Immunogenicity analysis of a composition of inactivated human rotavirus A strains in mice following immunisation

INTRODUCTION. Currently, rotavirus infection is prevented with live attenuated vaccines. However, international and Russian vaccination practices, as well as the physiological characteristics of paediatric patients, necessitate the development of inactivated rotavirus vaccines. Prerequisites for the development of such vaccines are the availability of virus strains capable of stable replication and the selection of optimal inactivation conditions providing for the required antigenicity and immunogenicity levels.

AIM. This study aimed to evaluate and compare the characteristics of the rotavirus-specific immune response to native strains and to a composition of inactivated rotavirus A strains in a mouse model.

MATERIALS AND METHODS. The study used human rotavirus A strains (RRV-4, RRV-5, RRV-6, and RRV-7), a standard rotavirus strain (SA-11 NVC 2364, National Virus Collection of the Russian Federation), and cultures of pig embryo kidney cells treated with Versene solution (SPEV) and Vero cells. Virus titration was used to determine the infectivity of the strains grown in Vero cells maintained in continuous culture. The authors monitored infected cell cultures up to the onset of the cytopathic effect, calculated the 50% tissue culture infectious dose (TCID₅₀) by the Kärber method modified by Ashmarin, and expressed the results as log₁₀ TCID₅₀/mL. Virus strains were inactivated with formaldehyde. To evaluate immuno­genicity, outbred white mice were immunised with native strains and the composition of inactivated strains (RRV-4, RRV-5, RRV-6, and RRV-7). After immunisation, blood was taken from the animals, and the serum titre of rotavirus A antibodies was determined by indirect heterogeneous enzyme immunoassay.

RESULTS. The infectivity of the rotavirus strains adapted to Vero cells ranged from 8.9 to 7.9 log₁₀ TCID₅₀/mL. When selecting inactivation conditions, the authors showed that inactiva­tion occurred at a temperature of 37 °C and a formaldehyde concentration of 0.05–0.025% (depending on the duration of treatment). The antigenicity analysis demonstrated that the antigen titre of the inactivated strain composition (1:16) was lower than that of native strains (1:32–1:64). The authors demonstrated comparability of immunogenicity profiles of the inacti­vated strain composition and native strains in mice.

CONCLUSIONS. The study generated candidate rotavirus A strains that exhibited stable replica­tion in continuous cultures of Vero cells. The authors selected optimal inactivation conditions for these rotavirus strains and developed an inactivated strain composition showing antigenicity and immunogenicity. The presented data suggest that the composition of inactivated rotavirus A strains can be considered as a basis for further development of an inactivated rotavirus vaccine.

1. Bondarev VP, Shevtsov VA, Indikova IN, Evreinova EE, Gorenkov DV. Rotavirus epidemiology and vaccination tactics. BIOpreparations. Prevention, Diagnosis, Treatment. 2019;19(2):81–7 (In Russ.). https://doi.org/10.30895/2221-996X-2019-19-2-81-87

2. De Grazia S, Filizzolo C, Bonura F, Pizzo M, Di Bernardo F, Collura A, et al. Identification of a novel intra-genotype reassortant G1P[8] rotavirus in Italy, 2021. Int J Infect Dis. 2024;140:113–8. https://doi.org/10.1016/j.ijid.2024.01.020

3. Mayansky NA, Kulichenko TV, Mayansky AN. Rotavirus infection: epidemiology, pathology, vaccination. Annals of the Russian Academy of Medical Sciences. 2015;70(1):47–54 (In Russ.). https://doi.org/10.15690/vramn.v70i1.1231

4. Crawford SE, Ramani S, Tate JE, Parashar UD, Svensson L, Hagbom M, et al. Rotavirus infection. Nat Rev Dis Primers. 2017;3:17083. https://doi.org/10.1038/nrdp.2017.83

5. Sadiq A, Bostan N, Khan J, Aziz A. Effect of rotavirus genetic diversity on vaccine impact. Rev Med Virol. 2022;32(1):e2259. https://doi.org/10.1002/rmv.2259

6. Jain S, Vashistt J, Changotra H. Rotaviruses: is their surveillance needed? Vaccine. 2014;32(27):3367–78. https://doi.org/10.1016/j.vaccine.2014.04.037

7. Korovkin AS, Ignatyev GM. Results and prospects of rotavirus immunisation in the Russian Federation. Biological Products. Prevention, Diagnosis, Treatment. 2023;23(4):499–512 (In Russ.). https://doi.org/10.30895/2221-996X-2023-23-4-499-512

8. Burke RM, Tate JE, Dahl RM, Aliabadi N, Parashar UD. Rotavirus vaccination is associated with reduced seizure hospitalization risk among commercially insured US children. Clin Infect Dis. 2018;67(10):1614–6. https://doi.org/10.1093/cid/ciy424

