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Comparative Immunogenicity in Rabbits of the Polypeptides Encoded by the 5' Terminus of Hepatitis C Virus RNA.

Sominskaya I, Jansons J, Dovbenko A, Petrakova N, Lieknina I, Mihailova M, Latyshev O, Eliseeva O, Stahovska I, Akopjana I, Petrovskis I, Isaguliants M - J Immunol Res (2015)

Bottom Line: The C-terminally truncated core was also weakly immunogenic on the T-cell level.To enhance core-specific cellular response, we immunized rabbits with the core aa 1-152 gene forbidding F-protein formation.Our data promotes the use of rabbits for the immunogenicity tests of prototype HCV vaccines.

View Article: PubMed Central - PubMed

Affiliation: Latvian Biomedical Research and Study Center, Ratsupites Street 1, Riga LV-1067, Latvia.

ABSTRACT
Recent studies on the primate protection from HCV infection stressed the importance of immune response against structural viral proteins. Strong immune response against nucleocapsid (core) protein was difficult to achieve, requesting further experimentation in large animals. Here, we analyzed the immunogenicity of core aa 1-173, 1-152, and 147-191 and of its main alternative reading frame product F-protein in rabbits. Core aa 147-191 was synthesized; other polypeptides were obtained by expression in E. coli. Rabbits were immunized by polypeptide primes followed by multiple boosts and screened for specific anti-protein and anti-peptide antibodies. Antibody titers to core aa 147-191 reached 10(5); core aa 1-152, 5 × 10(5); core aa 1-173 and F-protein, 10(6). Strong immunogenicity of the last two proteins indicated that they may compete for the induction of immune response. The C-terminally truncated core was also weakly immunogenic on the T-cell level. To enhance core-specific cellular response, we immunized rabbits with the core aa 1-152 gene forbidding F-protein formation. Repeated DNA immunization induced a weak antibody and sustained proliferative response of broad specificity confirming a gain of cellular immunogenicity. Epitopes recognized in rabbits overlapped those in HCV infection. Our data promotes the use of rabbits for the immunogenicity tests of prototype HCV vaccines.

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Related in: MedlinePlus

Expression of structural proteins encoded by the 5′ terminus of HCV RNA, HCV core aa 1–173 (lanes 4–6) and F-protein (lanes 7–9). E. coli were transformed with plasmids expressing core 1–173 and F-protein; cell lysates were resolved by 15% SDS-PAGE; gel was stained with Coomassie brilliant blue. HCV core 1–173 (0.5, 1, and 2.5 µg per well, lanes 4–6) and F-protein (0.5, 1, and 2.5 µg per well, lanes 7–9), respectively. Controls: His-tagged outer surface protein BB0689 of B. burgdorferi (2.5 µg, lane 1), lysozyme (2.5 µg, lane 2); HBcAg (2.5 µg, lane 3); PageRuler Plus Prestained Protein Ladder (Thermo Scientific, lane M). Position of molecular mass markers is given on the left.
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fig1: Expression of structural proteins encoded by the 5′ terminus of HCV RNA, HCV core aa 1–173 (lanes 4–6) and F-protein (lanes 7–9). E. coli were transformed with plasmids expressing core 1–173 and F-protein; cell lysates were resolved by 15% SDS-PAGE; gel was stained with Coomassie brilliant blue. HCV core 1–173 (0.5, 1, and 2.5 µg per well, lanes 4–6) and F-protein (0.5, 1, and 2.5 µg per well, lanes 7–9), respectively. Controls: His-tagged outer surface protein BB0689 of B. burgdorferi (2.5 µg, lane 1), lysozyme (2.5 µg, lane 2); HBcAg (2.5 µg, lane 3); PageRuler Plus Prestained Protein Ladder (Thermo Scientific, lane M). Position of molecular mass markers is given on the left.

Mentions: Core 1–173 and F-protein were expressed in E. coli with high yields (2–5 mg/L) and purified by His-tag chromatography. Coomassie staining of PAAG containing protein-rich fractions demonstrated the presence of proteins of expected molecular mass of 19 kDa for HCV core 1–173 (lanes 4–6) and of 16 kDa for F-protein (lanes 7–9) (Figure 1), in conformity with the observed products of translation of ARFs of HCV genotypes 1a, 1b, 1c, 2, and 3 [27, 51–55]. Proteins were of over 95% purity (Figure 1).


