Comparison of protective Antibody response in guinea pigs immunized with Wild type (Wt) BoHV-1 and ΔgE BoHV-1 mutant virus vaccine

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1 Comparison of protective Antibody response in guinea pigs immunized with Wild type (Wt) BoHV-1 and ΔgE BoHV-1 mutant virus vaccine L.R.ANANTHAKRISHNA 1*, MOHINI SAINI 2, PRAVEEN K GUPTA 3 and RIZWANA TABASSUM 4 1 Asst.Professor, 2, 3 Principal Scientist, 4 Research Associate 1 Department of Veterinary Physiology and Biochemistry; Karnataka Veterinary, Animal & Fisheries Sciences University; Veterinary College, Vinobanagar, Shimoga, India, 2 Division of Biochemistry, 3 & 4 Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India Abstract: Vaccination has been one of the most important interventions in disease prevention, control and eradication. The impact of vaccination largely depends on type of immune response and the extent of protective antibodies that would neutralize the infectious pathogen in subsequent exposure. BoHV-1 causes several clinical conditions in cattle including rhinotracheitis, vaginitis, balanoposthitis, abortion, conjunctivitis, and enteritis which impact a severe economic loss to dairy industry. When the ΔgE BoHV-1 mutant virus was intended to be used as vaccine one of the major immunological parameters such as virus neutralizing antibodies to protect animals against infected virus and/or immunized virus. To determine the suitability of a vaccine, in this study we performed in vitro test method to compare the level of neutralizing antibodies produced by ΔgE BoHV-1mutant virus and wild type BoHV-1 test vaccines in experimentally immunized groups of guinea pigs. Binary ethylenimine (BEI) used to inactivate virus and the vaccine was then prepared from ΔgE BoHV-1 and wild type BoHV-1 viruses by mixing separately with Freund s incomplete adjuvant (Immunocruz) in the ratio of 1:1. These vaccines were used to vaccinate guinea pigs (4 animals for each vaccine) and 4 animals were used as control. The neutralizing antibody titres in blood serum samples 90 days post vaccination (p.v.) were determined by a virus neutralization test. The VNT test results revealed that both wild type and ΔgE mutant virus were significantly more immunogenic reflected by high neutralizing antibodies to BoHV-1 virus contemplating the fact that ΔgE mutant virus generated (VD lab)can efficiently elicit protective immune response against BoHV-1 in the host. Key Words: VNT, BoHV-1, vaccine, neutralizing antibodies. INTRODUCTION: Infectious bovine rhinotracheitis/infectious pustular vulvovaginitis caused by Bovine Herpesvirus (BoHV-1) which is a member of genus Varicellovirus, subfamily Alphaherpesviriniae of the family Herpesviridae. BoHV-1 was first reported in the US in 1950s and in India in BoHV-1 causes several clinical conditions in cattle including rhinotracheitis (Misra and Mishra, 1987), vaginitis, balanoposthitis, abortion, conjunctivitis, and enteritis (Gibbs and Rweyemamu, 1977). The World Organization for Animal Health [OIE] lists BoHV-1 as a List B-notifiable disease. OIEs List B diseases are transmissible diseases that are of socioeconomic and/or public health importance and also significant in international trade. In India, extensive work has been carried out in cattle, buffalo and small ruminants. This virus has been found widely in Andhra Pradesh, Gujarat, Kerala, Orissa, Tamil Nadu, Karnataka and Uttar Pradesh (Singh et al., 1983). 65.3% exotic, 73% crossbred and 62% indigenous cattle have been found seropositive against BoHV-1 (Mallick et al., 1986). In breeding bulls, 95% seropositivity from Tamil Nadu, 41% from Karnataka, and 32.34% from Punjab have been reported from India (Renukaradhya et al., 1993). In a recent study, serum and semen of breeding bulls and training bulls was evaluated from Uttar Pradesh State for the presence of BoHV-1 antibodies using VNT and ELISA, virus shedding using a Taqman probe based qpcr, and virus isolation (Ravishankar et al., 2013). Very high percentage of breeding bulls (96.92%) and training bulls (52.63%) were found positive for BoHV-1 and a serious concern needs to be taken into consideration as semen will be collected from these bulls in mutant virus vaccine Page 52

