scholarly journals Using Plasmodium knowlesi as a model for screening Plasmodium vivax blood-stage malaria vaccine targets reveals new candidates

2021 ◽  
Vol 17 (7) ◽  
pp. e1008864
Author(s):  
Duncan N. Ndegwa ◽  
Prasun Kundu ◽  
Jessica B. Hostetler ◽  
Alejandro Marin-Menendez ◽  
Theo Sanderson ◽  
...  
Keyword(s):  
Plasmodium Vivax ◽  
Culture System ◽  
Vaccine Development ◽  
Expression System ◽  
Critical Role ◽  
Blood Stage ◽  
Effective Vaccine ◽  
Mammalian Expression ◽  
Close Phylogenetic Relationship

Plasmodium vivax is responsible for the majority of malaria cases outside Africa. Unlike P. falciparum, the P. vivax life-cycle includes a dormant liver stage, the hypnozoite, which can cause infection in the absence of mosquito transmission. An effective vaccine against P. vivax blood stages would limit symptoms and pathology from such recurrent infections, and therefore could play a critical role in the control of this species. Vaccine development in P. vivax, however, lags considerably behind P. falciparum, which has many identified targets with several having transitioned to Phase II testing. By contrast only one P. vivax blood-stage vaccine candidate based on the Duffy Binding Protein (PvDBP), has reached Phase Ia, in large part because the lack of a continuous in vitro culture system for P. vivax limits systematic screening of new candidates. We used the close phylogenetic relationship between P. vivax and P. knowlesi, for which an in vitro culture system in human erythrocytes exists, to test the scalability of systematic reverse vaccinology to identify and prioritise P. vivax blood-stage targets. A panel of P. vivax proteins predicted to function in erythrocyte invasion were expressed as full-length recombinant ectodomains in a mammalian expression system. Eight of these antigens were used to generate polyclonal antibodies, which were screened for their ability to recognize orthologous proteins in P. knowlesi. These antibodies were then tested for inhibition of growth and invasion of both wild type P. knowlesi and chimeric P. knowlesi lines modified using CRISPR/Cas9 to exchange P. knowlesi genes with their P. vivax orthologues. Candidates that induced antibodies that inhibited invasion to a similar level as PvDBP were identified, confirming the utility of P. knowlesi as a model for P. vivax vaccine development and prioritizing antigens for further follow up.

scholarly journals Using Plasmodium knowlesi as a model for screening Plasmodium vivax blood-stage malaria vaccine targets reveals new candidates

2020 ◽  
Author(s):  
Duncan N. Ndegwa ◽  
Jessica B. Hostetler ◽  
Alejandro Marin-Menendez ◽  
Theo Sanderson ◽  
Kioko Mwikali ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Plasmodium Vivax ◽  
Blood Cells ◽  
Culture System ◽  
Parasite Species ◽  
Vaccine Development ◽  
Blood Stage ◽  
Effective Vaccine ◽  
Human Red Blood Cells

ABSTRACTPlasmodium vivax is responsible for the majority of malaria cases outside Africa. Unlike P. falciparum, the P. vivax life-cycle includes a dormant liver stage, the hypnozoite, which can cause infection in the absence of mosquito transmission. An effective vaccine against P. vivax blood stages would limit symptoms and pathology from such recurrent infections, and therefore could play a critical role in the control of this species. Vaccine development in P. vivax, however, lags considerably behind P. falciparum, which has many identified targets with several having transitioned to Phase II testing. By contrast only one P. vivax blood-stage vaccine candidate based on the Duffy Binding Protein (PvDBP), has reached Phase Ia, in large part because the lack of a continuous in vitro culture system for P. vivax limits systematic screening of new candidates. We used the close phylogenetic relationship between P. vivax and P. knowlesi, for which an in vitro culture system in human erythrocytes exists, to test the scalability of systematic reverse vaccinology to identify and prioritise P. vivax blood-stage targets. A panel of P. vivax proteins predicted to function in erythrocyte invasion were expressed as full-length recombinant ectodomains in a mammalian expression system. Eight of these antigens were used to generate polyclonal antibodies, which were screened for their ability to recognize orthologous proteins in P. knowlesi. These antibodies were then tested for inhibition of growth and invasion of both wild type P. knowlesi and chimeric P. knowlesi lines modified using CRISPR/Cas9 to exchange P. knowlesi genes with their P. vivax orthologues. Candidates that induced antibodies that inhibited invasion to a similar level as PvDBP were identified, confirming the utility of P. knowlesi as a model for P. vivax vaccine development and prioritizing antigens for further follow up.AUTHOR SUMMARYMalaria parasites cause disease after invading human red blood cells, implying that a vaccine that interrupts this process could play a significant role in malaria control. Multiple Plasmodium parasite species can cause malaria in humans, and most malaria outside Africa is caused by Plasmodium vivax. There is currently no effective vaccine against the blood stage of any malaria parasite, and progress in P. vivax vaccine development has been particularly hampered because this parasite species cannot be cultured for prolonged periods of time in the lab. We explored whether a related species, P. knowlesi, which can be propagated in human red blood cells in vitro, can be used to screen for potential P. vivax vaccine targets. We raised antibodies against selected P. vivax proteins and testedtheir ability to recognize and prevent P. knowlesi parasites from invading human red blood cells, thereby identifying multiple novel vaccine candidates.

