Co-administration of toll-like receptor (TLR)-3 agonist Poly I:C with different infectious bursal disease (IBD) vaccines improves IBD specific immune response in chicken

Toll-like receptor (TLR) agonists are emerging as promising vaccine adjuvants and immunomodulators in poultry against many diseases. Infectious bursa disease (IBD) still remains as a major threat to poultry industry. Improving the vaccine mediated immune response would help in better protection against IBD virus infection. Adjuvant potential of TLR3 agonist, Polinosinic polycytidylic acid (Poly I:C) with different IBD vaccines has been analyzed in chicken in the present study. Intermediate, intermediate plus IBD vaccine, bursaplex vaccine and their respective poly I:C combinations were used for immunization of chicken. IBD specific antibody titers, bursa to body weight ratio, body weight gain and bursal lesion scores were evaluated at weekly interval in different immunization groups. Fold changes in cytokines IL-1β and IFN-γ mRNA expression levels in spleen were also analyzed in different groups. Intermediate plus IBD vaccine induced significantly (P ≤ 0.05) higher IBD specific antibody response at 35 days of age than other groups with comparatively lower body weight gain and moderate bursal lesion score. Poly I:C co-administration with intermediate IBD vaccine and bursaplex vaccine improved the IBD specific antibody titers, better body weight gain and moderately less bursal lesion score. However, Poly I:C combination with intermediate plus IBD vaccine did not improve the specific immune response. IL-1β levels were up-regulated in intermediate plus and bursaplex group, whereas IFN-γ m RNA expression levels were upregulated in intermediate IBD with Poly I:C group. In conclusion, poly I:C co-administration with intermediate IBD and bursaplex vaccine was beneficial and improved the specific immune response with least immunosuppression and bursal damage.


Introduction
TLR ligands or agonists are considered as promising adjuvant candidates due to their role in self/ non-self-differentiation, antigen presenting cell maturation and downstream cytokine stimulation (Janeway and Medzhitov 2002;Bendelac and Medzhitov 2002). Many TLR agonists including cytidine phosphate guanosine (CpG), flagellin, Polinosinic polycytidylic acid (Poly I:C) and S-[2,3bis(palmitoyloxy)-(2RS)-propyl] -(R)-cysteine (Pam2CSK4) are currently being tested or employed along with many vaccines to improve immune response (Sayers et al. 2012). The potential of TLR ligands as adjuvants and prophylactic agents has also been established with many poultry vaccines (Bhadouriya et al. 2019).
Infectious bursal disease (IBD) is one of the most important viral disease of poultry caused by IBD virus (IBDV), a non-enveloped ds RNA virus of Birnaviridae family (Eterradossi 2020;Muller et al. 1979). IBDV targets the bursa of Fabricius of 3-6 weeks old chicks resulting in immunosuppression and mortality (Hirai and Shimakura 1974). Biosecurity and vaccination are two major effective strategies for IBD control. At present, live attenuated, killed and immune-complex IBD vaccines are available for effective control of IBD (Liew et al. 2016). Live attenuated IBD vaccines are mainly used in broilers at early stages for protection; although killed vaccines are employed in breeders for the induction of maternal antibodies (MDA) and subsequent protection of chicks at early stages. Among the modified live IBV vaccines, intermediate and intermediate plus (hot) vaccines are widely used based on the disease incidences and maternal immunity levels. Although these vaccines are capable of inducing protective IBD specific immunity in host, immunosuppression due to bursal damage by the vaccine viruses is major drawback (Rautenschlein et al. 2007). Alleviating the immunosuppressive effect of IBD vaccines and potentiating their immunostimulation has more implications at field level.
Poly I:C, a dsRNA analogue, is a well characterized TLR3 agonists, induce type I IFNs in chickens and act as an antiviral agent (Karpala et al. 2008;Parvizi et al. 2012). Adjuvant effect of poly I:C have been demonstrated with inactivated avian influenza vaccine in chicken and ducks (Liang et al. 2013;Ichinohe et al. 2007;Zhang et al. 2017). Enhanced protection and reduced tumor development were observed in chicken immunized with HVT vaccine for Marek's disease along with poly I:C (Parvizi et al. 2012). Earlier we reported the improvement Newcastle disease (ND) specific immune response in chicken upon co-administration of Poly I:C with mesogenic R2B live ND vaccine (Kannaki et al. 2019). However, there is dearth of information on the effect of Poly I:C and its adjuvant potential with IBD vaccines. In the present study, we explored the immunomodulatory potential of poly I:C with different types of IBD vaccines in chicken.