Comparative Immunogenicity in Rabbits of the Polypeptides Encoded by the 5' Terminus of Hepatitis C Virus RNA.

Sominskaya I, Jansons J, Dovbenko A, Petrakova N, Lieknina I, Mihailova M, Latyshev O, Eliseeva O, Stahovska I, Akopjana I, Petrovskis I, Isaguliants M - J Immunol Res (2015)

Expression of structural proteins encoded by the 5′ terminus of HCV RNA, HCV core aa 1–173 (lanes 4–6) and F-protein (lanes 7–9). E. coli were transformed with plasmids expressing core 1–173 and F-protein; cell lysates were resolved by 15% SDS-PAGE; gel was stained with Coomassie brilliant blue. HCV core 1–173 (0.5, 1, and 2.5 µg per well, lanes 4–6) and F-protein (0.5, 1, and 2.5 µg per well, lanes 7–9), respectively. Controls: His-tagged outer surface protein BB0689 of B. burgdorferi (2.5 µg, lane 1), lysozyme (2.5 µg, lane 2); HBcAg (2.5 µg, lane 3); PageRuler Plus Prestained Protein Ladder (Thermo Scientific, lane M). Position of molecular mass markers is given on the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4644844&req=5

fig1: Expression of structural proteins encoded by the 5′ terminus of HCV RNA, HCV core aa 1–173 (lanes 4–6) and F-protein (lanes 7–9). E. coli were transformed with plasmids expressing core 1–173 and F-protein; cell lysates were resolved by 15% SDS-PAGE; gel was stained with Coomassie brilliant blue. HCV core 1–173 (0.5, 1, and 2.5 µg per well, lanes 4–6) and F-protein (0.5, 1, and 2.5 µg per well, lanes 7–9), respectively. Controls: His-tagged outer surface protein BB0689 of B. burgdorferi (2.5 µg, lane 1), lysozyme (2.5 µg, lane 2); HBcAg (2.5 µg, lane 3); PageRuler Plus Prestained Protein Ladder (Thermo Scientific, lane M). Position of molecular mass markers is given on the left.
Mentions: Core 1–173 and F-protein were expressed in E. coli with high yields (2–5 mg/L) and purified by His-tag chromatography. Coomassie staining of PAAG containing protein-rich fractions demonstrated the presence of proteins of expected molecular mass of 19 kDa for HCV core 1–173 (lanes 4–6) and of 16 kDa for F-protein (lanes 7–9) (Figure 1), in conformity with the observed products of translation of ARFs of HCV genotypes 1a, 1b, 1c, 2, and 3 [27, 51–55]. Proteins were of over 95% purity (Figure 1).

Bottom Line: The C-terminally truncated core was also weakly immunogenic on the T-cell level.To enhance core-specific cellular response, we immunized rabbits with the core aa 1-152 gene forbidding F-protein formation.Our data promotes the use of rabbits for the immunogenicity tests of prototype HCV vaccines.

View Article: PubMed Central - PubMed

Affiliation: Latvian Biomedical Research and Study Center, Ratsupites Street 1, Riga LV-1067, Latvia.

ABSTRACT
Recent studies on the primate protection from HCV infection stressed the importance of immune response against structural viral proteins. Strong immune response against nucleocapsid (core) protein was difficult to achieve, requesting further experimentation in large animals. Here, we analyzed the immunogenicity of core aa 1-173, 1-152, and 147-191 and of its main alternative reading frame product F-protein in rabbits. Core aa 147-191 was synthesized; other polypeptides were obtained by expression in E. coli. Rabbits were immunized by polypeptide primes followed by multiple boosts and screened for specific anti-protein and anti-peptide antibodies. Antibody titers to core aa 147-191 reached 10(5); core aa 1-152, 5 × 10(5); core aa 1-173 and F-protein, 10(6). Strong immunogenicity of the last two proteins indicated that they may compete for the induction of immune response. The C-terminally truncated core was also weakly immunogenic on the T-cell level. To enhance core-specific cellular response, we immunized rabbits with the core aa 1-152 gene forbidding F-protein formation. Repeated DNA immunization induced a weak antibody and sustained proliferative response of broad specificity confirming a gain of cellular immunogenicity. Epitopes recognized in rabbits overlapped those in HCV infection. Our data promotes the use of rabbits for the immunogenicity tests of prototype HCV vaccines.

Show MeSH
Related in: MedlinePlus