2 future. The Government of India has taken a step to screen all the breeding bull for the disease used either for natural or artificial insemination (Ravishankar et al., 2013). To determine the seroprevalence and carry out the epidemiological studies either in the field conditions or to ascertain the efficacy of newly developed vaccines, serological assays stand as a reliable and robust tool. Even though, enzyme-linked immunosorbent assay (ELISA) became available, a VNT remains a possible alternative as a screening test, but is also useful as a confirmation test or a semiquantitative measurement of protective antibodies generated by a vaccine to be determined. The test vaccines comprising wild type BoHV-1 virus and ΔgE BoHV-1 virus were inactivated by binary ethylineamine (BEI) compound and used for immunization experiment (Bahnemann et al, 1990). The experiment involves the immunisation of groups of guinea pig with the test vaccines constituting wild type BoHV-1 virus and ΔgE BoHV-1 virus that were mixed appropriately with Freund s incomplete adjuvant. After 90 th day post immunisation blood samples are collected and the sera are tested individually for the detection of BoHV-1 antibody using a virus neutralisation assay (Brito et, 2014). The vaccine may be passed if the antibody titres obtained with the ΔgE BoHV-1 vaccine were greater than or equal to the antibody titres obtained for the wild type BoHV-1 virus (P=0.95). METHODS: Virus propagation and titration The ΔgE BoHV-1 virus produced using homologous recombination between wild type BoHV-1 and ge-transfer vector expressing red fluorescent protein (RFP) in VD laboratory of Biotechnology Division, Indian Veterinary Research Institute by Pawar (2012). Wild type BoHV-1 and ΔgE BoHV-1 mutant virus with no RFP was used for immunization experiment in guinea pigs. After several rounds of virus propagation in MDBK cells the titres of wild type BoHV-1 and ΔgE BoHV-1 mutant virus were and units /ml, respectively as determined by serial dilution in 96 well plate (Reed and Muench). Dose volume calculation It has been calculated that 0.5 ml of Wild type BoHV-1 virus solution contains 2.5 X viruses and 0.5 ml of ΔgE BoHV-1 virus solution contains 5 X viruses. Virus inactivation Both the viruses are chemically inactivated using binary ethylineamine (BEI) compound. The stock solution of 0.1M BEI (100%) is prepared freshly and incubated at 37 o C for an hour. The virus solution (25ml) was inactivated by 1% of 0.1M BEI (250µl) solution by incubating at 37 o C for 12 h. Freshly prepared 1M Sodium thiosulphate solution was sterilized by 0.22 µm filters and stored at 4 o C. BEI was neutralized by adding 250 µl of 1M Sodium thiosulphate into BEI treated virus solution. Vaccine preparation Freund s incomplete adjuvant (Immunocruz) was used as adjuvant. The virus and adjuvant were mixed in the ratio of 1:1 for vaccine preparation. For immunization experimental studies in guinea pigs, the following vaccines were prepared as follows: 1.6 ml of Wt. virus solution mixed thoroughly with 6 ml of Freund s incomplete adjuvant. Similarly 2. 6 ml of ΔgE BoHV-1 virus solution mixed thoroughly with 6 ml of Freund s incomplete adjuvant. Both the preparations were kept at 4 o C until use. An aliquot of both vaccine preparations were kept at 37 o C overnight for sterility check. Immunization of guinea pigs with inactivated wild type and ΔgE BoHV-1 mutant grouping of animals Guinea pigs weighing approximately 500grams were procured from Laboratory Animal Resource section, IVRI. The guinea pigs were divided into following groups for immunization experiment. Animals were fed with concentrates (40grams/animal/day), green roughages (150grams/animal/day) and sufficient water. mutant virus vaccine Page 53

