Immunogenicity and E cacy Evaluation of Vero Cell-Adapted Infectious Bursal Disease Virus LC-75 Vaccine Strain in Ethiopia

Wakjira Kebebe National Veterinary Institute Molalegne Bitew Ethiopian Biotechnology Institute Fufa Dawo College of Veterinary Medicine and Agriculture Bedaso Mammo College of Veterinary Medicine and Agriculture Hawa Mohammed National Veterinary Institute Martha Yami National Veterinary Institute Belayneh Getachew National Veterinary Institute Takele Abayneh National Veterinary Institute Esayas Gelaye (  esayasgelaye@gmail.com ) National Veterinary Institute https://orcid.org/0000-0002-7329-8632

Results: Identity of the vaccine seed was con rmed using gene-speci c primers using reverse transcription polymerase chain reaction. Con uent monolayer of Vero cells was infected with vaccine virus and serial passage continued till passage ten. Characteristic virus induced cytopathic effect was observed starting from passage 2 on the third day post-infection. The infectious titer of adapted virus showed a linear increment along the passage level. Virus induced speci c antibody was determined using indirect ELISA after vaccination of 14 days old chicks through ocular route. Accordingly, the antibody titer measured from Vero cells vaccinated chicks revealed similar level with the currently available CEF cellbased vaccine. Chicks vaccinated with Vero cell adapted virus showed complete protection against very virulent IBDV, while unvaccinated group had 60% morbidity and 25% mortality.
Conclusions: The IBDV vaccine strain well adapted on Vero cells and found to be immunogenic induces antibodies development and successfully protects chicks against challenging with the circulating eld IBDV isolate. Hence, it is recommended to produce IBD vaccine using Vero cell culture with enough quantity to conquer the limitations using CEF cells and thus to vaccinate chicks to protect against IBDV infection.

Background
Infectious bursal disease (IBD also known as Gumboro) is an immunosuppressive disease of chicken and is the second priority chicken disease in Ethiopia [1] that needs to be controlled primarily through vaccination. The etiological agent is IBD virus (IBDV) belongs to the family Birnaviridae which is a nonenveloped, icosahedral in shape with double-stranded and bi-segmented ribonucleic acid (RNA) genome [2]. The majority of chicken production in Ethiopia is under small scale backyard management system and they serve as source of egg and meat consumptions and for generating cash income. In general, Ethiopia has around 60.5 million poultry [3]. Losses due to chicken mortality occur in different age groups due to presence of different poultry diseases including IBD. Among the causes of the poultry disease in Ethiopia, IBD is one of the diseases which is challenging chicken production and product quality. IBD was rst reported in Ethiopia in 2002 at privately owned commercial poultry farm in which 45-50% mortality rate was reported [4]. Subsequently, IBD has become a second priority problem next to Newcastle disease in both commercial and backyard poultry production systems in Ethiopia despite regular vaccination practices (in commercial farms) using IBDV LC-75 vaccine and improved biosecurity measures [5]. Since then, the circulation of very virulent [6,7] and/or classical virulent IBD viruses [6] were reported from different parts of Ethiopia. Vaccination is the cheapest, most practical and economical method of disease control globally. Most control strategies designed in the country do not take into consideration the local chickens which contribute to 99% of the chicken population. Despite that improved chicken genotypes are being distributed to smallholder farmers in the country by different organizations as a means to improve livelihood of the farmers and these development activities failed because of presence of priority endemic poultry diseases like IBD and Newcastle disease. Previously, Tadelle and Ogle [8] reported that chicken vaccination against prevailing diseases in Ethiopia did not take into account local chicken population found under smallholder farmers' management conditions. Starting from 2004, highly pathogenic IBDV strains emerged via mutation and genetic re-assortment [9]. IBD is a contagious and economically important poultry disease in Ethiopia [10]. Unless the chickens get vaccine during their early life (2 to 6 weeks of age), it leads to considerable economic loss. Therefore, vaccination is considered as an important means of protecting birds during their rst weeks of life [11]. One of the strategies to prevent and control IBD is to hyper-immunize breeders with vaccines [12]. Although passive immunity promotes good protection of chickens during the rst weeks of life, permanent protection against IBD requires the administration of live vaccines [13]. The live IBD vaccines so far developed and in use are categorized as "mild", "intermediate" and "hot" according to their degree of virulence [14]. Mild vaccines are safe for speci c pathogen-free (SPF) chickens but are not very effective in the presence of high levels of maternal antibodies or against very virulent strains of IBDV. Hence, the intermediate and hot vaccines are much more effective but may induce moderate to severe lesions in the Bursa of Fabricius [14]. Currently, Ethiopia is producing live intermediate plus IBDV vaccine using LC-75 strain using chicken embryo broblast cell (CEF) [7] at the National Veterinary Institute (NVI). Production of IBD vaccine based on CEF cells needs high human labor for preparation of primary cells, costly since it needs use of speci c pathogen-free eggs and is time-consuming with a minimum production capacity. Different cell lines such as Vero cells are also suitable for the growth of IBDV so that for vaccine production it is easily manageable and reproducible with a minimum cost but needs proper adaptation of the vaccine strain to the Vero cells. Therefore, in this paper we reported adaptation of IBDV vaccine strain (intermediate plus LC-75) on Vero cell, and evaluation of the immunogenicity and e cacy of Vero cell adapted vaccine strain in target host.

