Comparative Evaluation of HEP-2(Cincinnati) and Vero cell lines sensitivity to Bivalent Oral Polio Virus Vaccine (Type 1 and Type 3) by using In-vitro Microtitration Potency Assay

 

Surekha, Anoop Kumar, Harit Kasana, Jaipal Meena, Archana Sayal

National Institute of Biologicals, Sector - 62, Noida, Uttar Pradesh – 201309.

*Corresponding Author E-mail: jmeena@nib.gov.in

 

ABSTRACT:

Background: Poliomyelitis is a severe viral infectious disease caused by poliovirus and usually, children under the age of 5 years are affected mostly who are not immunized or fully vaccinated. For precise evaluation of quality control testing of the vaccine is based on three parameters i.e. Identity, efficacy and safety. One of the most important test is potency to know about the efficacy and well-founded results in quality control of vaccines. Objectives: To evaluate the and compare sensitivity of HEP-2(Cincinnati) and Vero cell lines to Bivalent Oral Polio Virus Vaccine (Type 1 and Type 3) Methods: The sensitivity of Hep 2-c and Vero cell lines were compared by using a cell culture-based potency test based on CCID 50 /SHD. Results: Present study showed that the Hep 2-C is most sensitive for bOPV (Type1 and Type3) and the Vero cell line is less sensitive for Type 3 Virus. The Vero cell line is similarly sensitive as Hep 2-c for Type1 virus and TVC (Total Virus Count) of bOPV. Conclusions: The present study, suggest that the Vero cell line can also be used for the Potency assay of TVC (Total Virus Count) and Type1 in bivalent oral polio vaccine or monovalent (Type1) OPV along with Hep 2-c cell line.

 

KEYWORDS: Poliomyelitis, Polio Vaccine, Hep 2-C, Potency, Sensitivity.

 

 

INTRODUCTION: 

Poliomyelitis is a severe viral infectious disease caused by three serotypes of poliovirus (Type1, Type2, and Type3). It affects the nervous system and identifies by flaccid paralysis (endure dizziness). India has had a wonderful journey of becoming a polio-free country. The last reported case of polio in India were in West Bengal and Gujarat on 13 January 2011. India has not had a single case of poliovirus since then. On 27th March 2014, WHO certified polio-free to India with the rest of the Southeast Asia region. India is free from polio but the diseases persist in some near countries (Pakistan and Afghanistan) and it may come back1. In 2019, Pakistan and Afghanistan reported 176 number of wild polio virus type 1 cases since 2014. Immunization with polio vaccine is the best prevention from poliomyelitis disease.

 

 

 

In WHO Expanded Program on Immunization (EPI) Polio Vaccine has been comprised in September 19772,3. Vaccines are still the most effective way of preventing infectious diseases4,5. Immunization for poliomyelitis diseases is done by using two types of polio vaccine one is Formalin Inactivated Salk Polio Vaccine (IPV) was developed by Jonas Salk in 1952 and Sabin attenuated Oral Polio Vaccine (OPV) which was developed by Albert Sabin in 19576. WHO has been preparing for the change in immunization prospects since, poliomyelitis eradication was initiated7. In 2006, the functioning group on Pre-and Post –Polio eradication was established to inspect all the related matters to the pre-and post-Polio eradication. The expert committee suggests in its final report to switch from trivalent oral polio vaccine (tOPV) to inactivated polio vaccine (IPV) and bivalent polio vaccine (bOPV) for use in the continuous immunization program. For the polio eradication and concluded strategic plan was developed in 2012 when the world health assembly announces the completion of the eradication of poliomyelitis8,9. The objective of the plan is the removal of oral polio vaccine (OPVs) starting with (OPV2) Poliovirus Type 2 and it was declared on Sep.20, 2015.