A Continuous, Long-Term Plasmodium vivax In Vitro Blood-Stage Culture: What Are We Missing?

2017 ◽  
Vol 33 (12) ◽  
pp. 921-924 ◽  
Author(s):  
Richard Thomson-Luque ◽  
Katy Shaw Saliba ◽  
Clemens H.M. Kocken ◽  
Erica M. Pasini
Keyword(s):  
Plasmodium Vivax ◽  
Blood Stage

scholarly journals Naturally induced humoral response against Plasmodium vivax reticulocyte binding protein 2P1

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Jenni Hietanen ◽  
Anongruk Chim-ong ◽  
Jetsumon Sattabongkot ◽  
Wang Nguitragool
Keyword(s):  
Plasmodium Vivax ◽  
Vaccine Development ◽  
Natural Infection ◽  
Humoral Response ◽  
Human Antibody ◽  
Asymptomatic Carriers ◽  
Erythrocyte Invasion ◽  
Human Proteins ◽  
Human Immune Response

Abstract Background Plasmodium vivax is the most prevalent malaria parasite in many countries. A better understanding of human immunity to this parasite can provide new insights for vaccine development. Plasmodium vivax Reticulocyte Binding Proteins (RBPs) are key parasite proteins that interact with human proteins during erythrocyte invasion and are targets of the human immune response. The aim of this study is to characterize the human antibody response to RBP2P1, the most recently described member of the RBP family. Methods The levels of total IgG and IgM against RBP2P1 were measured using plasmas from 68 P. vivax malaria patients and 525 villagers in a malarious village of western Thailand. The latter group comprises asymptomatic carriers and healthy uninfected individuals. Subsets of plasma samples were evaluated for anti-RBP2P1 IgG subtypes and complement-fixing activity. Results As age increased, it was found that the level of anti-RBP2P1 IgG increased while the level of IgM decreased. The main anti-RBP2P1 IgG subtypes were IgG1 and IgG3. The IgG3-seropositive rate was higher in asymptomatic carriers than in patients. The higher level of IgG3 was correlated with higher in vitro RBP2P1-mediated complement fixing activity. Conclusions In natural infection, the primary IgG response to RBP2P1 was IgG1 and IgG3. The predominance of these cytophilic subtypes and the elevated level of IgG3 correlating with complement fixing activity, suggest a possible role of anti-RBP2P1 antibodies in immunity against P. vivax.

scholarly journals A Combined Adjuvant TF–Al Consisting of TFPR1 and Aluminum Hydroxide Augments Strong Humoral and Cellular Immune Responses in Both C57BL/6 and BALB/c Mice

Vaccines ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 1408
Author(s):  
Qiao Li ◽  
Zhihua Liu ◽  
Yi Liu ◽  
Chen Liang ◽  
Jiayi Shu ◽  
...  
Keyword(s):  
Immune Responses ◽  
Vaccine Development ◽  
Inbred Mouse ◽  
Effective Vaccine ◽  
Mouse Strains ◽  
Cellular Immune Responses ◽  
Recombinant Hbsag ◽  
Cellular Immune

