Generic placeholder image

Current Pharmaceutical Biotechnology


ISSN (Print): 1389-2010
ISSN (Online): 1873-4316

Research Article

Rational Design and Evaluation of the Recombinant Multiepitope Protein for Serodiagnosis of Rubella

Author(s): Marilen Souza, Juliana Machado, Jonatas da Silva, Luana Ramos, Lais Nogueira, Patrícia Ribeiro, Daniel Dias, Josiane Santos, José Carlos Santos, Yanna Nóbrega, Amanda Souza, Sonia Freitas, Mariana Campos da Paz, Maria Felipe, Fernando Torres and Alexsandro Galdino*

Volume 23, Issue 8, 2022

Published on: 13 January, 2022

Page: [1094 - 1100] Pages: 7

DOI: 10.2174/1389201022666210907170921

Price: $65


Background: Rubella is an infection caused by rubella virus (RV) and is generally regarded as a mild childhood disease. The disease continues to be of public health importance mainly because when the infection is acquired during early pregnancy, it often results in fetal abnormalities, which are classified as congenital rubella syndrome (CRS). An accurate diagnosis of rubella is thus of pivotal importance for proper treatment.

Objectives: The aim of the study was to produce a recombinant multiepitope protein (rMERUB) for the diagnosis of rubella, based on conserved immunodominant epitopes of glycoprotein E1 and E2.

Methods: A synthetic gene was designed and cloned into vector pET21a with a 6xHis tag at the Cterminal for affinity purification and overexpressed in Escherichia coli cells. Biophysical analysis of rMERUB was performed by circular dichroism. Biological activity was assessed using an in-house ELISA assay.

Results: Expression in Escherichia coli showed a ~22 kDa protein that was purified and used to perform structural assays and an IgG ELISA. Structural analyses reveal that rMERUB has a β leaf pattern that promotes the exposure of epitopes, thus allowing antibody recognition. Evaluation of 33 samples (22=positive; 11=negative) was performed using in-house ELISA and this was compared with a commercial kit. The sensitivity was 100% (95% CI: 85-100) and specificity 90.91% (95% CI: 62-99). Excellent agreement (Kappa index = 0.9) was obtained between ELISA assays.

Conclusion: The careful choice of epitopes and the high epitope density, coupled with simple-step purification, pinpoints rMERUB as a promising alternative for rubella diagnosis, with potential for the development of a diagnostic kit.

Keywords: Rubella virus, multiepitope protein, diagnostic test, enzyme immunoassay, fetal abnormalities, glycoprotein.

