scholarly journals Shotgun Immunoproteomic Approach for the Discovery of Linear B-Cell Epitopes in Biothreat Agents Francisella tularensis and Burkholderia pseudomallei

2021 ◽  
Vol 12 ◽  
Author(s):  
Patrik D’haeseleer ◽  
Nicole M. Collette ◽  
Victoria Lao ◽  
Brent W. Segelke ◽  
Steven S. Branda ◽  
...  
Keyword(s):  
Immune Responses ◽  
B Cell ◽  
Francisella Tularensis ◽  
Burkholderia Pseudomallei ◽  
Outer Membrane Proteins ◽  
Screening Method ◽  
Cost Effective ◽  
Immune Memory ◽  
B Cell Epitopes ◽  
Linear Epitopes

Peptide-based subunit vaccines are coming to the forefront of current vaccine approaches, with safety and cost-effective production among their top advantages. Peptide vaccine formulations consist of multiple synthetic linear epitopes that together trigger desired immune responses that can result in robust immune memory. The advantages of linear compared to conformational epitopes are their simple structure, ease of synthesis, and ability to stimulate immune responses by means that do not require complex 3D conformation. Prediction of linear epitopes through use of computational tools is fast and cost-effective, but typically of low accuracy, necessitating extensive experimentation to verify results. On the other hand, identification of linear epitopes through experimental screening has been an inefficient process that requires thorough characterization of previously identified full-length protein antigens, or laborious techniques involving genetic manipulation of organisms. In this study, we apply a newly developed generalizable screening method that enables efficient identification of B-cell epitopes in the proteomes of pathogenic bacteria. As a test case, we used this method to identify epitopes in the proteome of Francisella tularensis (Ft), a Select Agent with a well-characterized immunoproteome. Our screen identified many peptides that map to known antigens, including verified and predicted outer membrane proteins and extracellular proteins, validating the utility of this approach. We then used the method to identify seroreactive peptides in the less characterized immunoproteome of Select Agent Burkholderia pseudomallei (Bp). This screen revealed known Bp antigens as well as proteins that have not been previously identified as antigens. Although B-cell epitope prediction tools Bepipred 2.0 and iBCE-EL classified many of our seroreactive peptides as epitopes, they did not score them significantly higher than the non-reactive tryptic peptides in our study, nor did they assign higher scores to seroreactive peptides from known Ft or Bp antigens, highlighting the need for experimental data instead of relying on computational epitope predictions alone. The present workflow is easily adaptable to detecting peptide targets relevant to the immune systems of other mammalian species, including humans (depending upon the availability of convalescent sera from patients), and could aid in accelerating the discovery of B-cell epitopes and development of vaccines to counter emerging biological threats.

scholarly journals Shotgun Immunoproteomic Approach for the Discovery of Linear B Cell Epitopes in Biothreat Agents Francisella tularensis and Burkholderia pseudomallei

2021 ◽  
Author(s):  
Patrik D'haeseleer ◽  
Nicole M Collette ◽  
Victoria Lao ◽  
Brent W Segelke ◽  
Steven S Branda ◽  
...  
Keyword(s):  
Immune Responses ◽  
B Cell ◽  
Francisella Tularensis ◽  
Burkholderia Pseudomallei ◽  
Outer Membrane Proteins ◽  
Screening Method ◽  
Immune Memory ◽  
B Cell Epitopes ◽  
Peptide Epitopes ◽  
Linear Epitopes