The routine immunization introduces a single dose of IPV, it boosts the immunity and reduces the risk of paralytic polio10,11. In India Sabin, the bivalent oral polio vaccine (sbOPV) is being used in the national immunization program after the switch over from trivalent polio vaccine (tOPV) to bivalent polio vaccine (bOPV) from April 201612. On 17th Oct., 2019 WHO has declared the eradication of type 3 virus following the last reported case in November 201213. As per the recommendation by WHO combination of bOPV and IPV is using for immunization, it has been cleared that Sabin vaccine strains are safe, vigorous, and                effective14,15. Vaccination is important to control diseases, but vigorous vaccines are more important. The efficacy of the vaccine is depending upon how the virus particles stimulate the cellular immune system16. Potency test is one of the most effective tests in quality control of vaccine. In quality control standardization of the test procedure and selection of the material/reagents is important to carry out potency testing of vaccine or biological products17,18. For the potency testing of the vaccine cell culture method based on CCID50 is used to get an exact estimate of viral content in the vaccine sample19,20.

 

In the present study, the sensitivity of potency assay for bivalent polio virus vaccine (Type1 and Type 3) was tested in Vero cell line and compared with WHO recommended Hep2-c cell line for Bivalent Polio Vaccine.

 

MATERIALS AND METHODS:

Sample Batch of bivalent oral polio vaccine was taken for the study of different cell lines sensitivity for the precise vaccine evaluation. The selection of the batch is dependent on the expiry date of the vaccine and storage temperature as per manufacturers21. bOPV batch was qualified based on acceptance criteria of the quality characteristics such as the potency of Type 1(not less the 1x106.00 infectious virus units), Type 3 (not less than then1x105.8 infectious virus units) and TOPV (not less than then 1x106.21 infectious virus units) CCID50 per single human dose and other parameters i.e. identification, Stability and sterility according to Indian pharmacopeia 201821.

 

In-Vitro Micro titration Potency Assay for Type1, Type3, and TVC of bOPV: To check the sensitivity of Hep2-c and Vero cell lines in bOPV (Type1 and Type3) by using cell culture-based in vitro micro titration potency assay. A confluent monolayer of Hep2-c and Vero cell line was used. The batch of sample and Reference standard (NIBSC code 02/306) was taken and for dilution medium EMEM supplemented with 2% FBS was used. The range of vaccine dilution was started with 10-1to 10-7.5. Vaccine and reference Sample were titrated in 1 log step from 10-1.0 to 10-3.0 than in 0.5 log steps till 10-7.5 in triplicates. Antisera Mixture P1 (1:4000) and P3 (1:1000) were prepared and used in micro titration potency assay. Plates were labelled for vaccine and reference samples (Type 1 and Type 3) separately for Hep2-c and Vero cell line. 50”l of antisera (Type 1 against type 3) and 50”l of antisera (Type 3 against type 1) was dispensed in all the 10 wells except the cell control where 100”l of 2% EMEM was used. 50”l each of diluted vaccine sample and reference was added in 10 wells, above labelled microtiter plates. Plating of the dilutions was performed from the highest dilution i.e.   10-7.5 backward to10-4.0. All the plates containing antisera and Vaccine dilutions were covered and incubated at 37șC in a CO2 incubator for 3hours.

 

TVC (Total Virus Count) plates were labelled separately. 50”l of 2% EMEM and 50”l of diluted vaccine sample and reference vaccine was dispensed in all the wells of respective plates except cell control where 100”l of cells were added. TVC (Total Virus Count) Plates were covered and store at 2-8șc until cell suspension was not prepared. Suspension was prepared from confluent monolayers of Hep2-c and Vero cell line in EMEM supplemented with 10% of Fetal Bovine Serum (FBS). Viable cell count was taken by using a haemocytometer and trypan blue or automated cell counter. The range of cell count was approximately 1 - 2 x 105 cells/ml (100”l/well). All the plates were covered and incubated at 37șc in a CO2 incubator for 7 days. After the 7th day of incubation, plates were observed for presence (P) and absence (A) of any cytopathic effect (CPE), by using an inverted microscope. Spearman Karbar method was used for the calculation of the final titre in log10CCID50 per SHD of Sample and Reference bOPV vaccine (Type1 and Type3). The test was valid as there was no cytopathic effect (CPE) was observed in cell control wells.

