Preparation and Evaluation of Immune Complexes as Vaccine Candidate for Fowl Adenovirus Infection associated with Hydropericardium Syndrome in Domestic Fowl

Immune complex (Icx) vaccine is an antigen-antibody complex which will engage all immunocytes with Fc binding capabilities, enhance early maturation and B memory cell formation. In vitro preformed Icx have been shown to be 100 times more efficient in inducing humoral immune response in vivo than the native protein antigen. In present study, immune complexes were prepared by mixing varying quantities of HPS virus with different concentrations of egg derived immunoglobulins (IgY). The efficacy of immune complex formulations as vaccine was checked in broiler birds. The Icx vaccinated birds exhibited protection in the range of 53.4% to 66.7% following challenge with virulent HPS virus. Birds vaccinated with 40 and 80 units of IgY with 100 TCID50 HPS virus did not shed virus in feces following challenge and induced better antibody response. No significant change in body weight gain was observed between survived birds from vaccinated and control groups. The study showed that immune complex vaccines significantly reduced the mortality and stopped the excretion of the virus in feces thus afforded protection and prevented the horizontal transmission of virus by oral-fecal route.

Hydropericardium syndrome (HPS), primarily a disease of broiler birds of 3-6 weeks of age, is caused by Fowl Adenovirus-4 (Mazaheri et al., 1998;Kataria et al., 2013). Disease has also been reported in 7-day-old broiler birds. The disease is characterized by sudden onset of death, with mortality up to 80% (Shane, 1996;Asthana et al., 2013;Kataria et al., 2013). The accumulation of straw/amber coloured jelly-like fluid in the pericardial sac mainly characterizes the disease. Other predominant lesions include enlarged and discoloured liver with foci of haemorrhage and/or necrosis and enlarged kidneys with distended tubules (Abe et al., 1998;Roy et al., 2004). Transmission of the virus occurs horizontally by oral-fecal route (Akhtar, 1995) and vertically to the offspring (Nagy et al., 2006). Inactivated vaccines produced in primary cell culture systems have shown promising results against HPS in broilers (Gupta et al., 2005;Asthana et al., 2013, Shah et al., 2017. However, inactivated vaccines have disadvantages like low level of immunity, requirement of additional immunizations, increased dependence on adjuvant and expensive production. Maternal antibodies neutralize the vaccine virus and thus play major role in vaccination failure. Therefore, there is a need for development of safe live virus vaccines which can boost humoral immune response at early period of chick life and at the same time shield the vaccine virus from maternal antibodies. The capacity of immune complexes to augment antibody (Ab) responses is well established. The enhancing effects of immune complexes have been attributed mainly to Fc-mediated adjuvant activity (Hioe et al., 2009). Immune complex vaccine improve the safety due to very low antigen involved which is complexed with antibody in immune complex vaccine, protective even in the face of maternal antibodies, offer long lasting immunity, lower effective dosing schedule and eliminate use of adjuvants (Haddad et al., 1994;Schat et al., 2011). Immune complex vaccine can be administered to day old chicks or in ovo; in absence of maternal antibodies it starts produce immune response by the age chicks susceptible to disease. In presence of maternal antibodies immune complex antibody is able to shield the vaccine virus and release slowly when maternal antibodies decline (Coletti et al., 2001;Chansiripornchai et al., 20119;Mazengia, 2012).
Present research work describes formulation of an immune complex vaccine, using chicken egg derived antibodies, against HPS virus infection in domestic fowl and level of protection it afforded in broiler chicks.

Virus isolates
A field isolate of FAV4 (HPS-G) (Asthana et al., 2011) was used to hyper immunize layer birds for production of egg yolk antibodies (IgY). Another field isolate of FAV4 (HPS-K) (Asthana et al., 2011) was used for challenge studies. Both isolates were propagated and titrated in chicken embryo liver cell culture and maintained in Department of Veterinary Microbiology, College of Veterinary and Animal Sciences, G.B. Pant University of Agriculture & Technology, Pantnagar.

Egg yolk antibodies (IgY)
IgY were raised against HPS virus in layer birds as per the method described by Rani et al. (2012). IgY were purified from eggs laid between days 35-40 post immunization using chicken IgY purification kit (Bangalore Genei, India) as per manufactures recommendations.
IgY thus obtained were titrated by virus neutralization test (beta procedure) as described by Haddad et al. (1994). Briefly, serially diluted purified HPS IgY suspension was tested against a fixed dose of HPS virus (100 TCID 50 ) which was incubated and residual virus producing cytopathic effects was assayed using Chicken embryo liver cell (CEL) culture. The mean neutralization dose (ND 50 ) of IgY suspension was calculated according to the method of Reed and Muench (1938). One unit of IgY activity was taken as the reciprocal of dilution that protects 50% of the CEL cells from the cytopathic effect of HPS virus.

