improve protein expression and consequently the immunogenicity of gene fragments in DNA vaccines for organisms whose codon usage differs substantially from that of mammals.

scholarly journals A Bicistronic DNA Vaccine Containing Apical Membrane Antigen 1 and Merozoite Surface Protein 4/5 Can Prime Humoral and Cellular Immune Responses and Partially Protect Mice against Virulent Plasmodium chabaudi adami DS Malaria

2004 ◽  
Vol 72 (10) ◽  
pp. 5565-5573 ◽  
Author(s):  
A. Rainczuk ◽  
T. Scorza ◽  
T. W. Spithill ◽  
P. M. Smooker
Keyword(s):  
Immune Responses ◽  
Dna Vaccine ◽  
Apical Membrane ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Delivery Systems ◽  
Plasmodium Chabaudi ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen

ABSTRACT The ultimate malaria vaccine will require the delivery of multiple antigens from different stages of the complex malaria life cycle. In order to efficiently deliver multiple antigens with use of DNA vaccine technology, new antigen delivery systems must be assessed. This study utilized a bicistronic vector construct, containing an internal ribosome entry site, expressing a combination of malarial candidate antigens: merozoite surface protein 4/5 (MSP4/5) (fused to a monocyte chemotactic protein 3 chemoattractant sequence) and apical membrane antigen 1 (AMA-1) (fused to a tissue plasminogen activator secretion signal). Transfection of COS 7 cells with bicistronic plasmids resulted in production and secretion of both AMA-1 and MSP4/5 in vitro. Vaccination of BALB/c mice via intraepidermal gene gun and intramuscular routes against AMA-1 and MSP4/5 resulted in antibody production and significant in vitro proliferation of splenocytes stimulated by both AMA-1 and MSP4/5. Survival of BALB/c mice vaccinated with bicistronic constructs after lethal Plasmodium chabaudi adami DS erythrocytic-stage challenge was variable, although significant increases in survival and reductions in peak parasitemia were observed in several challenge trials when the vaccine was delivered by the intramuscular route. This study using a murine model demonstrates that the delivery of malarial antigens via bicistronic vectors is feasible. Further experimentation with bicistronic delivery systems is required for the optimization and refinement of DNA vaccines to effectively prime protective immune responses against malaria.

scholarly journals Characterization of Glycoproteins of Native 19kDa C-Terminal Merozoite Surface Protein-1 from Native Antigen of Plasmodium falciparum

2019 ◽  
Author(s):  
Sahar Tajik ◽  
Sedigheh Sadeghi ◽  
Ayda Iravani ◽  
Mitra Khalili ◽  
Mohammad Arjmand ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Large Scale ◽  
Vaccine Development ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Surface Proteins ◽  
Ring Stage ◽  
Native Form ◽  
Sds Page

Background: Plasmodium falciparum is the protozoan parasite which causes malignant malaria of medical concern. Prime candidates for recombinant vaccine development are asexual stage antigens of P. falciparum, for example, merozoite surface proteins (MSP1 and MSP2) not given satisfactory results to date. In this study, the 19kDa C-terminal of MSP1, a vaccine candidate was purified in its native form in the ring stage, and its glycoproteins studied. Methods: The study was carried out at the Biochemistry Department of Pasteur Institute of Iran in the years 2015–2016. Large scale culture of P. falciparum was performed in vitro with 80% ring stage parasitemia. Isopycnic ultracentrifuga­tion with 36% sucrose and analytical SDS-PAGE on the supernatant and precipitate performed, and the 19kDa antigen was obtained by cutting it from strips of preparative SDS gels. Purified protein was concentrated and analyzed by SDS-PAGE and immunoblotting, using antibodies raised to recombinant C-terminal MSP1. Results: The purified protein gave a single band of 19kDa antigen as shown by silver staining of SDS-PAGE and a sin­gle bond in immunoblotting. Bioinformatics also confirmed the likelihood of the presence of glycans on the antigen. Conclusion: The presence of N and O-glycoproteins were detected by Q proteome kit. This work was done on the ring stage, and earlier workers confirmed the presence of glycoproteins on MSP1 in the other stages. This glycosylation is present in all stages, and maybe incomplete protection elicited by recombinant MSP1 antigens is due to lack of N and O-glycoproteins.