TFPR1 is a novel adjuvant for protein and peptide antigens, which has been demonstrated in BALB/c mice in our previous studies; however, its adjuvanticity in mice with different genetic backgrounds remains unknown, and its adjuvanticity needs to be improved to fit the requirements for various vaccines. In this study, we first compared the adjuvanticity of TFPR1 in two commonly used inbred mouse strains, BALB/c and C57BL/6 mice, in vitro and in vivo, and demonstrated that TFPR1 activated TLR2 to exert its immune activity in vivo. Next, to prove the feasibility of TFPR1 acting as a major component of combined adjuvants, we prepared a combined adjuvant, TF–Al, by formulating TFPR1 and alum at a certain ratio and compared its adjuvanticity with that of TFPR1 and alum alone using OVA and recombinant HBsAg as model antigens in both BALB/c and C57BL/6 mice. Results showed that TFPR1 acts as an effective vaccine adjuvant in both BALB/c mice and C57BL/6 mice, and further demonstrated the role of TLR2 in the adjuvanticity of TFPR1 in vivo. In addition, we obtained a novel combined adjuvant, TF–Al, based on TFPR1, which can augment antibody and cellular immune responses in mice with different genetic backgrounds, suggesting its promise for vaccine development in the future.

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 Resolution ofStaphylococcus aureusBiofilm Infection Using Vaccination and Antibiotic Treatment

2011 ◽  
Vol 79 (4) ◽  
pp. 1797-1803 ◽  
Author(s):  
Rebecca A. Brady ◽  
Graeme A. O'May ◽  
Jeff G. Leid ◽  
Megan L. Prior ◽  
J. William Costerton ◽  
...  
Keyword(s):  
Antibiotic Treatment ◽  
Vaccine Development ◽  
Hypothetical Protein ◽  
Quadrivalent Vaccine ◽  
Effective Vaccine ◽  
Vast Number ◽  
Protective Antigens ◽  
Biofilm Model

ABSTRACTStaphylococcus aureusinfections, particularly those from methicillin-resistant strains (i.e., MRSA), are reaching epidemic proportions, with no effective vaccine available. The vast number and transient expression of virulence factors in the infectious course of this pathogen have made the discovery of protective antigens particularly difficult. In addition, the divergent planktonic and biofilm modes of growth with their accompanying proteomic changes also demonstrate significant hindrances to vaccine development. In this study, a multicomponent vaccine was evaluated for its ability to clear a staphylococcal biofilm infection. Antigens (glucosaminidase, an ABC transporter lipoprotein, a conserved hypothetical protein, and a conserved lipoprotein) were chosen since they were found in previous studies to have upregulated and sustained expression in a biofilm, bothin vitroandin vivo. Antibodies against these antigens were first used in microscopy studies to localize their expression inin vitrobiofilms. Each of the four antigens showed heterogeneous production in various locations within the complex biofilm community in the biofilm. Based upon these studies, the four antigens were delivered simultaneously as a quadrivalent vaccine in order to compensate for this varied production. In addition, antibiotic treatment was also administered to clear the remaining nonattached planktonic cells since the vaccine antigens may have been biofilm specific. The results demonstrated that when vaccination was coupled with vancomycin treatment in a biofilm model of chronic osteomyelitis in rabbits, clinical and radiographic signs of infection significantly reduced by 67 and 82%, respectively, compared to infected animals that were either treated with vancomycin or left untreated. In contrast, vaccination alone resulted in a modest, and nonsignificant, decrease in clinical (34% reduction) and radiographic signs (9% reduction) of infection, compared to nonvaccinated animal groups untreated or treated with vancomycin. Lastly, MRSA biofilm infections were significantly cleared in 87.5% of vaccinated and antibiotic-treated animals, while antibiotics or vaccine alone could not significantly clear infection compared to controls (55.6, 22.2, and 33.3% clearance rates, respectively). This approach to vaccine development may lead to the generation of vaccines against other pathogenic biofilm bacteria.

scholarly journals In Vitro Growth-Inhibitory Activity and Malaria Risk in a Cohort Study in Mali