Peptide-based subunit vaccines are coming to the forefront of current vaccine approaches, with safety and cost-effective production among their top advantages. Peptide vaccine formulations consist of multiple synthetic linear epitopes that together trigger desired immune responses that can result in robust immune memory. The advantages of peptide epitopes are their simple structure, ease of synthesis, and ability to stimulate immune responses by means that do not require complex 3D conformation. Identification of linear epitopes is currently an inefficient process that requires thorough characterization of previously identified full-length protein antigens, or laborious techniques involving genetic manipulation of organisms. In this study, we apply a newly developed generalizable screening method that enables efficient identification of B cell epitopes in the proteomes of pathogenic bacteria. As a test case, we used this method to identify epitopes in the proteome of Francisella tularensis (Ft), a Select Agent with a well-characterized immunoproteome. Our screen identified many peptides that map to known antigens, including verified and predicted outer membrane proteins and extracellular proteins, validating the utility of this approach. We then used the method to identify seroreactive peptides in the less characterized immunoproteome of Select Agent Burkholderia pseudomallei (Bp). This screen revealed known Bp antigens as well as proteins that have not been previously identified as antigens. The present workflow is easily adaptable to detecting peptide targets relevant to the immune systems of other mammalian species, including humans (depending upon the availability of convalescent sera from patients), and could aid in accelerating the discovery of B cell epitopes and development of vaccines to counter emerging biological threats.

scholarly journals Paper-based ELISA Diagnosis Technology for Human Brucellosis Based on a Multiepitope Fusion Protein

2021 ◽  
Author(s):  
Dehui Yin ◽  
Qiongqiong Bai ◽  
Xiling Wu ◽  
Han Li ◽  
Jihong Shao ◽  
...  
Keyword(s):  
Fusion Protein ◽  
B Cell ◽  
Predictive Value ◽  
Outer Membrane Proteins ◽  
Diagnostic Technique ◽  
Human Brucellosis ◽  
B Cell Epitopes ◽  
Whatman Filter Paper ◽  
Nano Zinc Oxide ◽  
Nano Zinc

Abstract Background: At present, as a serious zoonotic infectious disease, the incidence of brucellosis is increasing each year worldwide, exhibiting signs of resurgence. Brucellosis seriously threatens the health of humans, and it is necessary to strengthen the methods utilized for its rapid and accurate diagnosis.Methods: Bioinformatic technology was used to predict B-cell epitopes of the main outer membrane proteins of Brucella and subsequently verified the antigenicity of these epitopes. Prepared a Brucella multiepitope fusion protein and verified the antigenicity of the protein by indirect ELISA. Whatman filter paper was then modified with nano-zinc oxide to construct a paper-based ELISA (p-ELISA) technology for the diagnosis of brucellosis.Results: A total of 22 linear B cell epitopes were predicted. Each epitope could recognize some brucellosis sera. The constructed multiepitope fusion protein had good antigenicity and significantly reduced cross-reaction compared with LPS. The sensitivity and specificity of the method were 92.38% and 98.35%, the positive predictive value was 98.26%, and the negative predictive value was 91.67%.Conclusions: A multiepitope fusion protein of Brucella was successfully prepared, and a rapid diagnostic technique for brucellosis was established. This technology has potential application value and can be used for the rapid diagnosis of brucellosis.

scholarly journals In SilicoPrediction of T and B Cell Epitopes of Der f 25 inDermatophagoides farinae

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Xiaohong Li ◽  
Hai-Wei Yang ◽  
Hao Chen ◽  
Jing Wu ◽  
Yehai Liu ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Triosephosphate Isomerase ◽  
Allergic Diseases ◽  
Major Allergen ◽  
House Dust Mites ◽  
T Cell Epitopes ◽  
B Cell Epitopes ◽  
Physiochemical Properties ◽  
Linear Epitopes