Experimental birds
Day old Vanaraja chicks (n = 320) hatched from hatchery of ICAR-Directorate of Poultry Research, Hyderabad were used in the present study. Chicks were housed, fed with standard nutrition and provided ad libitum water. All the experiments were carried out following ethical guidelines and approved by Institute animal ethics committee (IAEC/ DPR/2017/7). All the chicks received HVT vaccine for Marek's disease at day-old and LaSota vaccine for ND at 5 th and 28 th day through intranasal route at recommended dose.

IBD vaccines and TLR3 agonist
Commercially available vaccines viz., intermediate, intermediate plus (Venky's India Pvt. Ltd., India) and bursaplex (Zoetis India Pvt Ltd., India) IBD vaccines were procured and used. TLR3 agonist, Poly I:C (Sigma, MO, USA) dissolved in sterile nuclease free water was used in the study.

Immunization trial
One-day old vanaraja chicks were randomly divided into eight groups of 40 chicks each and immunized as follows. Bursaplex and poly I:C adjuvanted (10 µg/ chick) with bursaplex vaccine were given by subcutaneous route (s/c) at day old. Other vaccines viz., intermediate, intermediate plus IBD and poly I:C adjuvants of respective vaccines were given on 10 th day of age and booster dose on 16 th day of age. Intermediate and intermediate plus vaccine were given by oral route either alone or in combination with Poly I:C by intramuscular route (10 µg/ chick). Poly I:C control group received only Poly I:C by intramuscular route. Unimmunized control received only sterile PBS by i/m route (Table 1). All the vaccines were given at recommended dose as per the manufacturer's instruction.

Evaluation of humoral immune response
Blood samples from different groups of immunization trial (n = 6 /each group) were collected at weekly interval from 7D and up to 35D (7, 14, 21, 28 and 35D), serum separated and stored at -20ºC until further analysis. IBD specific serum antibody response from serum samples were analyzed by indirect ELISA (IDEXX laboratories, USA) by following manufacturer's instruction. Briefly, the test was performed on 96-well ELISA plate precoated with IBDV antigen. Diluted (1:500) test sera were dispended (100 µl/well) in duplicates. Undiluted positive and negative controls (each 100 µl/well) provided along with the kit were also dispended on the coated wells. After incubating for 30 min at 25ºC, the plates were washed with distilled water to remove any unbound material and followed by the addition of 100 µl Table 1 Immunization plan to assess the immunomodulatory potential of Poly I:C with different IBD vaccines in chicken + * : Immunization at day-old by i/m route a prime dose at 10 th day of age conjugate. After 30 min at 25ºC, the unbound conjugate was washed away and TMB substrate (100 µl) was added. The subsequent color developed was measured by spectrophotometer at 650 nm and corresponding OD values were recorded. The respective antibody titers were calculated as given below: The OD values were converted into titers using their software (xChekPlus, IDEXX). The titer greater than 396 is considered positive.
ND specific antibodies were also analyzed in the serum samples by haemagglutination inhibition (HI) test using 1% chicken RBCs, OIE protocol. The HI titer was determined as the highest dilution of serum samples that inhibited NDV agglutination of chicken RBCs.

Body weight gain, Bura bodyweight ratio and bursal lesion scoring
Body weight of three randomly selected birds from each group was recorded at weekly interval (7, 14, 21, 28, 35 & 42 D). The mean difference in body weight of different group birds (n = 3) at 7 D and 42 D was calculated as body weight gain during immunization trial. Three birds from each group were humanely sacrificed at weekly interval; bursa tissue was collected and weighed. Bursa to body weight ratio (B:BW) was calculated as: bursa of Fabricius weight (g) / Body weight (g) × 1000. Bursa tissue from different groups collected on 21 st day was subjected to histopathological lesion scoring. The scoring was performed as per Muskett et al. (1979) using following scale: No damage (0); mild necrosis (1); moderate and generalized lymphoid depletion (2); severe lymphoid depletion (3); atrophy of follicles and fibroplasia (4).

RNA extraction and cDNA synthesis
Spleen tissue from different groups of experimental birds were collected aseptically after humane sacrifice at 14 th day Titer = Antilog 1.09 (log 10 S∕P) + 3.36 of age (n = 3 /each group). Total RNA was extracted by using TRIzol (Invitrogen, CA) according to the manufacturer's protocol and treated with DNAse I (MBI Fermentas, USA) to remove traces of genomic DNA. The purity and concentration of extracted RNA were checked in Nanodrop™ -2000 spectrophotometer (Thermo Fisher Scientific, USA) Subsequently, 5 ng of purified RNA was reverse transcribed to cDNA using Superscript II first strand cDNA synthesis kit (Invitrogen, CA).