Graphical Abstract
Word Health Organization. Global measles and rubella strategic plan., 2012, 1-44.
Christianson, A.; Modell, B. Medical genetics in developing countries. Annu. Rev. Genomics Hum. Genet., 2004, 5(1), 219-265.
[] [PMID: 15485349]
Andrus, J.K.; de Quadros, C.A.; Solórzano, C.C.; Periago, M.R.; Henderson, D.A. Measles and rubella eradication in the Americas. Vaccine, 2011, 29(4)(Suppl. 4), D91-D96.
[] [PMID: 22185837]
Karasek, E.; Paradowska-Stankiewicz, I. Rubella in Poland in 2011. Przegl. Epidemiol., 2013, 67(2), 189-193, 313-315.
[PMID: 24040714]
Morikawa, M.; Cho, K.; Yamada, T.; Yamada, T.; Sato, S.; Minakami, H. Fetal macrosomia in Japanese women. J. Obstet. Gynaecol. Res., 2013, 39(5), 960-965.
[] [PMID: 23279000]
Nóbrega, Y.K.M.; de Carvalho, B.C.; Nitz, N.; Vital, T.E.; Leite, F.B.; Sequeira, I.J.; Moreira, E.E.; de Andrade, J.K.B.; Gandolfi, L.; Pratesi, R.; Hecht, M.M. Rubella seropositivity in pregnant women after vaccination campaign in Brazil’s federal district. Viral Immunol., 2017, 30(9), 675-677.
[] [PMID: 28972455]
Matthews, J.D.; Tzeng, W.P.; Frey, T.K. Determinants of subcellular localization of the rubella virus nonstructural replicase proteins. Virology, 2009, 390(2), 315-323.
[] [PMID: 19539969]
Clarke, D.M.; Loo, T.W.; Hui, I.; Chong, P.; Gillam, S. Nucleotide sequence and in vitro expression of rubella virus 24S subgenomic messenger RNA encoding the structural proteins E1, E2 and C. Nucleic Acids Res., 1987, 15(7), 3041-3057.
[] [PMID: 3562245]
Dominguez, G.; Wang, C.Y.; Frey, T.K. Sequence of the genome RNA of rubella virus: evidence for genetic rearrangement during togavirus evolution. Virology, 1990, 177(1), 225-238.
[] [PMID: 2353453]
Frey, T.K. Molecular biology of rubella virus. Adv. Virus Res., 1994, 44(C), 69-160.
[] [PMID: 7817880]
Wolinsky, J.S. Rubella. In: Virology, 3rd ed; Fields, B.N.; Knipe, D.M.; Howley, P.M.; Chanock, R.M.; Melnick, J.L.; Roizman, B., Eds.; Lipincott-Raven Publishers: Philadelphia, PA, 1996; pp. 899-929.
Mitchell, L.A.; Décarie, D.; Tingle, A.J.; Zrein, M.; Lacroix, M. Identification of immunoreactive regions of rubella virus E1 and E2 envelope proteins by using synthetic peptides. Virus Res., 1993, 29(1), 33-57.
[] [PMID: 7692685]
Chaye, H.; Chong, P.; Tripet, B.; Brush, B.; Gillam, S. Localization of the virus neutralizing and hemagglutinin epitopes of E1 glycoprotein of rubella virus. Virology, 1992, 189(2), 483-492.
[] [PMID: 1379391]
Law, L.J.; Ilkow, C.S.; Tzeng, W.P.; Rawluk, M.; Stuart, D.T.; Frey, T.K.; Hobman, T.C. Analyses of phosphorylation events in the rubella virus capsid protein: role in early replication events. J. Virol., 2006, 80(14), 6917-6925.
[] [PMID: 16809297]
Zrein, M.; Lacroix, M. Peptides, analogues and mixtures thereof for detecting and eliciting antibodies to the E1 and E2 protein of Rubella virus. U.S. Patent 5427792 A. 1992.
Giessauf, A.; Letschka, T.; Walder, G.; Dierich, M.P.; Würzner, R. A synthetic peptide ELISA for the screening of rubella virus neutralizing antibodies in order to ascertain immunity. J. Immunol. Methods, 2004, 287(1-2), 1-11.
[] [PMID: 15099751]
Zhou, Y.; Ushijima, H.; Frey, T.K. Genomic analysis of diverse rubella virus genotypes. J. Gen. Virol., 2007, 88(Pt 3), 932-941.
[] [PMID: 17325367]
Ho-Terry, ; Manfield, G.; Reginald, K. Rubella E1 glycoprotein antigens. EP Patent EP0299673A1. 1988.
Abernathy, E.; Cabezas, C.; Sun, H.; Zheng, Q.; Chen, M.H.; Castillo-Solorzano, C.; Ortiz, A.C.; Osores, F.; Oliveira, L.; Whittembury, A.; Andrus, J.K.; Helfand, R.F.; Icenogle, J. Confirmation of rubella within 4 days of rash onset: comparison of rubella virus RNA detection in oral fluid with immunoglobulin M detection in serum or oral fluid. J. Clin. Microbiol., 2009, 47(1), 182-188.
[] [PMID: 19005151]
Bellini, W.J.; Icenogle, J.P. Measles and Rubella viruses. In: Manual of clinical microbiology; 10th ed; Versalovic, J.; Carroll, K.; Funke, G.; Jorgensen, J.; Landry, M.; Warnock, D., Eds.; ASM Press: Washington, DC, 2015; p. 1372-1387.
Costello, M.; Yungbluth, M. Viral infections.Clinical diagnosis and management by laboratory methods; Henry, J.B., Ed.; W.B. Saunders: New York, 2001, pp. 1045-1071.
Agbede, O.O.; Adeyeni, O.O.; Olatinwo, A.W.O.; Salisu, T.J.; Kolawole, O.M. Sero-prevalence of antenatal Rubella in UITH. Open Public Health J., 2011, 4(1), 16.
Agbede, O.O.; Adeyemi, O.O.; Olatinwo, A.W.O. Significance of IgG-Avidity in Antenatal Rubella Diagnosis. J. Family Reprod. Health, 2013, 7(3), 131-137.