The house dust mites are major sources of indoor allergens for humans, which induce asthma, rhinitis, dermatitis, and other allergic diseases. Der f 25 is a triosephosphate isomerase, representing the major allergen identified inDermatophagoides farinae. The objective of this study was to predict the B and T cell epitopes of Der f 25. In the present study, we analyzed the physiochemical properties, function motifs and domains, and structural-based detailed features of Der f 25 and predicted the B cell linear epitopes of Der f 25 by DNAStar protean system, BPAP, and BepiPred 1.0 server and the T cell epitopes by NetMHCIIpan-3.0 and NetMHCII-2.2. As a result, the sequence and structure analysis identified that Der f 25 belongs to the triosephosphate isomerase family and exhibited a triosephosphate isomerase pattern (PS001371). Eight B cell epitopes (11–18, 30–35, 71–77, 99–107, 132–138, 173–187, 193–197, and 211–224) and five T cell epitopes including 26–34, 38–54, 66–74, 142–151, and 239–247 were predicted in this study. These results can be used to benefit allergen immunotherapies and reduce the frequency of mite allergic reactions.

scholarly journals A Multi-Epitope Fusion Protein-Based p-ELISA Method for Diagnosing Bovine and Goat Brucellosis

2021 ◽  
Vol 8 ◽  
Author(s):  
Dehui Yin ◽  
Qiongqiong Bai ◽  
Xiling Wu ◽  
Han Li ◽  
Jihong Shao ◽  
...  
Keyword(s):  
Fusion Protein ◽  
B Cell ◽  
Sensitivity And Specificity ◽  
Outer Membrane Proteins ◽  
Economic Losses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Method ◽  
Roc Curve Analysis ◽  
Predictive Values ◽  
B Cell Epitopes

In recent years, the incidence of brucellosis has increased annually, causing tremendous economic losses to animal husbandry in a lot of countries. Therefore, developing rapid, sensitive, and specific diagnostic techniques is critical to control the spread of brucellosis. In this study, bioinformatics technology was used to predict the B cell epitopes of the main outer membrane proteins of Brucella, and the diagnostic efficacy of each epitope was verified by an indirect enzyme-linked immunosorbent assay (iELISA). Then, a fusion protein containing 22 verified epitopes was prokaryotically expressed and used as an antigen in paper-based ELISA (p-ELISA) for serodiagnosis of brucellosis. The multi-epitope-based p-ELISA was evaluated using a collection of brucellosis-positive and -negative sera collected from bovine and goat, respectively. Receiver operating characteristic (ROC) curve analysis showed that the sensitivity and specificity of detection-ELISA in diagnosing goat brucellosis were 98.85 and 98.51%. The positive and the negative predictive values were 99.29 and 98.15%, respectively. In diagnosing bovine brucellosis, the sensitivity and specificity of this method were 97.85 and 96.61%, with the positive and negative predictive values being identified as 98.28 and 97.33%, respectively. This study demonstrated that the B cell epitopes contained in major antigenic proteins of Brucella can be a very useful antigen source in developing a highly sensitive and specific method for serodiagnosis of brucellosis.

scholarly journals Immunoinformatic Approach for the identification of T Cell and B Cell Epitopes in the Surface Glycoprotein and Designing a Potent Multiepitope Vaccine Construct Against SARS-CoV-2 including the new UK variant

2021 ◽  
Author(s):  
Kaveri Krishnasamy ◽  
Gracy Fathima Selvaraj ◽  
Kiruba Ramesh ◽  
Padmaoriya Padmanabhan ◽  
Vidya Gopalan ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
B Cell ◽  
T Lymphocyte ◽  
Vaccine Candidate ◽  
Surface Glycoprotein ◽  
T Cell Epitopes ◽  
B Cell Epitopes ◽  
New Variant