2009 ◽  
Vol 78 (2) ◽  
pp. 737-745 ◽  
Author(s):  
Peter D. Crompton ◽  
Kazutoyo Miura ◽  
Boubacar Traore ◽  
Kassoum Kayentao ◽  
Aissata Ongoiba ◽  
...  
Keyword(s):  
Inhibitory Activity ◽  
Vaccine Development ◽  
Critical Role ◽  
Malaria Risk ◽  
Activity Level ◽  
Asexual Blood Stage ◽  
Growth Inhibition Assay ◽  
Growth Inhibitory ◽  
Growth Inhibitory Activity

ABSTRACT Immunity to the asexual blood stage of Plasmodium falciparum is complex and likely involves several effector mechanisms. Antibodies are thought to play a critical role in malaria immunity, and a corresponding in vitro correlate of antibody-mediated immunity has long been sought to facilitate malaria vaccine development. The growth inhibition assay (GIA) measures the capacity of antibodies to limit red blood cell (RBC) invasion and/or growth of P. falciparum in vitro. In humans, naturally acquired and vaccine-induced P. falciparum-specific antibodies have growth-inhibitory activity, but it is unclear if growth-inhibitory activity correlates with protection from clinical disease. In a longitudinal study in Mali, purified IgGs, obtained from plasmas collected before the malaria season from 220 individuals aged 2 to 10 and 18 to 25 years, were assayed for growth-inhibitory activity. Malaria episodes were recorded by passive surveillance over the subsequent 6-month malaria season. Logistic regression showed that greater age (odds ratio [OR], 0.78; 95% confidence interval [95% CI], 0.63 to 0.95; P = 0.02) and growth-inhibitory activity (OR, 0.50; 95% CI, 0.30 to 0.85; P = 0.01) were significantly associated with decreased malaria risk in children. A growth-inhibitory activity level of 40% was determined to be the optimal cutoff for discriminating malaria-immune and susceptible individuals in this cohort, with a sensitivity of 97.0%, but a low specificity of 24.3%, which limited the assay's ability to accurately predict protective immunity and to serve as an in vitro correlate of antibody-mediated immunity. These data suggest that antibodies which block merozoite invasion of RBC and/or inhibit the intra-RBC growth of the parasite contribute to but are not sufficient for the acquisition of malaria immunity.

scholarly journals Theileria equi claudin like apicomplexan microneme protein contains neutralization-sensitive epitopes and interacts with components of the equine erythrocyte membrane skeleton

2021 ◽  
Author(s):  
Cynthia Onzere ◽  
Lindsay Fry ◽  
Richard Bishop ◽  
Marta Silva ◽  
Reginaldo Bastos ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Vaccine Development ◽  
Membrane Skeleton ◽  
Host Cells ◽  
Effective Vaccine ◽  
Theileria Equi ◽  
Anion Exchange Protein ◽  
Microneme Protein

Abstract Theileria equi (T. equi) is a widely distributed apicomplexan parasite that causes severe hemolytic anemia in equid species. There is currently no effective vaccine for control of the parasite and understanding the mechanism that T. equi utilizes to invade host cells may be crucial for vaccine development. Unlike most apicomplexan species studied to date, the role of micronemes in T. equi invasion of host cells is unknown. We therefore assessed the role of the T. equi claudin-like apicomplexan microneme protein (CLAMP) in the invasion of equine erythrocytes as a first step towards understanding the role of this organelle in the parasite. Our findings show that CLAMP is expressed in the merozoite and intra-erythrocytic developmental stages of T. equi and in vitro neutralization experiments suggest that the protein is involved in erythrocyte invasion. Proteomic analyses indicate that CLAMP interacts with the equine erythrocyte α-and β- spectrin chains in the initial stages of T. equi invasion and maintains these interactions while also associating with the anion-exchange protein, tropomyosin 3, band 4.1 and cytoplasmic actin 1 after invasion. Additionally, serological analyses show that T. equi-infected horses mount robust antibody responses against CLAMP indicating that the protein is immunogenic and therefore represents a potential vaccine candidate.