scholarly journals Polyclonal antibody responses to HIV Env immunogens resolved using cryoEM

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Aleksandar Antanasijevic ◽  
Leigh M. Sewall ◽  
Christopher A. Cottrell ◽  
Diane G. Carnathan ◽  
Luis E. Jimenez ◽  
...  
Keyword(s):  
High Resolution ◽  
Polyclonal Antibody ◽  
Vaccine Development ◽  
Mutational Analysis ◽  
Rhesus Macaques ◽  
Antibody Responses ◽  
Glycosylation Sites ◽  
High Resolution Analysis ◽  
Resolution Analysis ◽  
Resolution Mapping

AbstractEngineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms. In this study, we used rhesus macaques to evaluate the immunogenicity of several stabilized BG505 SOSIP constructs both as free trimers and presented on a nanoparticle. We applied a cryoEM-based method for high-resolution mapping of polyclonal antibody responses elicited in immunized animals (cryoEMPEM). Mutational analysis coupled with neutralization assays were used to probe the neutralization potential at each epitope. We demonstrate that cryoEMPEM data can be used for rapid, high-resolution analysis of polyclonal antibody responses without the need for monoclonal antibody isolation. This approach allowed to resolve structurally distinct classes of antibodies that bind overlapping sites. In addition to comprehensive mapping of commonly targeted neutralizing and non-neutralizing epitopes in BG505 SOSIP immunogens, our analysis revealed that epitopes comprising engineered stabilizing mutations and of partially occupied glycosylation sites can be immunogenic.

Plasmodium knowlesi: a relevant, versatile experimental malaria model

Parasitology ◽  
2016 ◽  
Vol 145 (1) ◽  
pp. 56-70 ◽  
Author(s):  
ERICA M. PASINI ◽  
ANNE-MARIE ZEEMAN ◽  
ANNEMARIE VOORBERG-VAN DER WEL ◽  
CLEMENS H. M. KOCKEN
Keyword(s):  
Parasite Species ◽  
Vaccine Development ◽  
Plasmodium Knowlesi ◽  
Host Interactions ◽  
Model Studies ◽  
Human Red Blood Cells ◽  
Malaria Model ◽  
Human Primate

SUMMARYThe primate malariaPlasmodium knowlesihas a long-standing history as an experimental malaria model. Studies using this model parasite in combination with its various natural and experimental non-human primate hosts have led to important advances in vaccine development and in our understanding of malaria invasion, immunology and parasite–host interactions. The adaptation to long-termin vitrocontinuous blood stage culture in rhesus monkey,Macaca fascicularisand human red blood cells, as well as the development of various transfection methodologies has resulted in a highly versatile experimental malaria model, further increasing the potential of what was already a very powerful model. The growing evidence thatP. knowlesiis an important human zoonosis in South-East Asia has added relevance to former and future studies of this parasite species.

scholarly journals Merozoite Surface Protein 1 of Plasmodium vivax Induces a Protective Response against Plasmodium cynomolgi Challenge in Rhesus Monkeys

2005 ◽  
Vol 73 (9) ◽  
pp. 5936-5944 ◽  
Author(s):  
Sheetij Dutta ◽  
Deep C. Kaushal ◽  
Lisa A. Ware ◽  
Sunil K. Puri ◽  
Nuzhat A. Kaushal ◽  
...  
Keyword(s):  
Plasmodium Vivax ◽  
Protective Efficacy ◽  
High Titer ◽  
Surface Protein ◽  
Merozoite Surface Protein ◽  
Control Group ◽  
Merozoite Surface Protein 1 ◽  
Significant Difference ◽  
Peak Parasitemia