 

RESULTS:

The optimum range of Reference vaccine and sample of bOPV (Type1 and Type3) and TVC (Total Virus Count) obtained by using two types of cell lines (Hep2-c and Vero) is shown in Table1 and Table 2.

 

Table 1: The appropriate range of titre in Reference vaccine by using cell culture based potency test.

Test No.

Cell line

Potency Titer (Type1) (log10CCID50)

Potency Titer (Type3) (log10CCID50)

TVC Titer

(log10CCID50

Variable Factors

1

Hep2-c

6.18

5.98

6.36

Virus (50”l)

EMEM + FBS (2%)

Antisera (Type1 and Type 3) 50 ”l

Cell Volume 100 ”l

CO2 (5%)

Incubation Temperature (37˚± 2˚ c)

Incubation Time (7 days)

Vero

6.08

4.86

6.25

2

Hep2-c

6.11

5.98

6.35

Vero

6.06

4.76

6.21

3

Hep2-c

6.11

5.95

6.40

Vero

6.06

4.90

6.26

 

4

Hep2-c

6.25

5.96

6.38

Vero

6.08

4.86

6.28

 

5

Hep2-c

6.16

5.95

6.36

Vero

6.13

4.85

 6.25

 

Table 2: The appropriate range of titre in vaccine sample by using cell culture based potency test.

Test No.

Cell line

Potency Titer (Type1) (log10CCID50)

Potency Titer (Type3) (log10CCID50)

TVC Titer

(log10CCID50

Variable Factors

1

Hep2-c

6.13

5.98

6.30

Virus (50”l)

EMEM + FBS (2%)

Antisera (Type1 and Type 3) 50 ”l

Cell Volume 100 ”l

CO2 (5%)

Incubation Temperature (37˚± 2˚ c)

Incubation Time (7 days)

Vero

6.08

4.85

6.25

2

Hep2-c

6.06

5.97

6.33

Vero

6.05

4.76

6.26

3

Hep2-c

6.10

5.93

6.25

Vero

6.06

4.86

6.23

 

4

Hep2-c

6.11

5.95

6.31

Vero

6.05

4.76

6.28

 

5

Hep2-c

6.13

5.93

6.28

Vero

6.11

4.83

6.23

 

COMPARISON BETWEEN HEP2-C AND VERO CELL LINE SENSITIVITY:

Batch of bOPV (Type1 and Type3) vaccine was repeated 5 times, each repeated test was performed in triplicate for the reliability of the results. The test was carried out as stated by IP (Indian Pharmacopeia, 2018) and WHO protocol. Comparison for Hep2-c cell line and Vero cell line sensitivity in bOPV (TVC, Type1 and Type 3) vaccine was carried out by using in vitro micro titration potency assay, results were calculated for each cell line. (Figure1)

 

As per the test performance of bOPV batch repeated 5 times no significant difference was found in the potency test of bOPV. For the sensitivity of Hep2-C cell line and Vero cell line on bOPV (Type1 and Type3) differences were found. The highest potency of bOPV (TVC, Type1 and Type3) was obtained by using Hep2-C cell line (10 6.33log10 CCID50, 10 6.13log10 CCID50,105.98 log10 CCID50) per single human dose. For VERO Cell line the highest potency of bOPV (TVC, Type1 and Type3) was obtained (106.28log10CCID50, 106.11log10CCID50, 104.86log10 CCID50.

 

Vero cell line show lowest potency as comparison to Hep2-c cell line for bOPV Type3, (Hep2-C 10 5.93 log10CCID50, Vero 104.76log10CCID50). Hep2-C and Vero cell lines were similar sensitive for bOPV Type1 (Hep2-C 106.13 log10CCID50, Vero 10 6.11 log10CCID50) and TVC (Hep2-C 106.31 log10CCID50, Vero 106.28log10CCID50).

 

Figure 1: Comparison for Hep2-c cell line and Vero cell line sensitivity in bOPV (TVC, Type1 and Type 3) vaccine was carried out by using in vitro micro titration potency assay.

