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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

Antibody responses raised after immunization of rabbits with polypeptides encoded by the 5′ terminus of HCV genomic RNA. Maximum titer of antibodies against the immunogens (a); kinetics of the development of specific antibody response; controls represent rabbits mock-immunized with adjuvant alone; serum reactivity was tested by ELISA on plates coated with core 1–173 and F-protein (b); reactivity in Western blotting of hyperimmune sera raised against F-protein (serum of rabbit 91, (c)) and HCV core 1–173 (serum of rabbit 93, (d)). Western blotting was done with hyperimmune sera of rabbits collected by the end of immunization and diluted 1 : 104. Lanes in panels (c and d) represent outer surface protein BB0689 of B. burgdorferi carrying 6xHis-tag (2.5 µg, lane 1), lysozyme (2.5 µg, lane 2), HBcAg (2.5 µg, lane 3), core 1–173 (0.5, 1, and 2.5 µg, lanes 4–6, resp.), and F-protein (0.5, 1, and 2.5 µg, lanes 7–9, resp.). PageRuler Plus Prestained Protein Ladder (Thermo Scientific, lane M). Position of molecular mass markers is given on the left.
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fig2: Antibody responses raised after immunization of rabbits with polypeptides encoded by the 5′ terminus of HCV genomic RNA. Maximum titer of antibodies against the immunogens (a); kinetics of the development of specific antibody response; controls represent rabbits mock-immunized with adjuvant alone; serum reactivity was tested by ELISA on plates coated with core 1–173 and F-protein (b); reactivity in Western blotting of hyperimmune sera raised against F-protein (serum of rabbit 91, (c)) and HCV core 1–173 (serum of rabbit 93, (d)). Western blotting was done with hyperimmune sera of rabbits collected by the end of immunization and diluted 1 : 104. Lanes in panels (c and d) represent outer surface protein BB0689 of B. burgdorferi carrying 6xHis-tag (2.5 µg, lane 1), lysozyme (2.5 µg, lane 2), HBcAg (2.5 µg, lane 3), core 1–173 (0.5, 1, and 2.5 µg, lanes 4–6, resp.), and F-protein (0.5, 1, and 2.5 µg, lanes 7–9, resp.). PageRuler Plus Prestained Protein Ladder (Thermo Scientific, lane M). Position of molecular mass markers is given on the left.

Mentions: Rabbits were immunized by the repeated injections of the polypeptides representing core aa 1–173 (core 173), core aa 1–152 (core 1–152), core aa 147–191 (core 147–191), and F-protein. All polypeptides were highly immunogenic on the humoral level; maximum antibody titers after completion of immunization cycle reached 106 and the titer of antibodies to aa 147–191 reached over 105 (Figure 2(a)). The strongest antibody response was achieved after immunization with HCV core 1–173 and F-protein (Figures 2(a) and 2(b)). HCV core 1–152 devoid of C-terminus generated a weaker antibody response with the maximum titer of 5 × 105 despite an identical immunization scheme and almost identical antigen structure of the proteins (except for the lack of C-terminus) (Figures 2(a) and 2(b)). A 44-amino-acid long core 147–191, although used in immunization without carriers (which normally ensure strong antibody response against the synthetic peptides), induced a strong specific immune response with the titers reaching 105 and the same kinetics of the antibody response as the longer polypeptides (Figures 3(a) and 3(b)). No anti-HCV core or anti-F-protein antibodies were detected in control rabbits 95, 96 receiving adjuvant alone (data not shown).


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)

Antibody responses raised after immunization of rabbits with polypeptides encoded by the 5′ terminus of HCV genomic RNA. Maximum titer of antibodies against the immunogens (a); kinetics of the development of specific antibody response; controls represent rabbits mock-immunized with adjuvant alone; serum reactivity was tested by ELISA on plates coated with core 1–173 and F-protein (b); reactivity in Western blotting of hyperimmune sera raised against F-protein (serum of rabbit 91, (c)) and HCV core 1–173 (serum of rabbit 93, (d)). Western blotting was done with hyperimmune sera of rabbits collected by the end of immunization and diluted 1 : 104. Lanes in panels (c and d) represent outer surface protein BB0689 of B. burgdorferi carrying 6xHis-tag (2.5 µg, lane 1), lysozyme (2.5 µg, lane 2), HBcAg (2.5 µg, lane 3), core 1–173 (0.5, 1, and 2.5 µg, lanes 4–6, resp.), and F-protein (0.5, 1, and 2.5 µg, lanes 7–9, resp.). 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

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fig2: Antibody responses raised after immunization of rabbits with polypeptides encoded by the 5′ terminus of HCV genomic RNA. Maximum titer of antibodies against the immunogens (a); kinetics of the development of specific antibody response; controls represent rabbits mock-immunized with adjuvant alone; serum reactivity was tested by ELISA on plates coated with core 1–173 and F-protein (b); reactivity in Western blotting of hyperimmune sera raised against F-protein (serum of rabbit 91, (c)) and HCV core 1–173 (serum of rabbit 93, (d)). Western blotting was done with hyperimmune sera of rabbits collected by the end of immunization and diluted 1 : 104. Lanes in panels (c and d) represent outer surface protein BB0689 of B. burgdorferi carrying 6xHis-tag (2.5 µg, lane 1), lysozyme (2.5 µg, lane 2), HBcAg (2.5 µg, lane 3), core 1–173 (0.5, 1, and 2.5 µg, lanes 4–6, resp.), and F-protein (0.5, 1, and 2.5 µg, lanes 7–9, resp.). PageRuler Plus Prestained Protein Ladder (Thermo Scientific, lane M). Position of molecular mass markers is given on the left.
Mentions: Rabbits were immunized by the repeated injections of the polypeptides representing core aa 1–173 (core 173), core aa 1–152 (core 1–152), core aa 147–191 (core 147–191), and F-protein. All polypeptides were highly immunogenic on the humoral level; maximum antibody titers after completion of immunization cycle reached 106 and the titer of antibodies to aa 147–191 reached over 105 (Figure 2(a)). The strongest antibody response was achieved after immunization with HCV core 1–173 and F-protein (Figures 2(a) and 2(b)). HCV core 1–152 devoid of C-terminus generated a weaker antibody response with the maximum titer of 5 × 105 despite an identical immunization scheme and almost identical antigen structure of the proteins (except for the lack of C-terminus) (Figures 2(a) and 2(b)). A 44-amino-acid long core 147–191, although used in immunization without carriers (which normally ensure strong antibody response against the synthetic peptides), induced a strong specific immune response with the titers reaching 105 and the same kinetics of the antibody response as the longer polypeptides (Figures 3(a) and 3(b)). No anti-HCV core or anti-F-protein antibodies were detected in control rabbits 95, 96 receiving adjuvant alone (data not shown).

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