[PMID: 24971115]
Olajide, O.M.; Aminu, M.; Randawa, A.J.; Adejo, D.S. Seroprevalence of rubella-specific IgM and IgG antibodies among pregnant women seen in a tertiary hospital in Nigeria. Int. J. Womens Health, 2015, 7, 75-83.
[] [PMID: 25610003]
Hobman, T.; Chantler, J.; Knipe, D.M. Rubella virus. In: Virology, 5th ed; Knipe, D.M.; Howley, P.M., Eds.; Lippincott Williams & Wilkins: Philadelphia, 2007; pp. 1069-1100.
Houghton, R.L.; Benson, D.R.; Reynolds, L.; McNeill, P.; Sleath, P.; Lodes, M.; Skeiky, Y.A.; Badaro, R.; Krettli, A.U.; Reed, S.G. Multiepitope synthetic peptide and recombinant protein for the detection of antibodies to Trypanosoma cruzi in patients with treated or untreated Chagas’ disease. J. Infect. Dis., 2000, 181(1), 325-330.
[] [PMID: 10608782]
Anandarao, R.; Swaminathan, S.; Fernando, S.; Jana, A.M.; Khanna, N. Recombinant multiepitope protein for early detection of dengue infections. Clin. Vaccine Immunol., 2006, 13(1), 59-67.
[] [PMID: 16426001]
Duthie, M.S.; Hay, M.N.; Morales, C.Z.; Carter, L.; Mohamath, R.; Ito, L.; Oyafuso, L.K.; Manini, M.I.; Balagon, M.V.; Tan, E.V.; Saunderson, P.R.; Reed, S.G.; Carter, D. Rational design and evaluation of a multiepitope chimeric fusion protein with the potential for leprosy diagnosis. Clin. Vaccine Immunol., 2010, 17(2), 298-303.
[] [PMID: 20016045]
Cheng, Z.; Zhao, J-W.; Sun, Z-Q.; Song, Y.Z.; Sun, Q.W.; Zhang, X.Y.; Zhang, X.L.; Wang, H.H.; Guo, X.K.; Liu, Y.F.; Zhang, S.L. Evaluation of a novel fusion protein antigen for rapid serodiagnosis of tuberculosis. J. Clin. Lab. Anal., 2011, 25(5), 344-349.
[] [PMID: 21919069]
Dai, J.; Jiang, M.; Wang, Y.; Qu, L.; Gong, R.; Si, J. Evaluation of a recombinant multiepitope peptide for serodiagnosis of Toxoplasma gondii infection. Clin. Vaccine Immunol., 2012, 19(3), 338-342.
[] [PMID: 22219311]
Faria, A.R.; de Castro Veloso, L.; Coura-Vital, W.; Reis, A.B.; Damasceno, L.M.; Gazzinelli, R.T.; Andrade, H.M. Novel recombinant multiepitope proteins for the diagnosis of asymptomatic Leishmania infantum-infected dogs. PLoS Negl. Trop. Dis., 2015, 9(1), e3429.
[] [PMID: 25569685]
Galdino, A.S.; Santos, J.C.; Souza, M.Q.; Nóbrega, Y.K.; Xavier, M.A.; Felipe, M.S.; Freitas, S.M.; Torres, F.A. A novel structurally stable multiepitope protein for detection of HCV. Hepat. Res. Treat., 2016, 2016, 6592143.
[] [PMID: 26942007]
de Souza, M.Q.; Galdino, A.S.; dos Santos, J.C.; Soares, M.V.; de Nóbrega, Y.C. Alvares, Ada.C.; de Freitas, S.M.; Torres, F.A.; Felipe, M.S. A recombinant multiepitope protein for hepatitis B diagnosis. BioMed Res. Int., 2013, 2013, 148317.
[] [PMID: 24294596]
Adler, A.J.; Greenfield, N.J.; Fasman, G.D. Circular dichroism and optical rotatory dispersion of proteins and polypeptides. In: Methods in enzymology; Academic Press: Elsevier, 1973; p. 27, pp. 675-735.
Böhm, G.; Muhr, R.; Jaenicke, R. Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Eng., 1992, 5(3), 191-195.
[] [PMID: 1409538]
Cohen, J. Weighted kappa: nominal scale agreement with provision for scaled disagreement or partial credit. Psychol. Bull., 1968, 70(4), 213-220.
[] [PMID: 19673146]
Landis, J.R.; Koch, G.G. An application of hierarchical kappa-type statistics in the assessment of majority agreement among multiple observers. Biometrics, 1977, 33(2), 363-374.
[] [PMID: 884196]
Ho-Terry, L.; Londesborough, P.; Cohen, A. Analysis of rubella virus complement-fixing antigens by polyacrylamide gel electrophoresis. Arch. Virol., 1986, 87(3-4), 219-228.
[] [PMID: 3947240]
Dipti, C.A.; Jain, S.K.; Navin, K. A novel recombinant multiepitope protein as a hepatitis C diagnostic intermediate of high sensitivity and specificity. Protein Expr. Purif., 2006, 47(1), 319-328.
[] [PMID: 16504539]
Lin, X.; Chen, Y.; Yan, J. Recombinant multiepitope protein for diagnosis of leptospirosis. Clin. Vaccine Immunol., 2008, 15(11), 1711-1714.
[] [PMID: 18827193]
Baschirotto, P.T.; Krieger, M.A.; Foti, L. Preliminary multiplex microarray IgG immunoassay for the diagnosis of toxoplasmosis and rubella. Mem. Inst. Oswaldo Cruz, 2017, 112(6), 428-436.
[] [PMID: 28591403]
Dimech, W.; Grangeot-Keros, L.; Vauloup-Fellous, C. Standardization of assays that detect anti-rubella virus IgG antibodies. Clin. Microbiol. Rev., 2016, 29(1), 163-174.
[] [PMID: 26607813]

Rights & Permissions Print Export Cite as
© 2024 Bentham Science Publishers | Privacy Policy