 

DISCUSSION:

One of the most effective human viral vaccine is oral poliomyelitis vaccine (OPV), has an important role in the eradication of polio in the world. The use of safe and vigorous vaccines for immunization is most essential. The efficacy of the vaccine has depends upon manufacturer consistency and the laboratory test of working virus seed. For this purpose, laboratories, should have effective and validated quality control tests such as potency, identity, and stability.

 

In virus vaccine development, cell culture for virus propagation show a major advancement. One of the most continuous cell lines accepted by the regulatory authorities is the Vero cell line and it has been used for over 30 years for the production of polio and rabies virus vaccine. Vero cell line-derived vaccines are rotavirus vaccine, Japanese encephalitis (JE), Dengue Vaccine, etc. These cell culture-based viral vaccines are used worldwide to immunize humans against infections. However, increased world demand and precise safety rules for novel vaccines to control and eradicate viral diseases have forced manufactures and researchers towards cell culture-based vaccines23.

 

In the worldwide initiative (WHO Technical Report Series, No. 910, 2002) for virological surveillance the culture of poliovirus is gold-standard. The study was aimed to examine the role of established laboratory tests for assuring the quality of live attenuated poliovirus vaccine. With the limitations, there are few cell culture studies about poliovirus and cell sensitivity for Oral polio vaccine samples24.

 

The purpose of present study to examine the cell lines (Hep2-C and Vero) sensitivity on (bOPV) bivalent oral polio vaccine (Type1 and Type3 Virus) for assuring the accurate evaluation of the polio vaccine for immunization by using a cell culture-based in vitro micro titration potency assay. This study showed Hep2-C is most sensitive for bOPV (Type1 and Type3) and the Vero cell line is less sensitive for Type 3 Virus. The Vero cell line is similarly sensitive as Hep2-c for Type1 virus and TVC (Total Virus Count) of bOPV.

 

The sensitivity of vero cell line was tested by using cell culture based potency assay and compared with Hep2-c cell line on bOPV vaccine. The outcome of the present study showed that the vero cell line can also be used for TVC (Total Virus Count) and Type 1 vaccine of bOPV or monovalent (Type1) vaccine.

 

REFERENCES:

1.      Madhekar A. Polio Endgame: How India Beat the Monster. Resonance. 2020; 25(12): 1753-1761.

2.      Baicus A. History of polio vaccination. World Journal of Virology. 2012; 1(4): 108.

3.      Rathore I. Rotavirus Vaccine: A Recent Innovation in Universal Immunization Programme (UIP). Int. J. Adv. Nur. Management. 2016; 4(4): 425-428.

4.      Nugraheni RW. Yusuf H. Dwi S. Design of Liposomes based Vaccine Adjuvant System. Asian J. Pharm. Tech. 2018; 8 (4):261-263.

5.      Yadav R. Vaccines Other Than Specified in National Immunization Schedule among Parents of under- five Children. Int. J. Adv. Nur. Management. 2015; 3(1): 64-68.

6.      Soleymani S. and ABEDI KB. Evaluation and comparison of Hela, Hep2C and Vero cell lines sensitivity to polio vaccinal virus using micro and macro vaccine potency tests. 2012

7.      Vysakh V. Binoy A. Sreenivas A. Abhinand B. Vijayan M. Vaccines-Pillars of Preventive Health. Research J. Pharm. and Tech. 2017; 10(9): 3205-3210. doi: 10.5958/0974-360X.2017.00569.8 Available on: https://www.rjptonline.org/AbstractView.aspx?PID=2017-10-9-69

8.      Tevi BC. Okeibunor J.Châtellier GM. Assefa A. Biey JNM. Cheikh D. Eshetu M. Anya BP. Dao H. Nasir Y. and Akanmori BD. Introduction of inactivated poliovirus vaccine and trivalent oral polio vaccine/bivalent oral polio vaccine switch in the African region. The Journal of Infectious Diseases. 2017; 216(suppl_1) 2: S66-S75.

