scholarly journals Assessment of Babesia bovis 6cys A and 6cys B as components of transmission blocking vaccines for babesiosis

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Heba F. Alzan ◽  
Reginaldo G. Bastos ◽  
Massaro W. Ueti ◽  
Jacob M. Laughery ◽  
Vignesh A. Rathinasamy ◽  
...  
Keyword(s):  
Babesia Bovis ◽  
Functional Domains ◽  
Inhibition Assay ◽  
B Cell Epitopes ◽  
Transmission Blocking ◽  
Tick Vector ◽  
Similar Work ◽  
Induction System ◽  
Transmission Blocking Vaccines

Abstract Background Babesia bovis reproduces sexually in the gut of its tick vector Rhipicephalus microplus, which involves expression of 6cys A and 6cys B proteins. Members of the widely conserved 6cys superfamily are candidates for transmission blocking vaccines (TBV), but intricacies in the immunogenicity of the 6cys proteins in the related Plasmodium parasites required the identification of transmission blocking domains in these molecules for vaccine design. Hereby, the immunogenic efficacy of recombinant (r) B. bovis 6cys A and B proteins as a TBV formulation was studied. Methods The immunogenicity of r6cys A and 6cys B proteins expressed in a eukaryotic system was evaluated in a cattle immunization trial (3 immunized and 3 control calves). A B. bovis sexual stage induction in vitro inhibition assay to assess the ability of antibodies to block the production of sexual forms by the parasite was developed. Results Immunized cattle generated antibodies against r6cys A and r6cys B that were unable to block sexual reproduction of the parasite in ticks. Additionally, these antibodies also failed in recognizing native 6cys A and 6cys B and peptides representing 6cys A and 6cys B functional domains and in inhibiting the development of sexual forms in an in vitro induction system. In contrast, rabbit antibodies generated against synthetic peptides representing predicted B-cell epitopes of 6cys A and 6cys B recognized recombinant and native forms of both 6cys proteins as well as peptides representing 6cys A and 6cys B functional domains and were able to neutralize development of sexual forms of the parasite in vitro. Conclusions These data, combined with similar work performed on Plasmodium 6cys proteins, indicate that an effective 6cys protein-based TBV against B. bovis will require identifying and targeting selected regions of proteins containing epitopes able to reduce transmission. Graphic abstract

Evaluation of Two Sexual-stage Antigens as Bivalent Transmission-blocking Vaccines in Rodent Malaria

2021 ◽  
Author(s):  
Fan Yang ◽  
Fei Liu ◽  
Xinxin Yu ◽  
Wenqi Zheng ◽  
Yudi Wu ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Chimeric Protein ◽  
Transmission Blocking ◽  
Native Proteins ◽  
Blocking Activity ◽  
Antibody Titers ◽  
Sexual Stages ◽  
Transmission Blocking Vaccines

Abstract Background: Transmission-blocking vaccine (TBV) is a promising strategy for malaria elimination. It is hypothesized that mixing or fusing two antigens targeting different stages of sexual development may provide higher transmission-blocking activity than these antigens are used individually.Methods: We designed a chimeric protein composed of fragments of Pbg37 and PSOP25 and expressed the recombinant protein in Escherichia coli Rosetta-gami B (DE3). After immunizing mice with mixing or fusing recombinant proteins, the antibody titers of sera were analyzed by ELISA. IFA and Western blot were performed to test the reactivity of the antisera with the native proteins of the parasite. The transmission blocking activity were assessed in vitro and in vivo assay. Results: When Pbg37 and PSOP25 were co-administered in a mixture or as a fusion protein, they elicited similar antibody responses in mice as single antigens without causing immunological interference with each other. Antibodies against the mixed or fused antigens recognized the target proteins in the gametocyte, gamete, zygote and ookinete stages. The two bivalent vaccines (mixed proteins or a fusion protein) produced the superior TBA compared to that of the antibodies against individual antigens.Conclusions: There was no immunological interference between the two antigens of bivalent vaccines. And the bivalent vaccines produced significantly stronger transmission-blocking activities than single antigens. Altogether, these data provide the theoretical basis for the development of combination TBVs targeting different sexual stages.