Adaptation of IBDV LC-75 on Vero Cells
The result of the present study showed that the LC-75 strain of IBDV vaccine strain successfully adapted in Vero cell culture starting from passage two to ten. At the rst passage, the infected cells remained intact on the surface of tissue culture ask and did not show any CPEs formation. During second passage, minor CPEs were observed starting from day four post-inoculation. However; starting from passage three up to passage ten, visible CPEs happened at day three post-infection by observing typical aggregation, rounding and clumping of large number of cells and detachment of infected cells with few cells oating in media as visualized by inverted microscope (Fig. 1).

Molecular detection and sequencing
The ampli ed PCR products were visualized on agarose gel electrophoresis that resulted in the generation of 645 bp expected size amplicon of the VP2 gene (Fig. 3). PCR amplicons obtained from virus-infected Vero cell homogenates were con rmed as IBDV (Fig. 3). The VP2 gene nucleotide sequences of the present LC75 vaccine strain viruses isolated from the different culture passages were deposited in the GenBank with accession numbers of IBDV/LC-75/passage 1 (MK798159), IBDV/LC-75/passage 5 (MK798160) and IBDV/LC-75/passage 10 (MK798161).

Immunogenicity of the developed vaccine
The indirect ELISA test result showed that all chicks contained fewer amount of maternally derived antibody that is within the negative range of the S/P ratio. Seven days post-inoculation of the antigen, the mean S/P ratio for Group 1 inoculated from passage 5 was 0.046 (antibody titer 141.86), for Group 2 inoculated from passage 10 was 0.068 (antibody titer 207.27) and for Group 3 inoculated with CEF cellbased vaccine was 0.094 (antibody titer 283.75). At day 14 post-inoculation S/P ratio was 1.28 (antibody titer 3572.68) for passage 5, 1.53 (antibody titer 4247.67) for passage 10 and 1.76 (antibody titer 4865.72) for CEF based vaccine (Gum 01/18). At day 21 post infections the S/P ratio or antibody titer seems equal with that of day 14 post-inoculation (Fig. 4). The chickens were boosted at day fourteen post-inoculation and, after 7 -14 day of boosting, the S/P ratio was strongly increased from 1.53 (antibody titer 4247.67) to 2.15 (antibody titer 6307.77) (Fig. 4).

