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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

Anti-HCV core immune response induced by single and repeated immunizations with synthetic gene encoding core aa 1–152 (DNAcore152). Rabbits were regularly bled; PBMCs were isolated and subjected to stimulation with HCV core-derived peptides and recombinant HCV core aa 1–152. Stimulation indexes (SI) observed after double priming (week 4) (a); boosting of T-cell response in rabbits receiving multiple injections of DNAcore152 illustrated by stimulation indexes exhibited by PBMCs of rabbits 101 and 102 before and after boost 5 at weeks 54 and 56, respectively (b); dynamics of antibody response to HCV core aa 1–152 in rabbits receiving repeated injections of DNAcore152 (c). All antigen stimulation tests were performed in triplicate; SI values represent an average with standard deviation. Test results were discarded if radioactivity incorporation values demonstrated by mitogen PHA were below 1000 counts per minute and if stimulation indexes in response to PHA were below 2. HCV core-specific antibodies response represent an average optical density exhibited by sera of each of the rabbits collected at given time points in two ELISA runs with standard deviations. OD of sera of rabbits immunized with empty vector DNA collected at the same time points did not exceed the optical density of 0.3 (data not shown).
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fig5: Anti-HCV core immune response induced by single and repeated immunizations with synthetic gene encoding core aa 1–152 (DNAcore152). Rabbits were regularly bled; PBMCs were isolated and subjected to stimulation with HCV core-derived peptides and recombinant HCV core aa 1–152. Stimulation indexes (SI) observed after double priming (week 4) (a); boosting of T-cell response in rabbits receiving multiple injections of DNAcore152 illustrated by stimulation indexes exhibited by PBMCs of rabbits 101 and 102 before and after boost 5 at weeks 54 and 56, respectively (b); dynamics of antibody response to HCV core aa 1–152 in rabbits receiving repeated injections of DNAcore152 (c). All antigen stimulation tests were performed in triplicate; SI values represent an average with standard deviation. Test results were discarded if radioactivity incorporation values demonstrated by mitogen PHA were below 1000 counts per minute and if stimulation indexes in response to PHA were below 2. HCV core-specific antibodies response represent an average optical density exhibited by sera of each of the rabbits collected at given time points in two ELISA runs with standard deviations. OD of sera of rabbits immunized with empty vector DNA collected at the same time points did not exceed the optical density of 0.3 (data not shown).

Mentions: To evade both the potential pitfalls as immune suppression induced by an excess of ROS and the immune competition from the ARF products, we exploited a synthetic gene encoding HCV core devoid of the C-terminus (DNAcore152) with a forbidden frameshift (not supporting F-protein formation [36]). Rabbits were immunized with DNAcore152 by two closely spaced priming injections (double prime, four rabbits), in two rabbits followed by a series of boosts performed first with one-month and then with four-month intervals. The latter scheme was applied in view of earlier experiments in chimpanzees which demonstrated gradual increase if there were proliferative responses to HCV core after repeated boosts performed under long period of time [4]. Contrary to the HCV core 1–152 immunized rabbits, rabbits receiving injections of DNAcore152 exhibited low but consistent proliferative response to both HCV core and core-derived peptides (Figure 5(a)), boosted by the booster DNA injections (Figure 5(b); Supplementary Figure S2). Rabbits immunized with repeated injections of DNAcore152 developed also a low-level humoral response to HCV core, weakly boosted after the repeated gene administrations (Figure 5(c)). No anticore responses were registered in rabbits mock-immunized with empty vector DNA (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)

Anti-HCV core immune response induced by single and repeated immunizations with synthetic gene encoding core aa 1–152 (DNAcore152). Rabbits were regularly bled; PBMCs were isolated and subjected to stimulation with HCV core-derived peptides and recombinant HCV core aa 1–152. Stimulation indexes (SI) observed after double priming (week 4) (a); boosting of T-cell response in rabbits receiving multiple injections of DNAcore152 illustrated by stimulation indexes exhibited by PBMCs of rabbits 101 and 102 before and after boost 5 at weeks 54 and 56, respectively (b); dynamics of antibody response to HCV core aa 1–152 in rabbits receiving repeated injections of DNAcore152 (c). All antigen stimulation tests were performed in triplicate; SI values represent an average with standard deviation. Test results were discarded if radioactivity incorporation values demonstrated by mitogen PHA were below 1000 counts per minute and if stimulation indexes in response to PHA were below 2. HCV core-specific antibodies response represent an average optical density exhibited by sera of each of the rabbits collected at given time points in two ELISA runs with standard deviations. OD of sera of rabbits immunized with empty vector DNA collected at the same time points did not exceed the optical density of 0.3 (data not shown).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig5: Anti-HCV core immune response induced by single and repeated immunizations with synthetic gene encoding core aa 1–152 (DNAcore152). Rabbits were regularly bled; PBMCs were isolated and subjected to stimulation with HCV core-derived peptides and recombinant HCV core aa 1–152. Stimulation indexes (SI) observed after double priming (week 4) (a); boosting of T-cell response in rabbits receiving multiple injections of DNAcore152 illustrated by stimulation indexes exhibited by PBMCs of rabbits 101 and 102 before and after boost 5 at weeks 54 and 56, respectively (b); dynamics of antibody response to HCV core aa 1–152 in rabbits receiving repeated injections of DNAcore152 (c). All antigen stimulation tests were performed in triplicate; SI values represent an average with standard deviation. Test results were discarded if radioactivity incorporation values demonstrated by mitogen PHA were below 1000 counts per minute and if stimulation indexes in response to PHA were below 2. HCV core-specific antibodies response represent an average optical density exhibited by sera of each of the rabbits collected at given time points in two ELISA runs with standard deviations. OD of sera of rabbits immunized with empty vector DNA collected at the same time points did not exceed the optical density of 0.3 (data not shown).
Mentions: To evade both the potential pitfalls as immune suppression induced by an excess of ROS and the immune competition from the ARF products, we exploited a synthetic gene encoding HCV core devoid of the C-terminus (DNAcore152) with a forbidden frameshift (not supporting F-protein formation [36]). Rabbits were immunized with DNAcore152 by two closely spaced priming injections (double prime, four rabbits), in two rabbits followed by a series of boosts performed first with one-month and then with four-month intervals. The latter scheme was applied in view of earlier experiments in chimpanzees which demonstrated gradual increase if there were proliferative responses to HCV core after repeated boosts performed under long period of time [4]. Contrary to the HCV core 1–152 immunized rabbits, rabbits receiving injections of DNAcore152 exhibited low but consistent proliferative response to both HCV core and core-derived peptides (Figure 5(a)), boosted by the booster DNA injections (Figure 5(b); Supplementary Figure S2). Rabbits immunized with repeated injections of DNAcore152 developed also a low-level humoral response to HCV core, weakly boosted after the repeated gene administrations (Figure 5(c)). No anticore responses were registered in rabbits mock-immunized with empty vector DNA (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