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

2008 ◽  
Vol 76 (5) ◽  
pp. 2018-2024 ◽  
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.

scholarly journals Differential expression of calcium-dependent protein kinase 4, tubulin tyrosine ligase, and methyltransferase by xanthurenic acid-induced Babesia bovis sexual stages

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Hala E. Hussein ◽  
Wendell C. Johnson ◽  
Naomi S. Taus ◽  
Janaina Capelli-Peixoto ◽  
Carlos E. Suarez ◽  
...  
Keyword(s):  
Babesia Bovis ◽  
Xanthurenic Acid ◽  
Bovine Babesiosis ◽  
Dependent Protein Kinase ◽  
Tick Vector ◽  
Calcium Dependent ◽  
Dependent Protein ◽  
Sexual Stages ◽  
Calcium Dependent Protein Kinase

Abstract Background Babesia bovis is one of the most significant tick-transmitted pathogens of cattle worldwide. Babesia bovis parasites have a complex lifecycle, including development within the mammalian host and tick vector. Each life stage has developmental forms that differ in morphology and metabolism. Differentiation between these forms is highly regulated in response to changes in the parasite’s environment. Understanding the mechanisms by which Babesia parasites respond to environmental changes and the transmission cycle through the biological vector is critically important for developing bovine babesiosis control strategies. Results In this study, we induced B. bovis sexual stages in vitro using xanthurenic acid and documented changes in morphology and gene expression. In vitro induced B. bovis sexual stages displayed distinctive protrusive structures and surface ruffles. We also demonstrated the upregulation of B. bovis calcium-dependent protein kinase 4 (cdpk4), tubulin-tyrosine ligase (ttl), and methyltransferase (mt) genes by in vitro induced sexual stages and during parasite development within tick midguts. Conclusions Similar to other apicomplexan parasites, it is likely that B. bovis upregulated genes play a vital role in sexual reproduction and parasite transmission. Herein, we document the upregulation of cdpk4, ttl, and mt genes by both B. bovis in vitro induced sexual stages and parasites developing in the tick vector. Understanding the parasite's biology and identifying target genes essential for sexual reproduction will enable the production of non-transmissible live vaccines to control bovine babesiosis. Graphical abstract

In vitro antimalarial interaction and transmission-blocking activity of cryptolepine

Planta Medica ◽  
2016 ◽  
Vol 81 (S 01) ◽  
pp. S1-S381
Author(s):  
A Donkor Forkuo ◽  
C Ansah ◽  
B Gyan ◽  
D Mancama ◽  
A Theron
Keyword(s):  
Transmission Blocking ◽  
Blocking Activity

scholarly journals Catalytic activity and stereoselectivity of engineered phosphotriesterases towards structurally different nerve agents in vitro

2021 ◽  
Author(s):  
Anja Köhler ◽  
Benjamin Escher ◽  
Laura Job ◽  
Marianne Koller ◽  
Horst Thiermann ◽  
...  
Keyword(s):  
Plasma Clearance ◽  
Nerve Agents ◽  
Inhibition Assay ◽  
Ache Inhibition ◽  
Catalytic Activities ◽  
Chiral Lc ◽  
Hydrolysis Of ◽  
Medical Countermeasures

AbstractHighly toxic organophosphorus nerve agents, especially the extremely stable and persistent V-type agents such as VX, still pose a threat to the human population and require effective medical countermeasures. Engineered mutants of the Brevundimonas diminuta phosphotriesterase (BdPTE) exhibit enhanced catalytic activities and have demonstrated detoxification in animal models, however, substrate specificity and fast plasma clearance limit their medical applicability. To allow better assessment of their substrate profiles, we have thoroughly investigated the catalytic efficacies of five BdPTE mutants with 17 different nerve agents using an AChE inhibition assay. In addition, we studied one BdPTE version that was fused with structurally disordered PAS polypeptides to enable delayed plasma clearance and one bispecific BdPTE with broadened substrate spectrum composed of two functionally distinct subunits connected by a PAS linker. Measured kcat/KM values were as high as 6.5 and 1.5 × 108 M−1 min−1 with G- and V-agents, respectively. Furthermore, the stereoselective degradation of VX enantiomers by the PASylated BdPTE-4 and the bispecific BdPTE-7 were investigated by chiral LC–MS/MS, resulting in a several fold faster hydrolysis of the more toxic P(−) VX stereoisomer compared to P(+) VX. In conclusion, the newly developed enzymes BdPTE-4 and BdPTE-7 have shown high catalytic efficacy towards structurally different nerve agents and stereoselectivity towards the toxic P(−) VX enantiomer in vitro and offer promise for use as bioscavengers in vivo.