Immunogenicity and e cacy evaluation
The comparison of IBD virus antibodies produced against Vero cell adapted virus from passages 5 and 10 with that of CEF cell based NVI vaccine (Batch Gum 01/18) showed the absence of signi cant difference between the two IBD vaccines used.
Chick groups vaccinated with Vero cell adapted LC-75 IBDV strain from passages 5 and 10 and from CEF cell based vaccine (Batch Gum 01/18) was found to be safe as no clinical signs and death were observed as compared to the unvaccinated control group (Fig. 5). Starting from day 4 post-inoculations of challenge virus, 60% of chicks from control (unvaccinated) group showed clinical signs such as ru ed feather, inappetence, di cult movement, yellowish diarrhea and dehydration, while 25% of the unvaccinated chickens died starting from day 6 post-challenge.

Discussion
The CEF cell-based vaccine production needs SPF chicken eggs, nevertheless obtaining of SPF eggs and processing of the embryo needs hard currency to import the SPF eggs and also incurs additional labor cost as its processing is labor-intensive and cannot be easily scaled up for large scale vaccine production. The use of Vero cell line for the IBDV vaccine production could be the best alternative.
In the present study, IBDV LC-75 vaccine strain was successfully adapted in Vero cells and the titer was compared with the IBD vaccine produced using chicken embryo broblast cells using the same vaccine strain. The Vero cell adapted IBDV LC-75 vaccine strain at different passages were proved to be immunogenic and protected chickens against challenge with vvIBDV eld strain (MB/263/17). Hence, the Vero cell based IBDV vaccine is recommended for ultimate vaccine production and use in Ethiopia.
Previously, it was suggested that the use of Vero cells in growing avian viruses could become an economical, less laborious, and continuous and e cient tool with an advantage of measuring virus effects outside the host animal [21] as compared to the laborious preparation of primary CEF cell. We have tested that con uent monolayer of Vero cells, following 36 hours of sub-culturing in growth medium, infected with IBDV LC-75 remained fully intact in passage 1 (P1) up to six days post-infection. When the rst passage virus was blindly harvested and passaged (second passage or P2), some changes in Vero cell monolayer began to develop on fourth day of incubation. Monolayer showed rounding of infected cells but complete CPEs of IBDV on Vero cells were not found in this passage too. CPE was observed starting from day three post-infection after the virus became adapted to the cell. This indicated that the virus had adjusted itself to Vero cell culture environment and started replication with good and observable CPEs. Hussain and Rasool [15] also reported that the passage level and the time at which the CPE observed and completed was similar to the present study. On the other hand, Isabela et al. [22] reported that detection of IBD virus titer inoculated on Vero cell was not observed and this could be TCID/ml) is higher than recommended by Code of American Federal Regulation [23] for protecting chickens against the disease. This suggests that the 3 rd passage could be used as a vaccine to protect the chicken against vvIBDV. Previously, Hussain and Rasool [15] also reported similar ndings in growth pattern of vvIBDV in Vero cells at passage 3 after 72 hours of infection. To con rm identity of the Vero-cell adapted IBDV LC-75 vaccine strain, RT-PCR test conducted amplifying the VP2 region. As expected, we ampli ed and sequenced the expected 645 bp band of the VP2 gene fragment. Sequencing of the VP2 gene was recommended for checking absence of nucleotide and/or amino acid change since VP2 gene is responsible for the antigenicity [24], antigenic variation [25], and pathogenicity of the IBD virus [1]. Immune response status following vaccination was assessed using iELISA at adapted virus is e cacious to protect the vvIBDV circulating in Ethiopia similar to that of CFC based prepared vaccine as described previously by OIE [13].

Conclusion
The present study con rmed that Vero cell adapted attenuated IBDV vaccine could be successfully produced similar with that of the CEF cell vaccine. The IBDV vaccine strain LC-75 was adapted well on Vero cells as early as passage 3 with a good virus titer/ml. The adapted vaccine is equally immunogenic and e cacious with no difference between passages and CEF cells based prepared vaccine. It is more economical to produce IBDV vaccine using Vero cells. Based on the above conclusions, production of IBDV vaccine using Vero cell line is recommended. In the future, a closely related cell type like avian stem cells or cell line developed from chicken could replace Vero cells and CEF cells for better vaccine production of avian pathogens as it allows use of host-related cell type for vaccine production that overcomes adaptation problem and also facilitates better vaccine production.

