Enzyme-linked immunosorbent assay to determine the potency of a rotavirus vaccine based on virus-like particles: analytical procedure development and validation

INTRODUCTION. Rotavirus vaccines based on virus-like particles (VLPs), non-infectious recombinant proteins of human rotavirus A that mimic the structure of the native virus, show promise for preventive vaccination. Presumably, the optimal method to determine the potency of VLP-based rotavirus vaccines is the enzyme-linked immunosorbent assay (ELISA) method used to measure the titre of specific IgG antibodies to rotavirus A proteins in serum samples from vaccinated animals.

AIM. This study aimed at assessing the potency of a VLP-based rotavirus vaccine by developing and validating an analytical procedure using ELISA to determine the levels of antibodies to the VP2 and VP6 proteins of rotavirus A in serum samples from vaccinated guinea pigs.

MATERIALS AND METHODS. The potency of the VLP-based rotavirus vaccine was determined in vivo in three types of experimental animals, including BALB/c mice, agouti guinea pigs, and newborn minipigs. The animals received three intramuscular injections of the vaccine at a dose of 30 µg. This study used the indirect ELISA method to quantify VP2- and VP6-specific immunoglobulin G (IgG) antibodies and the virus-neutralisation test to measure neutralising antibodies (nAbs) to rotavirus A in animal serum samples. The study involved calculating the geometric mean titres (GMTs) of antibodies. The authors validated the analytical procedure for potency assessment on two batches of the vaccine using standard statistical analysis methods.

RESULTS. The study compared VP2- and VP6-specific IgG and nAb levels 14 days after the first, second, and third vaccinations. The authors observed a significant increase in antibody titres and statistically significant (p<0.05) differences between groups as early as after the second vaccination. Double vaccination induced rotavirus-specific IgGs and nAbs in newborn minipigs (GMTs of 200.0 and 108.9, respectively) and guinea pigs (GMTs of 12,800 and 2,600, respectively). Vaccinated mice demonstrated a significant increase in rotavirus-specific IgG levels (GMT of 572,440). Guinea pigs were selected as a relevant model for validating the ELISA-based potency assessment procedure. The validation study used a double vaccination scheme. The validation using two batches of the VLP-based rotavirus vaccine indicated that the ELISA-based analytical procedure met the acceptance criteria for specificity, repeatability, and intermediate precision. The repeatability assessment resulted in a coefficient of variation (CV) of 12.4% for batch 1 and a CV of 7.7% for batch 2, whereas the intermediate precision assessment showed a CV of 6.9% for batch 1 and a CV of 10.2% for batch 2, which were within the acceptance criteria for both validation parameters (CV≤15%).

CONCLUSIONS. The authors developed and validated an ELISA-based analytical procedure for assessing the potency of VLP-based preventive rotavirus vaccines. According to the study results, ELISA is applicable to the control of the potency of VLP-based preventive rotavirus vaccines.

1. Estes MK. Rotaviruses and their replication. In: Knipe DN, Howeley PM, Griffin DE, eds. Fields virology. Philadelphia: Lippicott Williams and Wilkins; 2001. P. 1747–86.

2. Hallowell BD, Chavers T, Parashar U, Tate JE. Global estimates of rotavirus hospitalizations among children below 5 years in 2019 and current and projected impacts of rotavirus vaccination. J Pediatric Infect Dis Soc. 2022;11(4):149–58. https://doi.org/10.1093/jpids/piab114

3. Troeger C, Khalil IA, Rao PC, Cao S, Blacker BF, Ahmed T, et al. Rotavirus vaccination and the global burden of rotavirus diarrhea among children younger than 5 years. JAMA Pediatr. 2018;172(10):958–65. https://doi.org/10.1001/jamapediatrics.2018.1960

4. Yen C, Healy K, Tate JE, Parashar UD, Bines J, Neuzil K, et al. Rotavirus vaccination and intussusception — science, surveillance, and safety: a review of evidence and recommendations for future research priorities in low- and middle-income countries. Hum Vaccin Immunother. 2016;12(10):2580–9. https://doi.org/10.1080/21645515.2016.1197452

5. Cherepushkin SA, Tsibezov VV, Yuzhakov AG, Latyshev OE, Alekseev KP, Altayeva EG, et al. Synthesis and characterization of human rotavirus A (Reoviridae: Sedoreovirinae: Rotavirus: Rotavirus A) virus-like particles. Problems of Virology. 2021;66(1):55–64 (In Russ.). https://doi.org/10.36233/0507-4088-27

6. Bachmann MF, Jennings GT. Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns. Nat Rev Immunol. 2010;10(11):787–96. https://doi.org/10.1038/nri2868

7. Li C, Luo G, Zeng Y, Song F, Yang H, Zhang S, et al. Establishment of sandwich ELISA for quality control in rotavirus vaccine production. Vaccines (Basel). 2022;10(2):243. https://doi.org/10.3390/vaccines10020243

