Adjuvant effect of dispersed fullerene C60 on the immune response to constructs harboring amino acid and nucleotide sequences of hepatitis C virus nonstructural NS5B protein

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Abstract

Introduction. A vaccine against hepatitis C has not yet been developed. Recombinant proteins and plasmids encoding hepatitis C virus (HCV) proteins, the components of candidate vaccines, induce a weak immune response and require the use of adjuvants.

The aim of the work was to study the adjuvant action of an aqueous solution of fullerene C60 during immunization of mice with HCV recombinant protein NS5B (rNS5B) that is an RNA-dependent RNA polymerase, or with NS5B-encoding pcNS5B plasmid.

Materials and methods. An aqueous solution of dispersed fullerene (dnC60) was obtained by ultrafiltration. C57BL/6 mice were immunized with rNS5B subcutaneously, pcNS5B – intramuscularly mixed with different doses of dnC60 three times, then the humoral and cellular response to HCV was evaluated.

Results. Mice immunization with rNS5B in a mixture with dnC60 at doses of 2–50 µg/mouse significantly induced humoral response: a dose-dependent increase in IgG1 antibody titers was 7–20 times higher than in the absence of fullerene. There was no increase in the cellular response to rNS5B when administered with dnC60. The humoral response to DNA immunization was weak in mice of all groups receiving pcNS5B. The cellular response was suppressed when the plasmid was injected in a mixture with dnC60.

Conclusions. Dispersed fullerene dnC60 is a promising adjuvant for increasing the immunostimulating activity of weakly immunogenic proteins including surface and other HCV proteins, important for a protective response. Further research is needed to enhance the ability of dnC60 to boost the cellular immune response to the components of the candidate vaccine.

About the authors

Olga V. Masalova

Gamaleya NRC of Epidemiology and Microbiology, Ministry of Health of the Russian Federation

Email: ol.mas@mail.ru
ORCID iD: 0000-0001-5571-5669
SPIN-code: 7210-0870

Dr. Sci. (Biology), Leading Researcher, Head of the Laboratory

Russian Federation, 123098, Moscow

Ekaterina I. Lesnova

Gamaleya NRC of Epidemiology and Microbiology, Ministry of Health of the Russian Federation

Email: wolf252006@yandex.ru
ORCID iD: 0000-0002-2801-6843
SPIN-code: 9901-8607

Researcher

Russian Federation, 123098, Moscow

Sergey M. Andreev

NRC Institute of Immunology FMBA of Russia

Email: sm.andreevj@nrcii.ru
ORCID iD: 0000-0001-8297-579X
SPIN-code: 2542-5260

PhD, Head of the Laboratory

Russian Federation, 115522, Moscow

Nadezhda N. Shershakova

NRC Institute of Immunology FMBA of Russia

Email: nn.shershakova@nrcii.ru
ORCID iD: 0000-0001-6444-6499
SPIN-code: 7555-5925

PhD, Head of the Laboratory

Russian Federation, 115522, Moscow

Vyacheslav V. Kozlov

Gamaleya NRC of Epidemiology and Microbiology, Ministry of Health of the Russian Federation

Email: hyperslava@yandex.ru
ORCID iD: 0000-0002-0502-4824
SPIN-code: 6821-2115

Researcher

Russian Federation, 123098, Moscow

Kristina Yu. Permyakova

Gamaleya NRC of Epidemiology and Microbiology, Ministry of Health of the Russian Federation; Federal State Budgetary Educational Institution of Higher Education «Moscow State Academy of Veterinary Medicine and Biotechnology — MVA by K.I. Skryabin»

Email: kristusha164@mail.ru
ORCID iD: 0000-0002-3579-4416
SPIN-code: 6220-3425

Junior Researcher, Senior Lecturer

Russian Federation, 123098, Moscow; 109472, Moscow

Natalia A. Demidova

Gamaleya NRC of Epidemiology and Microbiology, Ministry of Health of the Russian Federation

Email: ailande@yandex.ru
ORCID iD: 0000-0003-1961-9789
SPIN-code: 8759-0277

Researcher

Russian Federation, 123098, Moscow

Vladimir T. Valuev-Elliston

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: gansfaust@mail.ru
ORCID iD: 0000-0003-0365-570X
SPIN-code: 3492-4501