Methods
Virus and cell culture

Adaption of vaccine strain using Vero cells
Vero cells were grown in 75cm 2 cell culture ask until con uent monolayer of cells was obtained.
Con uent Vero cell monolayer was washed three times using prewarmed PBS pH7.4 to remove dead cells and suspended using pre-warmed 0.25% trypsin/EDTA solution. Cell viability was checked with trypan blue stain and the number of viable cells was counted using cell counter EVE TM Automatic cell counter (E16041-016, NanoEnTekInc, Korea). The viable cells were counted and seeded into 75cm 2 tissue culture ask containing pre-warmed DMEM (Sigma-Aldrich) with sterile 10% fetal calf serum (FCS, Gibco) at a seeding density of 2.1x10 6 cells. The cells were incubated at 37°C with 5% CO 2 . The cells were observed under inverted microscope for the formation of con uent monolayer [15].

Infectious Bursal Disease virus inoculation into Vero cell
Con uent monolayer of Vero cells at 36 hours after sub-culturing was washed three times using prewarmed sterile PBS and used for virus infection using adsorption method. The cells were infected by adding 0.5 ml of IBDV LC-75 vaccine strain and incubated at 37°C for 1 hour with intermittent tilting of the ask to allow adsorption. After one-hour incubation, 10ml DMEM with 2% FCS was added into an infected ask and placed into 37°C incubator with 5% CO 2 . One ask of fresh cells with con uent monolayer was kept as control under similar condition. The infected cells were observed daily for the development of virus induced cytopathic effects (CPE) for six days. At six days of post-inoculation the infected cells with the virus were harvested, labelled and stored at -80°C for 24 hrs. The harvested virus was freeze-thawed alternatively three times at -80ºC/+30ºC and inoculated to fresh monolayer of Vero cells. This process was repeatedly conducted up to passage 10 (P10). At each passage, the harvested asks were labeled and stored at -80°C until subsequent experiments.

Titration of infectious bursal disease virus
The viral suspension kept at -80ºC was diluted 10-fold in sterile tubes (10 -1 to 10 -10 ) of passages 2, 3, 5, 7, 9 and 10. Then 100μl viral dilutions were dispensed into 96 well micro plates containing 100μl Vero cells per well with ten replicates for each dilution. Column 11 was left empty and column 12 was inoculated only with cells to serve as negative control. Finally, the plate was sealed by micro plate sealer and incubated at 37°C in 5% CO 2 . The inoculated plates were monitored under inverted microscope daily starting from 72 hours post-inoculation for eight days. The titre for each virus passage was determined according to Spearman Karber method [16].

