Expression, immunogenicity and diagnostic value of envelope proteins from an Egyptian hepatitis C virus isolate

Expression, immunogenicity and diagnostic value of envelope proteins from an Egyptian hepatitis C... The present work aimed at 1) characterization of the E1 and E2 proteins (HCV-E) from an Egyptian hepatitis C virus genotype 4a (HCV-4a) isolate at the molecular and immunological level, 2) in silico identification of the B- and T-cell epitopes responsible for the immunogenicity of HCV-E, and 3) evaluation of the diagnostic potential of both the recombinant HCV-E and antibodies raised using mammalian expression constructs encoding the protein. The region encoding the E1 and E2 proteins was amplified by RT-PCR from RNA isolated from blood of a human infected with HCV-4 and cloned into the pSC-TA plasmid, and the sequence was verified and used to construct a neighbor-joining phylogenetic tree. The translated nucleotide sequence was used to predict the HCV-E secondary structure using the PREDICT-PROTEIN server and PSI-PRED. A 3D model of HCV-E was generated using the online tool 3Dpro. B- and T-cell epitopes were predicted using the online tools BCPred and Epijen v1.0, respectively. The HCV-E-encoding sequence was later subcloned into the mammalian expression plasmid pQE, and the constructs that were generated were used to immunize mice in the absence and presence of adjuvants of plant origin. The maximum sequence identity obtained by nucleotide and protein BLAST analysis with previously published HCV-E sequences was 85 and 77 %, respectively. The B-cell epitope CFTPSPVVV at position 203 and the T-cell epitope ALSTGLIHL at position 380 were found to be highly conserved among all HCV genotypes. Both ELISA and Western blotting experiments on crude and purified recombinant HCV envelope proteins using mouse antisera raised using the HCV-E mammalian expression construct confirmed the specific antigenicity of the expressed protein. The antibodies raised in mice using the HCV-E-encoding construct could efficiently capture circulating antigens in patients’ sera with good sensitivity that correlated with liver enzyme levels (r = 0.4052, P < 0.0001 for ALT; r = −0.5439, P = 0.0019 for AST). Moreover, combining the HCV-E-encoding construct with extracts prepared from Echinacea purpurea and Nigella sativa prior to immunizing mice significantly (P < 0.05) increased both the humoral (14.9- to 20-fold increase in antibodies) and the cellular (CD4 + and cytotoxic CD8 + - T lymphocytes) responses compared to mice that received the DNA construct alone or PBS-treated mice. Both recombinant HCV-E protein preparations and antibodies raised using the HCV-E-encoding mammalian expression construct represent useful diagnostic tools that can report on active HCV infection. Also, the immunostimulatory effects induced by the two plant extracts used at the cellular and humoral level highlight the potential of natural products for inducing protection against HCV infection. The neutralizing capacity of the induced antibodies is a subject of future investigations. Furthermore, the predicted B- and T-cell epitopes may be useful for tailoring future diagnostics and candidate vaccines against various HCV genotypes. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Virology Springer Journals

Expression, immunogenicity and diagnostic value of envelope proteins from an Egyptian hepatitis C virus isolate

Loading next page...
 
/lp/springer_journal/expression-immunogenicity-and-diagnostic-value-of-envelope-proteins-COVz0f2CXk
Publisher
Springer Journals
Copyright
Copyright © 2015 by Springer-Verlag Wien
Subject
Biomedicine; Virology; Medical Microbiology; Infectious Diseases
ISSN
0304-8608
eISSN
1432-8798
D.O.I.
10.1007/s00705-015-2334-1
Publisher site
See Article on Publisher Site

Abstract

The present work aimed at 1) characterization of the E1 and E2 proteins (HCV-E) from an Egyptian hepatitis C virus genotype 4a (HCV-4a) isolate at the molecular and immunological level, 2) in silico identification of the B- and T-cell epitopes responsible for the immunogenicity of HCV-E, and 3) evaluation of the diagnostic potential of both the recombinant HCV-E and antibodies raised using mammalian expression constructs encoding the protein. The region encoding the E1 and E2 proteins was amplified by RT-PCR from RNA isolated from blood of a human infected with HCV-4 and cloned into the pSC-TA plasmid, and the sequence was verified and used to construct a neighbor-joining phylogenetic tree. The translated nucleotide sequence was used to predict the HCV-E secondary structure using the PREDICT-PROTEIN server and PSI-PRED. A 3D model of HCV-E was generated using the online tool 3Dpro. B- and T-cell epitopes were predicted using the online tools BCPred and Epijen v1.0, respectively. The HCV-E-encoding sequence was later subcloned into the mammalian expression plasmid pQE, and the constructs that were generated were used to immunize mice in the absence and presence of adjuvants of plant origin. The maximum sequence identity obtained by nucleotide and protein BLAST analysis with previously published HCV-E sequences was 85 and 77 %, respectively. The B-cell epitope CFTPSPVVV at position 203 and the T-cell epitope ALSTGLIHL at position 380 were found to be highly conserved among all HCV genotypes. Both ELISA and Western blotting experiments on crude and purified recombinant HCV envelope proteins using mouse antisera raised using the HCV-E mammalian expression construct confirmed the specific antigenicity of the expressed protein. The antibodies raised in mice using the HCV-E-encoding construct could efficiently capture circulating antigens in patients’ sera with good sensitivity that correlated with liver enzyme levels (r = 0.4052, P < 0.0001 for ALT; r = −0.5439, P = 0.0019 for AST). Moreover, combining the HCV-E-encoding construct with extracts prepared from Echinacea purpurea and Nigella sativa prior to immunizing mice significantly (P < 0.05) increased both the humoral (14.9- to 20-fold increase in antibodies) and the cellular (CD4 + and cytotoxic CD8 + - T lymphocytes) responses compared to mice that received the DNA construct alone or PBS-treated mice. Both recombinant HCV-E protein preparations and antibodies raised using the HCV-E-encoding mammalian expression construct represent useful diagnostic tools that can report on active HCV infection. Also, the immunostimulatory effects induced by the two plant extracts used at the cellular and humoral level highlight the potential of natural products for inducing protection against HCV infection. The neutralizing capacity of the induced antibodies is a subject of future investigations. Furthermore, the predicted B- and T-cell epitopes may be useful for tailoring future diagnostics and candidate vaccines against various HCV genotypes.

Journal

Archives of VirologySpringer Journals

Published: Apr 1, 2015

References

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off