SUSTAINED EXPRESSION OF FULL LENGTH AND VARIANT RECOMBINANT FACTOR VIII IN GENETICALLY ENGINEERED CELLS

1987 ◽  
Author(s):  
Nava Sarver ◽  
George A Ricca
Keyword(s):  
Factor Viii ◽  
Recombinant Dna ◽  
Expression System ◽  
Genetically Engineered ◽  
Full Length ◽  
Cdna Insert ◽  
Mammalian Expression ◽  
Coagulant Activity ◽  
Recombinant Fviii

A major effort is presently underway to provide factor VIII (FVIII) in a form free of viral pathogens via a recombinant DNA approach. We have constructed two chimeric FVIII cDNA vectors based on the bovine papillomavirus mammalian expression system. The first vector (FVIII) contained a full length FVIII cDNA; the second vector (AFVIII) contained a cDNA insert with an extensive deletion, corresponding to amino acid residues 747 to 1560 in the region encoding the "B" domain. This internal region is removed during activation of the parental FVIII molecule and is believed not to be required for coagulant activity. We have found that recombinant FVIII produced by stable cell lines harboring either the full length or the variant FVIII was capable of restoring coagulant activity to FVIII deficient plasma in. vitro. This expressed activity was neutralized by anti-FVIII antibodies. Similar to observations with FVIII derived from human plasma, the two recombinant FVIII forms were (i) inactivated by the chelating agent EDTA, (ii) demonstrated a biphasic response of an initial activation followed by a decay in activity when treated with thrombin, and (iii) presented the expected peptide banding pattern by western blot analyses. A higher percentage of ΔFVIII transformants were isolated expressing coagulant activity compared to transformants harboring the complete FVIII cDNA. Among the positive transformants isolated, those harboring ΔFVIII produced higher levels of coagulant activity than their full length counterparts. Comparable steady state levels of FVIII specific transcripts were detected in FVIII and ΔFVIII transformants indicating that the difference in expression levels is due to a post transcriptional event(s). Our study demonstrates the efficacy of a full length and an abridged recombinant FVIII produced by stably transformed cells in correcting coagulation deficiency in. vitro. It further suggests the potential usefulness of other molecular variants for efficient expression in genetically engineered cells.

scholarly journals Development of a 3D Co-Culture System as a Cancer Model Using a Self-Assembling Peptide Scaffold

Gels ◽  
2018 ◽  
Vol 4 (3) ◽  
pp. 65 ◽  
Author(s):  
Nausika Betriu ◽  
Carlos Semino
Keyword(s):  
Cell Proliferation ◽  
Cancer Cells ◽  
Culture System ◽  
Critical Role ◽  
Tumor Stroma ◽  
Pharmaceutical Drugs ◽  
Cancer Model ◽  
Proliferating Cells ◽  
Self Assembling

Cancer research has traditionally relied on two-dimensional (2D) cell culture, focusing mainly on cancer cells and their abnormal genetics. However, over the past decade, tumors have been accepted as complex tissues rather than a homogenous mass of proliferating cells. Consequently, cancer cells’ behavior can only be deciphered considering the contribution of the cells existing in the tumor stroma as well as its complex microenvironment. Since the tumor microenvironment plays a critical role in tumorigenesis, it is widely accepted that culturing cells in three-dimensional (3D) scaffolds, which mimic the extracellular matrix, represents a more realistic scenario. In the present work, an in vitro 3D co-culture system based on the self-assembling peptide scaffold RAD16-I (SAPS RAD16-I) was developed as a cancer model. For that, PANC-1 cells were injected into a RAD16-I peptide scaffold containing fibroblasts, resulting in a 3D system where cancer cells were localized in a defined area within a stromal cells matrix. With this system, we were able to study the effect of three well-known pharmaceutical drugs (Gemcitabine, 5-Fluorouracil (5-FU), and 4-Methylumbelliferone (4-MU)) in a 3D context in terms of cell proliferation and survival. Moreover, we have demonstrated that the anti-cancer effect of the tested compounds can be qualitatively and quantitatively evaluated on the developed 3D co-culture system. Experimental results showed that Gemcitabine and 5-FU prevented PANC-1 cell proliferation but had a high cytotoxic effect on fibroblasts as well. 4-MU had a subtle effect on PANC-1 cells but caused high cell death on fibroblasts.

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