The emergence of a novel coronavirus in China in late 2019 has turned into a SARS-CoV-2 pandemic affecting several millions of people worldwide in a short span of time with high fatality. The crisis is further aggravated by the emergence and evolution of new variant SARS-CoV-2 strains in UK during December, 2020 followed by their transmission to other countries. A major concern is that prophylaxis and therapeutics are not available yet to control and prevent the virus which is spreading at an alarming rate, though several vaccine trials are in the final stage. As vaccines are developed through various strategies, their immunogenic potential may drastically vary and thus pose several challenges in offering both arms of immunity such as humoral and cell-mediated immune responses against the virus. In this study, we adopted an immunoinformatics-aided identification of B cell and T cell epitopes in the Spike protein, which is a surface glycoprotein of SARS-CoV-2, for developing a new Multiepitope vaccine construct (MEVC). MEVC has 575 amino acids and comprises adjuvants and various cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes that possess the highest affinity for the respective HLA alleles, assembled and joined by linkers. The computational data suggest that the MEVC is non-toxic, non-allergenic and thermostable with the capability to elicit both humoral and cell-mediated immune responses. The population coverage of various countries affected by COVID-19 with respect to the selected B and T cell epitopes in MEVC was also investigated. Subsequently, the biological activity of MEVC was assessed by bioinformatic tools using the interaction between the vaccine candidate and the innate immune system receptors TLR3 and TLR4. The epitopes of the construct were analyzed with that of the strains belonging to various clades including the new variant UK strain having multiple unique mutations in S protein. Due to the advantageous features, the MEVC can be tested in vitro for more practical validation and the study offers immense scope for developing a potential vaccine candidate against SARS-CoV-2 in view of the public health emergency associated with COVID-19 disease caused by SARS-CoV-2.

scholarly journals Prediction and Evolution of B Cell Epitopes of Surface Protein in SARS-CoV-2

2020 ◽  
Author(s):  
Jerome R Lon ◽  
Yunmeng Bai ◽  
Bingxu Zhong ◽  
Fuqaing Cai ◽  
Hongli Du
Keyword(s):  
B Cell ◽  
Vaccine Development ◽  
Surface Protein ◽  
The Other ◽  
Reference Sequence ◽  
Prediction Methods ◽  
Whole Genome ◽  
B Cell Epitopes ◽  
Conformational Epitopes ◽  
Linear Epitopes

AbstractThe discovery of epitopes is helpful to the development of SARS-CoV-2 vaccine. The sequences of the surface protein of SARS-CoV-2 and its proximal sequences were obtained by BLAST, the sequences of the whole genome of SARS-CoV-2 were obtained from the GenBank. Based on the NCBI Reference Sequence: NC_045512.2, the conformational and linear B cell epitopes of the surface protein were predicted separately by various prediction methods. Furthermore, the conservation of the epitopes, the adaptability and other evolutionary characteristics were also analyzed. 7 epitopes were predicted, including 5 linear epitopes and 2 conformational epitopes, one of the linear and one of the conformational were coincide. The epitope D mutated easily, but the other epitopes were very conservative and the epitope C was the most conservative. It is worth mentioning that all of the 6 dominated epitopes were absolutely conservative in nearly 1000 SARS-CoV-2 genomes, and they deserved further study. The findings would facilitate the vaccine development, had the potential to be directly applied on the treatment in this disease, but also have the potential to prevent the possible threats caused by other types of coronavirus.

scholarly journals Prediction and Evolution of B Cell Epitopes of Surface Protein in SARS-CoV-2

2020 ◽  
Author(s):  
Jerome Rumdon Lon ◽  
Yunmeng Bai ◽  
Bingxu Zhong ◽  
Fuqiang Cai ◽  
Hongli Du
Keyword(s):  
B Cell ◽  
Vaccine Development ◽  
Surface Protein ◽  
Conformational Epitope ◽  
Reference Sequence ◽  
Effective Vaccine ◽  
Antigenic Determinants ◽  
B Cell Epitopes ◽  
Linear Epitopes ◽  
Long Acting