9.      Patil SM. Maske AP. Sapkale GN. Kure AB. Unique Approaches to Vaccine Development Formulation and Delivery. Research J. Pharmacology and Pharmacodynamics. 2010; 2(2):99-102.

10.   Anya BPM. Moturi E. Aschalew T. Tevi-Benissan MC. Akanmori BD. Poy AN. Mbulu KL. Okeibunor J. Mihigo R. Zawaira F. Contribution of polio eradication initiative to strengthening routine immunization: Lessons learnt in the WHO African region. Vaccine. 2016; 34(43): 5187-5192.

11.   Ahmed S. Othman NH. Sulaiman SA. Rao US. Mahadeva NS, Baig AA. Resistance to Polio Vaccination in Some Muslim Communities and the Actual Islamic Perspectives – A Critical Review. Research J. Pharm. and Tech. 2014; 7(4): 494-495. Available on: https://www.rjptonline.org/AbstractView.aspx?PID=2014-7-4-10

12.   Kado G. Relative sensitivity of three cell substrates to the Sabin poliovirus strains. Developments in Biological Standardization. 1976; 37: 261-264.

13.   Barrett PN. Mundt W. Kistner O. Howard MK. Vero cell platform in vaccine production: moving towards cell culture-based viral vaccines. Expert Review of Vaccines. 2009; 8(5): 607-618.

14.   Kumar A.Tomar, V. Development of potent and stable lyophilized Sabin formulated live attenuated bivalent oral polio vaccine. European Journal of Biomedical and Pharmaceutical Sciences. 2019; 6(11): 218-226.

15.   Soumya M. Deepa R. Herd Immunity. Int. J. Nur. Edu. and Research. 2021; 9(1):125-127.

16.   Dhanashri U. Gadhave P. Gaikwad S. Nayana VP. Swati B. Udugade. DNA vaccines: A hope full ray in Immunology. Asian J. Res. Pharm. Sci. 5(2): 2015; 126-131.

17.   Kumar A. Vyas J. Patel SM. Patel AB. Patel AI. Patel NK. Shah S. Sheth D. Stability testing: An Essential study for Vaccine Formulation Development. Asian Journal of Pharmaceutical Research. 2022; 12(1):29-6.

18.   Roshani P. Kumbhar S. Suryawanshi S. Patil P. Kumar S. Patil V. An Opportunities and Challenges in Vaccine Development. Asian Journal of Research in Pharmaceutical Sciences. 2022; 12(1):83-7.

19.   WHO Expert Committee on Biological Standardization and World Health Organization, 2002. WHO Expert Committee on Biological Standardization: fifty-first report. World Health Organization.

20.   Soleimani S. Lotfi M. Hatami AR and Bordbar N. Comparison of sensitivity of Hela, Hep2c and Vero cell cultures to poliomyelitis vaccinal virus 2008.

21.   Blake IM. Pons SM. Molodecky NA. Diop OM. Chenoweth P. Bandyopadhyay AS. Zaffran M. Sutter RW. and Grassly NC. Type 2 poliovirus detection after global withdrawal of trivalent oral vaccine. New England Journal of Medicine. 2018; 379(9): 834-845.

22.   Jegede AS. What led to the Nigerian boycott of the polio vaccination campaign?. PLoS Med. 2007; 4(3): 73.

23.   Eswaran SP. Praharaj AK. Chander Y. Nagendra A. Potency titration of oral polio vaccine by estimation of live virus content using tissue culture technique. Medical Journal Armed Forces India. 2003; 59(2):  105-107.

24.   Dhanashri U. Gadhave P.Gaikwad S. Nayana V. Pimpodkar, Swati B. Udugade. DNA vaccines: A hope full ray in Immunology. Asian J. Res. Pharm. Sci. 2015; 5(2):  126-131.

 

 

Received on 29.04.2022            Modified on 20.10.2022

Accepted on 23.03.2023           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(10):4719-4722.

DOI: 10.52711/0974-360X.2023.00766