Real time PCR quantification (qRT-PCR) of IL-1β and IFN-γ cytokine mRNA levels
Cytokine mRNA expression levels of interleukon-1β (IL-1β) and IFN-γ were analyzed by real -time PCR using Maxima SYBR Green qPCR kit (MBI Fermentas, USA) in diluted cDNA samples using Insta Q96™ real time PCR machine (Himedia, India). The guidelines of Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) were followed. Primer amplification efficiency was assessed for each gene from the standard curve generated by using serial tenfold dilution of transcribed RNA. Regression analysis of the Ct values of standard curve was done to calculate slope and amplification efficiency of the primers. The efficiency for the primers ranged from 94 to 96%. For real time PCR, each 10 μl reaction mixture contained 5 μl of 2X Maxima SYBR Green PCR Master Mix, 1 μl of cDNA, and gene specific primers (20 pmol/µl) listed in Table 2. Briefly, each reaction involved a pre-incubation step at 94ºC for 30 s, followed by 40 cycles of 95ºC for 30 s, 55ºC for 30 s and extension at 72ºC for 30 s. Subsequently melting curve analysis was performed to check the specific product. Each sample was run in triplicate with no template control (NTC). β actin was used as housekeeping gene for normalizing the expression data.

Statistical analysis
Humoral immune response data including IBD and ND titers were analyzed by one-way analysis of variance (ANOVA) with Tukey's post-hoc test to analyze the difference between mean titer between groups at different time intervals. Mean titers were considered significantly different when P ≤ 0.05. Statistical analysis was performed using SPSS v. 14. Body  Fan et al. (2013) weight gain, bursa to body weight ratio and bursal lesion scores were also analyzed by one-way ANOVA for any significant differences between groups. Expression levels of IL-1β and IFN-γ mRNA were calculated after normalizing with housekeeping gene and fold-changes in gene expressions were calculated as 2 −ΔΔ Ct (Livak and Schmittgen 2001). Comparison of fold changes in gene expression between groups were performed using General Linear Model in version 9.2 of SAS software with a P value < 0.05 considered significant.

IBD specific antibody titer
Mean maternal antibody level of day-old chicks was 3800 ± 356. In poly I:C and unimmunized control group the maternal antibody levels declined and reached to unprotective (< 396) levels from 21 st day of age (Fig. 1). However, the groups which received IBD vaccine either alone or in combination with Poly I:C induced seroconversion and protective antibody titers from 28-35 th day (Protective titer cutoff: 396). Among the immunized groups, intermediate IBD plus vaccine induced significantly (Mean titer 3912 ± 1385; P ≤ 0.05) higher levels of IBD specific antibodies on 35 th day of age than other vaccine groups; followed by intermediate plus co-administered with poly I:C (1481 ± 914). Intermediate IBD vaccine, bursaplex vaccine and their combination with poly I:C also induced higher and protective antibody titers on 35 th day of age. Poly I:C combination with intermediate and bursaplex vaccine induced better IBD specific antibody titers than the vaccine alone. However, for intermediate plus vaccine, poly I:C combination did not improve the titers.

HI titer
Significant differences (P ≤ 0.05) in mean HI titer were observed between different groups on 4 th and 5 th week of age (Fig. 2). Intermediate IBD and intermediate plus vaccines in combination with Poly I:C groups showed significantly lower HI titers on both 4 th ad 5 th week in comparison to other groups. Protective antibody titers were observed in all groups from 5 th week onwards. Control group that did not receive any ND vaccine showed waning of ND antibody titer from 2 nd to 3 rd week and become unprotective for ND from 4 th week onwards (data not shown).

Body weight gain, Bursa to Body weight ratio and bursal lesion score
Body weight gain calculated between the period of 7-days to 42 days of age during immunization trial showed significantly lower (P ≤ 0.05) in intermediate plus IBD and Intermediate plus IBD with Poly I:C group in comparison to other groups and control (Fig. 3). Bursa to body weight ratio between different groups ranged from 1.72 to 4.96 (Table 3). No significantly difference was observed among the different groups on different intervals. Histopathological bursal lesion scoring for different groups on 21 st day of age did not show any significant   (Table 4).

Cytokine mRNA expression levels
Fold changes in cytokines IL-1β and IFN-γ mRNA expression levels in spleen tissue of different groups on 14 th day of age are presented in Table 5. IL-1β levels were up-regulated in intermediate plus and bursaplex group in comparison to other groups. Whereas IFN-γ m RNA expression levels were upregulated in intermediate IBD with Poly I:C group than others.