Abstract Background In order to obtain antibodies that recognize natural proteins, it is possible to predict the antigenic determinants of natural proteins, which are eventually embodied as polypeptides. The polypeptides can be coupled with corresponding vectors to stimulate the immune system to produce corresponding antibodies, which is also a simple and effective vaccine development method. The discovery of epitopes is helpful to the development of SARS-CoV-2 vaccine. Methods The analyses were related to epitopes on 3 proteins, including spike(S), envelope(E) and membrane(M) proteins, which were associated with the lipid envelope of the SARS-CoV-2. Based on the NCBI Reference Sequence: NC_045512.2, the conformational and linear B cell epitopes of the surface protein were predicted separately by various prediction methods. Furthermore, the conservation of the epitopes, the adaptability and other evolutionary characteristics were also analyzed, the sequences of the whole genome of SARS-CoV-2 were obtained from the GISAID. Results 7 epitopes were predicted, including 6 linear epitopes and 1 conformational epitope. One of the linear and one of the conformational consist of identical sequence, but represent different forms of epitopes. It is worth mentioning that all of the 6 dominated epitopes were conservative in nearly 3500 SARS-CoV-2 genomes, and they showed a phenomenon which is helpful to obtain stable and long-acting epitopes under the condition of high frequency of amino acid mutation, and deserved further study at the experiment level. Conclusion The findings would facilitate the vaccine development, had the potential to be directly applied on the prevention in this disease, but also have the potential to prevent the possible threats caused by other types of coronavirus.

scholarly journals Francisella tularensis Vaccines Elicit Concurrent Protective T- and B-Cell Immune Responses in BALB/cByJ Mice

PLoS ONE ◽  
2015 ◽  
Vol 10 (5) ◽  
pp. e0126570 ◽  
Author(s):  
Roberto De Pascalis ◽  
Lara Mittereder ◽  
Alicia Y. Chou ◽  
Nikki J. Kennett ◽  
Karen L. Elkins
Keyword(s):  
Immune Responses ◽  
B Cell ◽  
Francisella Tularensis

scholarly journals Paper-based ELISA diagnosis technology for human brucellosis based on a multiepitope fusion protein

2021 ◽  
Vol 15 (8) ◽  
pp. e0009695
Author(s):  
Dehui Yin ◽  
Qiongqiong Bai ◽  
Xiling Wu ◽  
Han Li ◽  
Jihong Shao ◽  
...  
Keyword(s):  
Fusion Protein ◽  
B Cell ◽  
Sensitivity And Specificity ◽  
Predictive Value ◽  
Outer Membrane Proteins ◽  
Diagnostic Methods ◽  
Human Brucellosis ◽  
Testing Method ◽  
Animal Populations ◽  
Linear Epitopes

Background Brucellosis, as a serious zoonotic infectious disease, has been recognized as a re-emerging disease in the developing countries worldwide. In china, the incidence of brucellosis is increasing each year, seriously threatening the health of humans as well as animal populations. Despite a quite number of diagnostic methods currently being used for brucellosis, innovative technologies are still needed for its rapid and accurate diagnosis, especially in area where traditional diagnostic is unavailable. Methodology/Principal findings In this study, a total of 22 B cell linear epitopes were predicted from five Brucella outer membrane proteins (OMPs) using an immunoinformatic approach. These epitopes were then chemically synthesized, and with the method of indirect ELISA (iELISA), each of them displayed a certain degree of capability in identifying human brucellosis positive sera. Subsequently, a fusion protein consisting of the 22 predicted epitopes was prokaryotically expressed and used as diagnostic antigen in a newly established brucellosis testing method, nano-ZnO modified paper-based ELISA (nano-p-ELISA). According to the verifying test using a collection of sera collected from brucellosis and non-brucellosis patients, the sensitivity and specificity of multiepitope based nano-p-ELISA were 92.38% and 98.35% respectively. The positive predictive value was 98.26% and the negative predictive value was 91.67%. The multiepitope based fusion protein also displayed significantly higher specificity than Brucella lipopolysaccharide (LPS) antigen. Conclusions B cell epitopes are important candidates for serologically testing brucellosis. Multiepitope fusion protein based nano-p-ELISA displayed significantly sensitivity and specificity compared to Brucella LPS antigen. The strategy applied in this study will be helpful to develop rapid and accurate diagnostic method for brucellosis in human as well as animal populations.

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