3 Sl.no. No of lab animals Vaccine type Dose per animal route 1. 4 Wild type virus + Freund s incomplete adjuvant 2. 4 ΔgE BoHV-1 virus +Freund s incomplete adjuvant 3. 4 Freund s incomplete adjuvant (Control) The Blood (1-1.5ml) was collected via retro-orbital route from all animals on day 90 followed by booster immunization and serum was separated and stored at -80 o C. Virus Neutralizing (VN) antibody response VNT was performed from three groups of sera (Wt, ΔgE, control) collected 90 th day post immunization (4 animals/per group). VN test was performed according to OIE s standard protocol. The test sera were inactivated at 56 C for 30 min in water bath. The test sera was diluted two folds in cell culture medium starting with undiluted serum and continued to 1/1024 horizontally in a 96-well flat-bottomed cell culture grade microtitre plate in at least two wells per dilution and 50 µl volumes per well. The test sera raised in guinea pig against wild type BoHV-1 virus, ΔgE BoHV-1 mutant virus and oil adjuvant (Control) were used in the experiment. The volume of 50 µl per well of BoHV-1 stock in culture medium was added at a dilution calculated to provide 100 TCID50 per well. In the toxicity control (virus control) wells, 50 µl of culture medium was added in place of virus to 4 empty wells. For cell controls 100 µl of culture medium was added to each of 4 empty wells and the plates were incubated at 37 C for 24 h. Following incubation 100 µl of cell suspension ( cells/well) was added to all the wells and the plates were incubated for 4-6 days at 37 C. The plates were read microscopically for CPEs from 4-6 days. In the cell control wells, the monolayer should be intact. The test sera results were expressed as the reciprocal of the dilution of serum that neutralised the virus in 50% of the wells. If 50% of the wells with undiluted serum neutralised the virus, the (initial dilution) titre was read as 1 (1/2 using the final dilution convention). If all the undiluted and 50% of the wells with 1/2 diluted serum neutralised the virus, the (initial dilution) titre is 2 (final dilution 1/4). For qualitative results, any neutralisation at a titre of 1 or above (initial dilution convention) is considered to be positive. RESULTS AND DISCUSSION: The ΔgE BoHV-1 mutant virus and the wild type BoHV-1 were propagated by infecting MDBK cells. When titrated by TCID50 method the virus titres were found to be units/ml and units /ml respectively. There was no growth in vaccine preparations which was kept for sterility check and the vaccine was used for immunization experiments in guinea pigs. Blood was collected on 90th day from the Guinea pigs immunized with vaccine preparation (Wt, ΔgE BoHV-1 and control). Sera were separated and the immune response was analyzed by VNT. The VNT for sera against wild type BoHV-1, ΔgE BoHV-1 mutant virus and control was done in microtitration plates following the procedure described by O.I.E manual using 2-fold dilutions of heat inactivated serum sample (at 56ºC for 30 min) incubated with 100 TCID 50 of BoHV-1 and 100 µl of MDBK cells. All samples were run in duplicate and final observations were recorded after 5 days of incubation at 37ºC. Virus neutralizing antibody titers of the serum samples were expressed as log10 of the reciprocal serum dilution that protected e 50% of cells in 96 plate wells in other words the reciprocal of the dilution of serum that neutralized the virus in 50% of the wells. A serum sample of a neutralizing antibody titer <1:4 (<0.6 log10) was regarded as negative. The results of virus neutralization test revealed that both wild type and ΔgE BoHV-1virus were significantly more immunogenic (Figure.1). It has been reported that the minimal immunogenic virus neutralizing antibody titer for IBR (BoHV- 1) virus was e log (Zuffa and Feketeova, 1980) or 0.9 (USA Code of Federal Regulation, 1987). Thus VNT test revealed that both wild type and ΔgE BoHV-1virus were significantly more immunogenic reflected by high mutant virus vaccine Page 54