The EYTEX™ System and the Neutral Red Uptake Inhibition Assay in Cultured Hep G2 Cells as Alternative Methods for In Vivo Eye Irritation Following the EEC Protocol

1990 ◽  
Vol 17 (4) ◽  
pp. 325-333
Author(s):  
Paul J. Dierickx ◽  
Virginia C. Gordon
Keyword(s):  
Neutral Red ◽  
Alternative Methods ◽  
Inhibition Assay ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Eye Irritation ◽  
Uptake Inhibition ◽  
In Vitro Methods ◽  
Neutral Red Uptake

The neutral red uptake inhibition assay and the EYTEX™ system were investigated as alternative methods for the assessment of eye irritation, determined according to the EEC protocol. The 17 test chemicals used were mainly organic solvents. The xenobiotics were applied to Hep G2 cells for 24 hours at different concentrations. Neutral red uptake inhibition was then measured. The results are expressed as the NI50 value, which is the concentration of test compound required to induce a 50% reduction in neutral red uptake. The same chemicals were also tested as coded samples by the EYTEX™ test according to the manufacturer's directions. A nearly identical quantitative correlation was found for both in vitro methods with corneal opacity scores: r = 0.84 for EYTEX™ scores and r = 0.83 for log NI50, expressed in μg/ml. Whilst these correlations are certainly not perfect, it is clear that both in vitro methods can be used as valuable prescreening methods.

scholarly journals In VitroAnti-Plasmodial Activity ofTrigonella foenum–graecumL.

2010 ◽  
Vol 7 (4) ◽  
pp. 441-445 ◽  
Author(s):  
M. Palaniswamy ◽  
B. V. Pradeep ◽  
R. Sathya ◽  
J. Angayarkanni
Keyword(s):  
Ethanol Extract ◽  
Traditional Healers ◽  
Active Principle ◽  
Test Plant ◽  
Moderate Activity ◽  
Inhibition Assay ◽  
Phytochemical Screening ◽  
Solvent Systems ◽  
Fenugreek Leaves

Developing countries, where malaria is one of the most prevalent diseases, still rely on traditional medicine as a source for the treatment of this disease. For the present study,Trigonella foenum-graecumL. (fenugreek) were collected from Coimbatore, Tamilnadu, India. The test plant has been used in India by traditional healers for the treatment of fever as well as other diseases. The active principle was extracted out in different solvent systems to assess the anti-plasmodial potential, with an aim that they can further be utilized to formulate drugs.In vitroanti-plasmodial assay of the extracted fractions of fenugreek leaves was carried out using laboratory adapted chloroquine sensitive and resistantPlasmodium falciparumisolates. Schizont maturation inhibition assay was adopted to analyze the potential of the extracts. Ethanol extract (50%) seemed to possess profound anti-plasmodial activity with IC50value of 8.75 ± 0.35 µg ml−1and 10.25 ± 0.35 µg ml−1against chloroquine sensitive and resistantP. falciparumisolates, respectively. Among the investigated six fractions of the plant extracts, two were found to have significant anti-plasmodial activity with IC50values <10 µg ml−1, namely ethanol and butanol extracts. Two extracts chloroform and ethyl acetate showed moderate activity with IC50values ranging from 10 to 20 µg ml−1, and the other two extracts, hexane and water appeared to be inactive with IC50values >85 µg ml−1. In addition, preliminary phytochemical screening of the various extracts indicated the presence of alkaloids, saponin, tannin like phenolic compounds, flavonoids and steroids.