Molecular characterization of IBD virus adapted in Vero cell
The identity of the IBD vaccine strain virus adapted in Vero cell was checked by ampli cation of the viral hypervariable core protein (VP2) gene of the IBD virus using gene-speci c primers by reverse transcription polymerase chain reaction (RT-PCR) [17]. The VP2 gene ampli cation was done on vaccine working seed and from passages 1, 3, 5, 7, 9 and 10.
RNA extraction, cDNA synthesis and RT-PCR Virus RNA extracted from the harvested virus using the RNeasy® Mini Kit (Qiagen) following the manufacturer's instruction. The extracted RNA was subjected to two steps RT-PCR. A 10μl reaction mix was prepared from 3μl RNase free water, 1μl Oligo dT, and 1μl of 10 Mm dNTP mix. Then, 5μl template RNA was added and incubated at 65ºC for 5 minutes and placed at 4ºC. A 10μl volume for cDNA synthesis mix was prepared from 1μl DEPC treated water, 2μl of 1X RT buffer, 4μl of 25 mM MgCl 2 , 2μl of 0.1 MDTT, and 1μl superscript III-RT enzyme and incubated at 55ºC for 50 minutes. The reactions were terminated at 85ºC for 5 minutes and chilled on ice and then collected by brief centrifugation to which 1μl RNase was added per sample and incubated for 20 minutes at 37ºC. Finally, the obtained cDNA was immediately used for PCR. For running PCR, a total of 20μl master mix was prepared by using 3μl of RNase free water, 2μl of forward primer, 2μl of reverse primer, 10μl of IQ super mix (Bio-Rad) and 3μl of template cDNA. The ampli cation forward and reverse primers used for the RT-PCR reaction to amplify the segment A, VP2 gene of the virus were used from the previously described method [18]. The two primers are expected to amplify 645 base pairs of the VP2 gene. Master mix was prepared and ampli ed using touchdown PCR (thermal cycler 2720, applied Bio-system). The PCR cycles involved initial denaturation at 95ºC for 5 minutes, followed by 15 cycles of denaturation at 95ºC for 30sec, annealing at 60ºC for 30sec, and elongation at 72ºC for 30sec. The 15 cycles of initial PCR followed by 20 cycles of denaturation at 95ºC for 30sec, annealing at 56ºC for 30sec, extension at 72ºC for 30sec and nal extension at 72ºC for 7 minutes for 1 cycle. The ampli ed PCR products were stained with GelRed ® (Biotium) and visualized using 1.5% agarose gel electrophoresis in 1x TAE buffer. A 100 bp DNA ladder (Fermentas) was used and IBD virus positive samples yielded a PCR product of 645 bp band size.
Immunogenicity and e cacy tests A total of eighty fourteen days old Koko breed speci c pathogen free chicks of both sex were used for vaccine evaluation study. Chicks obtained from the Ethiopian Institute of Agricultural Research, Bishoftu and screened for presence of maternal antibody against IBD virus using indirect ELISA test (IDvet, France) before commencing the animal experiment. All maternal antibody free chicks aged fourteen-day were grouped randomly into four groups (group 1-4); the sample size (number of chicks) per group were 20 chicks (total 80 chicks) according to OIE recommendation [13] to be included in one experimental group. The chicks were managed under the same animal facilities of separated room for each treatment group with similar management to avoid any confounding factors among the different groups. Group 1 and 2 were inoculated with Vero cell adapted IBDV passage 5 and 10, respectively.  [19]. The challenged birds were observed for 10 days for morbidity and mortality and the results were recorded as per OIE recommendation [13]. The chicks were identi ed using unique identi cation number attached on the wing and grouped and follow up till data analysis blindly.

Indirect ELISA
Indirect ELISA test was done at the NVI following the manufacturers' instruction (IDvet, France). Optical density (OD) of the test result was read by using microtiter plate reader at 450nm absorbance. The percentage positivity for test samples in relation to the negative and the positive controls was calculated as per the formula given by the kit manufacturer. The result was interpreted with reference to the cut-off value recommended by the manufacturer as the OD PC (positive control) should be >0.25, and OD PC /OD NC should be >3.0; formula indicated at the data management section.

Animals management
All experimental chicks handled according to the NVI animal handling and research ethics guideline. Feed and water provided ad libitum. After the end of the experiment, the chicks were euthanized humanly using manual cervical dislocation involved stretching and separating the cervical vertebrae by hand and properly incinerated using the incinerator located inside the institute compound considering all the biosafety and biosecurity measures. Ethical clearance obtained from the animal research ethical review committee, certi cate Ref. No: AAU/CVMA/VM/ERC/28/05/10/2018.

Data management and statistical analysis
All the collected data were entered into Microsoft Excel spread sheet program and analyzed using STATA version 12 [20]. A one-way analysis of variance (ANOVA) was used to differentiate effect of passage levels on immunogenicity of vaccines in chicks. Statistical signi cance was based on p < 0.05. The formula used for calculation of sample antibody level was taken from the kit manufacturer as described below.
SP value= OD sample -OD NC / OD PC -OD NC Where; SP: sample to positive ratio, OD: optical density, NC: negative control, PC: positive control.

Declarations
Ethics approval and consent to participate Ethical clearance for this study was obtained from animal research ethics and review committee, College of Veterinary Medicine and Agriculture, Addis Ababa University, Bishoftu, Ethiopia (AAU/CVMA/VM/ERC/28/05/10/2018).

Consent for publication
Not applicable.
Availability of data and material