4 neutralizing antibodies to BoHV-1 virus contemplating the fact that ΔgE BoHV-1 mutant virus generated (Vaccine Development lab) can efficiently elicit protective immune response against BoHV-1 in the host. CONCLUSION: The animals immunized with ΔgE mutant of BoHV-1 showed neutralizing antibodies against BoHV-1 virus to protect the animal efficiently from BoHV-1 infection. ACKNOWLEDGEMENT: The authors thanks to the Director and Joint Director (Academic), Indian Veterinary Research Institute, Izatnagar for providing necessary facilities. The authors are also grateful to the National Fund for Basic, Strategic and Frontier Application Research in Agriculture (NFBSFARA), Indian Council of Agricultural Research (ICAR), Government of India for financial support. REFERENCES: 1. P.K. Misra and A. Mishra, Infectious bovine rhinotracheitis virus infection and infertility in cows, heifers and bulls. Indian J Anim Sci, 57(4), , E.P.J Gibbs and M.M. Rweyemamu, Bovine herpesviruses. Part I. Bovine herpesvirus 1. Vet Bull, 47, , B.K. Singh, S.S. Ramakant, and S.S.Togaonkar, Adaptation of infectious bovine rhinotracheitis virus in MDBK cell line and testing buffalo sera for neutralizing antibodies. Indian J.Microbiol. Immunol. Infect. Dis., 4, 6-10, B.B.Mallick, S.K Das and T. Sribabu, Importance of bovine herpes virus 1 in India. Proceedings of National Symposium on current status of herpes virus infection in man and animals in India. Haryana Agricultural University, Hisar, 54-59, G.J. Renukaradhya, Sero-surveillance of cattle and buffaloes for infectious bovine rhinotracheitis infection by avidin-biotin ELISA. MVSc thesis. University of Agricultural Sciences, Bangalore. (1993) 6. C. Ravishankar, S. Nandi, V. Chander, and T.K. Mohapatra, Concurrent testing of breeding bulls for bovine herpesvirus 1 infection (BHV-1) in India. Veterinaria Italiana, 49 (2), , H G Bahnemann, Inactivation of viral antigens for vaccine preparation with particular reference to the application of binary ethylenimine,vaccine, 8, , BP Brito, AM Perez, AV Capozzo, Accuracy of traditional and novel serology tests for predicting crossprotection in foot-and-mouth disease vaccinated cattle, Vaccine, 32, , S.S. Pawar, Construction of bovine herpesvirus-1 (BoHV-1) deletion of mutant using homologous recombination and its characterization. Ph.D. thesis.,indian Veterinary Research Institute, Izatnagar(2012). 10. A. Zuffa, N. Feketeova, Protective action of inactivated adjuvanted IBR vaccine against experimental infection. Vet. Med., 25,51 56, USA Code of Federal Regulation, Animal Products, Number 9, Parts 1 to 199.Office of Federal Registration National Archives and Records Administration, USA,9,1-199,1987. mutant virus vaccine Page 55

5 Figure.1 Figure.1: Comparison of host-humoral immune response in the guinea pigs immunized with Wt BoHV-1 and ge BoHV-1 mutant AUTHOR S BIOGRAPHY: Dr.Anantha Krishna.L.R pursed his M.V.Sc (Master of Veterinay Science) and Ph.D in Animal Biochemistry from India s premier institute Indian Veterinary Research Institute (IVRI) Izatnagar, Bareilly, U.P. His area of research is molecular virology, immunology with respect to molecular biology of Bovine Herpesvirus-1 and validation of marker vaccine developed for IBR disease. Presently he is working as Assistant Professor in the department of Veterinary Physiology and Biochemistry in Veterinary College, Shivamogga. mutant virus vaccine Page 56