9. Bibera GL, Chen J, Pereira P, Benninghoff B. Dynamics of G2P[4] strain evolution and rotavirus vaccination: a review of evidence for Rotarix. Vaccine. 2020;38(35):5591–600. https://doi.org/10.1016/j.vaccine.2020.06.059

10. Kondakova OA, Nikitin NA, Trifonova EA, Atabekov IG, Karpova OV. Rotavirus vaccines: new strategies and approaches. Moscow University Biological Sciences Bulletin. 2017;72(4): 169–78. https://doi.org/10.3103/S0096392517040071

11. Wang Y, Li J, Liu P, Zhu F. The performance of licensed rotavirus vaccines and the development of a new generation of rotavirus vaccines: a review. Hum Vaccin Immunother. 2021;17(3):880–96. https://doi.org/10.1080/21645515.2020.1801071

12. Burke RM, Tate JE, Kirkwood CD, Steele AD, Parashar UD. Current and new rotavirus vaccines. Curr Opin Infect Dis. 2019;32(5):435–44. https://doi.org/10.1097/qco.0000000000000572

13. Zhou Y, Wu J, Hu X, Chen R, Lin X, Yin N, et al. Immunogenicity of inactivated rotavirus in rhesus monkey, and assessment of immunologic mechanisms. Hum Vaccin Immunother. 2023;19(1):2189598. https://doi.org/10.1080/21645515.2023.2189598

14. Kostina LV, Filatov IE, Eliseeva OV, Latyshev OE, Chernoryzh YaYu, Yurlov KI, et al. Study of the safety and immunogenicity of VLP-based vaccine for the prevention of rotavirus infection in neonatal minipig model. Problems of Virology. 2023;68(5):415–27 (In Russ.). https://doi.org/10.36233/0507-4088-194

15. Fellows T, Page N, Fix A, Flores J, Cryz S, McNeal M, et al. Association between immunogenicity of a monovalent parenteral P2-VP8 subunit rotavirus vaccine and fecal shedding of rotavirus following Rotarix challenge during a randomized, double-blind, placebo-controlled trial. Viruses. 2023;15(9):1809. https://doi.org/10.3390/v15091809

16. Seidakhmetova BA, Zhapparova GA, Marakhovskaya LG, Terebay AA, Nakhanov AK. Scaling of Vero cell culture for the production of biological products. Biosafety and Biotechnology. 2022;(9):44–52 (In Russ.). https://doi.org/10.58318/2957-5702-2022-9-44-52

17. Kaa KV, Ignatyev GM, Sinyugina AA, Ishmukhametov AA. Suscepti­bility of various cell lines to the Chikungunya virus and method selection for commercial-scale production of viral material. Biological Products. Prevention, Diagnosis, Treatment. 2023;23(1):111–120 (In Russ.). https://doi.org/10.30895/2221-996X-2023-23-1-111-120

18. Grachev VP, Khapchaev YuKh. Use of continuous human and animal cell lines for the production of viral vaccines. Journal of Microbiology, Epidemiology and Immunobiology. 2008;(1):82–90 (In Russ.). EDN: IJPFAR

19. Barrett PN, Mundt W, Kistner O, Howard MK. Vero cell platform in vaccine production: moving towards cell culture-based viral vaccines. Exp Rev Vaccines. 2009;8(5):607–18. https://doi.org/10.1586/erv.09.19

20. Louis KS, Siegel AC. Cell viability analysis using trypan blue: manual and automated methods. Methods Mol Biol. 2011;740:7–12. https://doi.org/10.1007/978-1-61779-108-6_2

21. Belousova RV, Trotsenko NI, Preobrazhenskaya EA. Workshop on veterinary virology. Moscow: KolosS; 2013 (In Russ.).

22. Ashmarin IP, Vorobiev AA. Statistical methods in microbiological research. Leningrad: Medgiz; 1962 (In Russ.).

23. Jiang B, Gentsch JR, Glass RI. Inactivated rotavirus vaccines: a priority for accelerated vaccine development. Vaccine. 2008;26(52):6754–8. https://doi.org/10.1016/j.vaccine.2008.10.008

24. Evreinova EE, Khantimirova LM, Shevtsov VA, Merkulov VA, Bondarev VP. Promising opportunities to improve polio vaccines. Biological Products. Prevention, Diagnosis, Treatment. 2022;22(2):142–53 (In Russ.). https://doi.org/10.30895/2221-996X-2022-22-2-336

25. Otrasheuskaja EV, Trukhin VP, Merkulov VA, Ignatyev GM. Chikungunya vaccines: advances in the development and prospects for marketing approval. Biological Products. Prevention, Diagnosis, Treatment. 2023;23(1):42–64 (In Russ.). https://doi.org/10.30895/2221-996X-2023-23-1-42-64

26. Coffin SE, Moser KA, Cohen S, Clark HF, Offit PA. Immu­nologic correlates of protection against rotavirus challenge after intramuscular immunization of mice. J Virol. 1997;71(10):7851–6. https://doi.org/10.1128/jvi.71.10.7851-7856.1997