PhD, Researcher

Russian Federation, 119991, Moscow

Evgeny A. Turetskiy

NRC Institute of Immunology FMBA of Russia; Sechenov First Moscow State Medical University (Sechenov University)

Email: ea.turetskiy@nrcii.ru
Scopus Author ID: 1006824

PhD

Russian Federation, 115522, Moscow; 119991 Moscow

Alexander V. Ivanov

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: aivanov@yandex.ru
ORCID iD: 0000-0002-5659-9679
SPIN-code: 5776-5496

PhD, Leading Researcher

Russian Federation, 119991, Moscow

Tatyana N. Nikolaeva

Gamaleya NRC of Epidemiology and Microbiology, Ministry of Health of the Russian Federation

Email: tatyananik.55@mail.ru
ORCID iD: 0000-0001-6226-7251
SPIN-code: 6679-1727

Dr. Sci. (Medicine), Head of the Laboratory

Russian Federation, 123098, Moscow

Musa R. Khaitov

NRC Institute of Immunology FMBA of Russia; Pirogov Russian National Research Medical University

Email: mr.khaitov@nrcii.ru
SPIN-code: 3199-9803

RAS Corr. Member, Professor, Dr. Sci. (Medicine), Director

Russian Federation, 115522, Moscow; 119997, Moscow

Alexander V. Pronin

Gamaleya NRC of Epidemiology and Microbiology, Ministry of Health of the Russian Federation

Email: proninalexander@yandex.ru
ORCID iD: 0000-0001-5266-9783
SPIN-code: 5736-5260

Professor, Dr. Sci. (Biology), Director

Russian Federation, 123098, Moscow

Alla A. Kushch

Gamaleya NRC of Epidemiology and Microbiology, Ministry of Health of the Russian Federation

Author for correspondence.
Email: vitallku@mail.ru
ORCID iD: 0000-0002-3396-5533
SPIN-code: 6964-1715

Professor, Dr. Sci. (Biology), Leading Researcher

Russian Federation, 123098, Moscow

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2. Fig. 1. Activity of IgG1 antibodies to the recombinant HCV rNS5B protein in the sera of mice immunized with rNS5B in a mixture with dispersed fullerene: a – sera titration curves in ELISA; b – the increase of antibody titers in groups of mice, the antibody titer in group 1 (1 : 105) was taken as a unit. OD – optical density at 450 nm.

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3. Fig. 2. Cellular response to recombinant NS5B protein in splenocytes of mice immunized with rNS5B in a mixture with dispersed fullerene. The lymphocyte proliferation is expressed as a stimulation index (SI); IFN-γ secretion is a cytokine concentration in culture fluids by ELISA, in pg/ml; SI was calculated as the ratio of radioactivity (in cpm/min) in wells with stimulants to radioactivity in wells with medium alone. The control group (Control) was administered saline alone.

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4. Fig. 3. Reduction of lymphocyte proliferation of mice immunized with pcNS5B plasmid with dispersed fullerene in response to in vitro stimulation by rNS5B protein. The stimulation index (SI) was calculated as the ratio of radioactivity (in cpm/min) in wells with stimulants to radioactivity in wells with medium alone.

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5. Fig. 4. Differences in the number of IFN-γ-synthesizing cells obtained from mice immunized with pcNS5B plasmid in the presence/absence of dnC60. The results are presented as a difference in the number of spot-forming cells (SFC) per 106 cells in wells with stimulants and in control wells without stimulants (with medium alone); p1–p6 are peptides listed in Table representing HCV sequences.

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6. Fig. 5. Determination of the proportion of CD4+ and CD8+ lymphocytes in the spleen of mice immunized with pcNS5B plasmid in combination with dispersed fullerene by flow cytometry. *p < 0.05 compared to the control group (Control), which was administered saline alone.

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Copyright (c) 2023 Masalova O.V., Lesnova E.I., Andreev S.M., Shershakova N.N., Kozlov V.V., Permyakova K.Y., Demidova N.A., Valuev-Elliston V.T., Turetskiy E.A., Ivanov A.V., Nikolaeva T.N., Khaitov M.R., Pronin A.V., Kushch A.A.

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