ABSTRACT The 42-kDa fragment of the merozoite surface protein 1 (MSP-142) is a leading candidate for the development of a vaccine to control malaria. We previously reported a method for the production of Plasmodium vivax MSP-142 (PvMSP-142) as a soluble protein (S. Dutta, L. W. Ware, A. Barbosa, C. F. Ockenhouse, and D. E. Lanar, Infect. Immun. 69:5464-5470, 2001). We report here a process to manufacture the same PvMSP-142 protein but as an insoluble inclusion body-derived protein which was then refolded in vitro. We compared the immunogenicity and protective efficacy of the soluble and refolded forms of PvMSP-142 protein by using a heterologous but closely related P. cynomolgi-rhesus monkey challenge model. As comparative controls we also expressed, purified, and immunized rhesus with the soluble and refolded forms of the P. cynomolgi MSP-142 (PcMSP-142) proteins. All proteins induced equally high-titer, cross-reacting antibodies. Upon challenge with P. cynomolgi, none of the MSP-142-vaccinated groups demonstrated sterile protection or a delay in the prepatent period. However, following an initial rise in parasitemia, all MSP-1-vaccinated animals had significantly lower parasite burdens as indicated by lower cumulative parasitemia, lower peak parasitemia, lower secondary peak parasitemia, and lower average daily parasitemia compared to the adjuvant control group (P < 0.05). Except the soluble PcMSP-142 group, monkeys in all other groups had fewer numbers of days with parasitemia of >10,000 parasites mm−3. Interestingly, there was no significant difference in the level of partial protection observed in the homologous and heterologous groups in this challenge model. The soluble and refolded forms of PcMSP-142 and PvMSP-142 proteins also appeared to have a similar partially protective effect.

scholarly journals Identification of Rotavirus VP6-Specific CD4+ T Cell Epitopes in a G1P[8] Human Rotavirus-Infected Rhesus Macaque

2008 ◽  
Vol 1 ◽  
pp. VRT.S563 ◽  
Author(s):  
Wei Zhao ◽  
Bapi Pahar ◽  
Karol Sestak
Keyword(s):  
T Cell ◽  
Rhesus Macaques ◽  
Immunoglobulin A ◽  
Cd4 T Cell ◽  
T Cell Epitopes ◽  
Primate Model ◽  
Human Rotavirus ◽  
Ifn Γ ◽  
Rotavirus Vp6

A non-human primate model was used to evaluate its potential for identification of rotavirus viral protein 6 (VP6) CD4+ T cell epitopes. Four juvenile rhesus macaques were inoculated with a mixed inoculum (G1P[8] and G9P[8]) of human rotaviruses. Infection accompanied by G1P[8] shedding was achieved in the two macaques that had no rotavirus immunoglobulin A (IgA) in plasma. To measure the interferon gamma (IFN-γ) and tumor necrosis factor (TNF) anti-viral cytokines produced by peripheral CD4+ cells that recognize VP6 epitopes, whole blood cells from one infected macaque were stimulated in vitro with VP6 peptides. Stimulation with peptide pools derived from the simian rotavirus VP6161–395 region revealed reactivity of CD4+ T cells with the VP6281–331 domain. A VP6301–315 region was identified as the epitope responsible for IFN-γ production while a broader VP6293–327 domain was linked to TNF production. These results suggest that human rotavirus-infected macaques can be used for identification of additional epitopes and domains to address specific questions related to the development of pediatric vaccines.

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

2019 ◽  
Vol 295 (2) ◽  
pp. 403-414 ◽  
Author(s):  
Susheel K. Singh ◽  
Jordan Plieskatt ◽  
Bishwanath Kumar Chourasia ◽  
Vandana Singh ◽  
Judith M. Bolscher ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Lactococcus Lactis ◽  
Malaria Vaccine ◽  
Vaccine Development ◽  
Expression System ◽  
Surface Protein ◽  
Circumsporozoite Protein ◽  
Full Length

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.

scholarly journals Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasite Plasmodium knowlesi

2016 ◽  
Vol 113 (26) ◽  
pp. 7231-7236 ◽  
Author(s):  
Robert W. Moon ◽  
Hazem Sharaf ◽  
Claire H. Hastings ◽  
Yung Shwen Ho ◽  
Mridul B. Nair ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Plasmodium Knowlesi ◽  
Binding Protein ◽  
Cynomolgus Macaque ◽  
Genomic Analysis ◽  
Human Infection ◽  
Comparative Genomic ◽  
Gene Encoding ◽  
Human Rbcs

The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen.

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