Discussion
IBD is one of the re-emerging threats to poultry industry worldwide. IBD causes severe acute infections in young broilers leading to bursal atrophy, immunosuppression and mortality in the range of 30% and 60% in broilers and layers respectively (Eterradossi 2020). Although vaccines are available to combat the IBD infection, certain issues like maternal antibodies and induction of bursal damage by live vaccines exists. Poly I:C, a TLR3 agonist is a safe and effective adjuvant for improving vaccine induced immune response in birds and animals. Poly I:C binds with TLR3 and activates MyD88 independent pathway through TIR-domain containing adapter-inducing interferon-β (TRIF) and activating the downstream molecules IRF-3 and NF-κb and results in the production of type I IFN and inflammatory cytokines (Kawai and Akira 2010). Its immunomodulatory potential has been demonstrated with killed avian influenza vaccine, Marek's vaccine and ND vaccine in chicken (Kannaki et al. 2019). Poly I:C alone or in combination with Pam3CSK4, a TLR3 and 2 agonists respectively, alleviated hot IBD vaccine induced immunosuppression in chicken (Bashir et al. 2019).
In the present study, we evaluated the effect of poly I:C upon co-administration with different IBD vaccines in chicken. Humoral antibodies play a major role in the protection of IBDV infection. Positive correlation exists between the IBD antibody titer and the protection (Nakamura et al. 1994). The experimental chicks had intermediate levels of maternal antibodies on day-old. Maternally derived antibodies (MDAs) usually start wane after first week and becomes unprotective from 2 to 3 weeks of age without any vaccines (Le Gros et al. 2009). We also observed waning of MDAs to unprotective levels around 3 rd week in unimmunized control group and Poly I:C group. In the present study, all three vaccines namely intermediate, intermediate plus and bursaplex vaccine alone and in combination with Poly I:C induced seroconversion and protective IBD specific antibody titers at 35 days of age even in the presence of higher levels of maternal antibodies. Rebound of antibody titers were observed at 35 D of age, around two weeks after booster vaccination, which is generally observed with prime-boost live vaccines. It is in conformity with earlier studies, wherein the seroconversion was observed around 18 days post-vaccination (Wyeth and Chettle 1990;Bose et al. 2003). Among the vaccines studied, intermediate plus vaccine could induce significantly higher antibody levels than other vaccine groups, nevertheless with moderate bursal lesions and growth suppression indicated by lower body weight gain during the trial period. Body weight gain was significantly lower in intermediate plus vaccine group similar to earlier studies (Ashash et al. 2019).
All these vaccines are shown to induce protective antibody titers in the presence of maternal antibodies (MDA) (Sedeik et al. 2018). Immune-complex vaccine is a mixture of the intermediate plus strain and specific antibodies, that is released after MDA drops off. Moreover, these vaccines offer the practical advantage of hatchery administration as they are given at day-old by parenteral route (Haddad et al. 1997). Further, the recommended single dose avoids handling of birds frequently as in conventional vaccines. We observed poly I:C co-administration improved the IBD specific immune response in intermediate and bursaplex IBD vaccine at 35 day of age, however, it is not the case in intermediate plus vaccine. Replication of vaccine virus in bursa and induction of bursal damages were directly correlated with their induction of humoral immunity (Rautenschlein et al. 2005). However, TLR agonist might reduce the replication of vaccine virus by upregulation of type I IFNs that are antiviral in nature (Cai et al. 2012). The effect may be more pronounced with hot vaccine compared to mild to moderate vaccines or immune complex vaccine that are slowly released. In an earlier study also, similar trend was observed and authors speculated the same (Bashir et al. 2019). Intermediate plus and intermediate IBD vaccines induce transient suppression of ND antibody titers (Rautenschlein et al. 2007). Transient suppression in ND titers was observed in the present study too around 35 th day of age supporting the earlier notion. In vitro stimulation of chicken PBMCs and in vivo administration of poly I:C upregulated type I IFN transcripts (Karpala et al. 2008;Parvizi et al. 2012). Upregulation of IFN-γ was observed in spleen tissue of poly I:C immunized groups, however, it was not significantly different from IBD vaccine group. Upregulation of IFN-γ along with other genes IL-6, MHC-II and CD-4 were observed in spleen tissue of ducks immunized with H9N2 avian influenza inactivated vaccine adjuvanted with Poly I:C (Zhang et al. 2017). In addition, upregulation of IFN-γ was reported in chicken splenocytes stimulated with Poly I:C (Cornelissen et al. 2012). In summary, poly I:C co-administration with different IBD vaccines showed that it improved specific immune response in intermediate IBD vaccine and bursaplex along with comparatively less immunosuppression and bursal damage. Hence, use of poly I: C along with these vaccinations would be beneficial to improve the vaccine response in broilers. Single injection of bursaplex with poly I:C is practically more useful for attaining better vaccine induced immunity.