Advancing RNA Virus Discovery and Biology with Whole Genome Sequencing

2021 ◽  
Author(s):  
◽  
Mariah Taylor ◽  
Keyword(s):  
Genetic Variation ◽  
Amino Acid ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Rna Virus ◽  
Animal Health ◽  
Functional Domains ◽  
Whole Genome ◽  
Coding Region

Two RNA virus families that pose a threat to human and animal health are Hantaviridae and Coronaviridae. These RNA viruses which originate in wildlife continue and will continue to cause disease, and hence, it is critical that scientific research define the mechanisms as to how these viruses spillover and adapt to new hosts to become endemic. One gap in our ability to define these mechanisms is the lack of whole genome sequences for many of these viruses. To address this specific gap, I developed a versatile amplicon-based whole-genome sequencing (WGS) approach to identify viral genomes of hantaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within reservoir and spillover hosts. In my research studies, I used the amplicon-based WGS approach to define the genetic plasticity of viral RNA within pathogenic and nonpathogenic hantavirus species. The standing genetic variation of Andes orthohantavirus and Prospect Hill orthohantavirus was mapped out and amino acid changes occurring outside of functional domains were identified within the nucleocapsid and glycoprotein. I observed several amino acid changes in functional domains of the RNA-dependent RNA polymerase, as well as single nucleotide polymorphisms (SNPs) within the 3’ non-coding region (NCR) of the S-segment. To identify whether virus adaptation would occur within the S- and L-segments we attempted to adapt hantaviruses in vitro in a spillover host model through passaging experiments. In early passages we identified few mutations in the M-segment with the majority being identified in the S-segment 3’ NCR and the L-segment. This work suggests that hantavirus adaptation occurs in the S- and L-segments although the effect of these mutants on pathology is yet to be determined. While sequencing laboratory isolates is easily accomplished, sequencing low concentrations of virus within the reservoir is a formidable task. I further translated our amplicon-based WGS approach into a pan-oligonucleotide amplicon-based WGS approach to sequence hantavirus vRNA and mRNA from reservoir and spillover hosts in Ukraine. This approach successfully identified a novel Puumala orthohantavirus (PUUV) strain in Ukraine and using Bayesian phylogenetics we found this strain to be associated with the PUUV Latvian lineage. Early during the SARS-CoV-2 pandemic, I applied the knowledge gained in the hantavirus WGS efforts to sequencing of SARS-CoV-2 from nasopharyngeal swabs collected in April 2020. The genetic diversity of 45 SARS-CoV-2 isolates was evaluated with the methods I developed. We identified D614G, a notable mutation known for increasing transmission, in over 90% of our isolates. Two major lineages distinguish SARS-CoV-2 variants worldwide, lineages A and B. While most of our isolates were found within B lineage, we also identified one isolate within lineage A. We performed in vitro work which confirmed A lineage isolates as having poor replication in the trachea as compared to the nasal cavity. Five of these isolates presented a unique array of mutations which were assessed in the keratin 18 human angiotensin-converting enzyme 2 (K18-hACE2) mouse model for its response immunologically and pathogenically. We identified a distinction of pathogenesis between the A and B lineages with emphysema being common amongst A lineage isolates. Additionally, we discovered a small cohort of likely SNPs that defined the late induction of eosinophils during infection. In summary, this work will further define the dynamics of genetic variation and plasticity within virus populations that cause disease outbreaks and will allow a deeper understanding of the virus-host relationship.

In-vitro and in vivo studies of traditionally prepared Zingiber officinale juice for Antimitotic and Lethality activity

2021 ◽  
pp. 4652-4656
Author(s):  
Nadar Sowmya ◽  
Chouhan Raghavendra Singh ◽  
Kosha Patel ◽  
Harshil Patel ◽  
Tanvi Dodiya ◽  
...  
Keyword(s):  
Zingiber Officinale ◽  
In Vivo Studies ◽  
Root Tip ◽  
Inhibition Assay ◽  
Lc50 Value ◽  
Antimitotic Activity ◽  
Ic50 Value ◽  
Traditionally Prepared

According to the literature survey most of the studies done on Adarak (Zingiber officinale rhizome) were performed on alcoholic extracts or isolated entities of ginger but no profound work has been done on the traditionally prepared or commonly consumed way of ginger. So, in this current study, fresh ginger rhizomes were traditionally pounded to make Adarak juice. The different concentration (0.05%, 0.5%, 1%, 2.5%, 5%, 10%) were taken as sample to perform onion root tip inhibition assay and brine shrimp lethality bioassay. It showed antimitotic inhibition in the range of 0.05-10% concentration with an IC50 value of 0.37 % on number of rootlets was 0.08 % on length of rootlets. The traditionally prepared Adarak juice showed cytotoxicity in the range of 0.05-2.5% concentration with LC50 value of 1.59%. The traditionally prepared Adarak juice possesses cytotoxic and antimitotic activity.

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