8. Gupta R, Arora K, Singha Roy S, Joseph A, Rastogi R, Mehrotra Arora N. Platforms, advances, and technical challenges in virus-like particles-based vaccines. Front Immunol. 2023;14:1123805. https://doi.org/10.3389/fimmu.2023.1123805

9. Yagovkin EA, Onishchenko GG, Popova AYu, Ezhlova EB, Melnikova AA, Soloviev MYu, et al. Condition and prospects of development of vaccines for specific prevention of enterovirus (nonpolio) infection. Epidemiology and Vaccinal Prevention. 2016;15(4):74–82 (In Russ.). https://doi.org/10.31631/2073-3046-2016-15-4-74-82

10. Vorobieva MS, Afonina OS, Barhaleva OA, Shcherbinina MS, Sarkisyan KA, Rukavishnikov AV, et al. The analysis of a long-term experience of studying inactivated cell-culture vaccines for preventing tick-borne encephalitis of domestic and foreign manufacture in terms of specific activity (immunogenicity). BIOpreparations. Prevention, Diagnosis, Treatment. 2015;(4):4–10 (In Russ.). EDN: VAEGZN

11. Filatov IE, Tsibezov VV, Balandina MV, Norkina SN, Latyshev OE, Eliseeva OV, et al. Virus-like particles based on rotavarus A recombinant VP2/VP6 proteins for assessment the antibody immune response by ELISA. Problems of Virology. 2023;68(2):161–171 (In Russ.). https://doi.org/10.36233/0507-4088-169

12. Khametova KM, Alekseev KP, Yuzhakov AG, Kostina LV, Raev SA, Musienko MI, et al. Evaluation of the molecular-biological properties of human rotavirus A strain Wa. Problems of Virology. 2019;64(1):16–22 (In Russ.). https://doi.org/10.18821/0507-4088-2019-64-1-16-22

13. Sullivan EJ, Rosenbaum MJ. Methods for preparing tissue culture in disposable microplates and their use in virology. Am J Epidemiol. 1967;85(3):424–37.

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):415–28 (In Russ.). https://doi.org/10.36233/0507-4088-194

15. Ward RL, McNeal MM, Sheridan JF. Evidence that active protection following oral immumization of mice with live rotavirus is not dependent on neutralizing antibody. Virology. 1992;188(1):57–66. https://doi.org/10.1016/0042-6822(92)90734-7

16. McAdams D, Estrada M, Holland D, Singh J, Sawant N, Hickey JM, et al. Concordance of in vitro and in vivo mea­sures of non-replicating rotavirus vaccine potency. Vaccine. 2022;40(34):5069–78. https://doi.org/10.1016/j.vaccine.2022.07.017

17. Choi AH, McNeal MM, Basu M, Flint JA, Stone SC, Cle­ments JD et al. Intranasal or oral immunization of inbred and outbred mice with murine or human rotavirus VP6 proteins protects against viral shedding after challenge with murine rotaviruses. Vaccine. 2002;20(27–28):3310–21. https://doi.org/10.1016/s0264-410x(02)00315-8

18. El-Attar L, Oliver SL, Mackie A, Charpilienne A, Poncet D, Cohen J, Bridger JC. Comparison of the efficacy of rotavirus VLP vaccines to a live homologous rotavirus vaccine in a pig model of rotavirus disease. Vaccine. 2009;27(24):3201–8. https://doi.org/10.1016/j.vaccine.2009.03.043

19. Franco MA, Greenberg HB. Immunity to rotavirus infection in mice. J Infect Dis. 1999;179(Suppl. 3):466–9. https://doi.org/10.1086/314805

20. Azevedo MP, Vlasova AN, Saif LJ. Human rotavirus virus-like particle vaccines evaluated in a neonatal gnotobiotic pig model of human rotavirus disease. Expert Rev Vaccines. 2013;12(2):169–81. https://doi.org/10.1586/erv.13.3

21. Niu X, Liu Q, Wang P, Zhang G, Jiang L, Zhang S, et al. Establishment of an indirect ELISA method for the detection of the bovine rotavirus VP6 protein. Animals (Basel). 2024;14(2):271. https://doi.org/10.3390/ani14020271

22. Afchangi A, Jalilvand S, Mohajel N, Marashi SM, Shoja Z. Rotavirus VP6 as a potential vaccine candidate. Rev Med Virol. 2019;29(2):e2027. https://doi.org/10.1002/rmv.2027

23. Shoja Z, Jalilvand S, Latifi T, Roohvand F. Rotavirus VP6: involvement in immunogenicity, adjuvant activity, and use as a vector for heterologous peptides, drug deli­very, and production of nano-biomaterials. Arch Virol. 2022;167(4):1013–23. https://doi.org/10.1007/s00705-022-05407-9