Preparation of HPS immune complex (HPS-Icx) vaccine
The HPS-Icx vaccine was prepared as per the method of Whitfill et al. (1995) with some modifications. HPS-G isolate having a titre of 6.5 log 10 TCID 50 /ml was used to prepare HPS-Icx vaccine. The neutralizing antibody titre of purified IgY was 2500 units/50µl, which was suitably diluted in serum free medium to get 80 and 40 units of IgY per 50µl.
In experiment 1, two formulations of each dose (100μl) of the complex vaccine was prepared by mixing 50µl each of 100 TCID 50 /50µl of HPS virus antigen with 40 and 80 units of HPS IgY/50µl.
In experiment 2, two formulations of each dose (100μl) of the complex vaccine was prepared by mixing 50 µl each of 200 TCID 50 /50μl of HPS virus antigen with 40 and 80 units of HPS IgY/50μl. Vaccine formulations were incubated at room temperature for one hour with gentle shaking every 15 minutes. These formulations were administered to one week old broiler chickens.

Experimental evaluation of HPS-Icx Vaccine
Ninety-day-old broiler chicks were procured from local commercial hatchery and randomly assigned to 6 groups of 15 chicks each. Chicks in Group I and II were vaccinated at one week age with 100 TCID 50 HPSV with 40 and 80 units of IgY, respectively. Group III and IV chicks were vaccinated at one week age with 200 TCID 50 HPSV mixed with 40 and 80 units of IgY. Chicks of Group V were unvaccinated, challenged at 21 st day of age and served as positive control and Group VI chicks were unvaccinated and unchallenged and served as negative control group.
All 15 birds in group I-V were challenged intramuscularly with 1.0 ml of HPS-K virus isolate (7.5 log 10 TCID 50 /ml) at 14 day post vaccination.
All the vaccinated groups were observed for mortality, body weight changes, faecal shedding of virus and level of antibody titers post vaccination at weekly interval. Mortality patterns of different groups were obtained by calculating total no. of birds died in each group post challenge. Body weight changes of birds in different groups following post challenge were obtained by taking body weight of all the birds at weekly interval till the end of experiment. The antibody levels were assessed by indirect ELISA (2003). Fecal samples were collected, with the help of sterilized swabs and then diluted in HBSS (pH 7.2), and stored at -20 o C until tested by sandwich ELISA for the presence of viral antigens.

Detection of viral antigens in fecal samples
HPS virus antigens in fecal samples were detected by sandwich ELISA (Saifuddin and Wilks, 1991). Briefly, Wells of ELISA plate were coated with 100µl of rabbit anti-HPSV sera diluted to 1:400 in coating buffer (carbonate buffer, pH 9.6) and incubated at room temperature for 90 minutes. Coated wells were washed 5 times each with washing buffer for 5 minutes and tapped thoroughly. The unreacted sites of the wells were blocked with 100 µl of 5% skimmed milk powder in PBS-T for 90 minutes at room temperature and washed as mentioned above.
Then test fecal samples, known negative fecal and tissue samples were added in 100µl volume in triplicate. Following incubation at room temperature for 90 minutes the plate was washed and tapped thoroughly. Then chicken anti-HPSV sera was diluted (1:200) and dispensed at 100 µl per well followed by incubation at room temperature for 90 minutes. The plate was washed and anti-chicken HRPO conjugates 1:500 dilution was dispensed @ 100 µl per well and incubated at room temperature for 90 minutes. After washing and tapping, 100µl of freshly prepared substrate solution ortho-phenylenediamine dihydrochloride (OPD) in citrate buffer (pH 4.6) was added and incubated at room temperature for 30 min in dark. The enzyme substrate reaction was stopped by adding 100µl of 1N H 2 SO 4 to each well and optical density of substrate reaction was measured spectrophotometrically at 492 nm wavelength in ELISA reader. The results of the ELISA were interpreted by comparing 6 negative controls fecal samples with that of test fecal samples collected from birds survived in the Group I and II. The mean absorbance value and standard deviation (SD) were calculated for negative fecal samples. A test sample was considered positive if the absorbance value was greater than the mean value of the negative fecal samples plus 3 x standard deviation (Saifudin and Wilks, 1991).

Production of IgY and preparation of Immune complexes
In present investigation four white leghorn layer birds were used for production of IgY against a local isolate HPS virus. Use of hens represents both refinement and reduction in animal use for large scale production of polyclonal antibody (Schade et al., 2011) which can be employed for therapeutic and diagnostic purposes (Lemamy et al., 1999). Rani et al. (2012) used purified HPS IgY for treatment of experimentally induced HPS in broilers. Purification of IgY from yolk is convenient and simple using different purification methods. IgY purification kits are also available commercially for obtaining high yield and purity.
Virus Neutralization Test was carried out using CEL cells as indicator system to calculate neutralizing antibody titre of IgY and it was observed that IgY was efficient to neutralize the virus in vitro and form immune complex which prevent the CEL cells from cytopathic effects of HPS virus. An Immune complex vaccine against HPS virus prepared by mixing cell culture HPS virus antigen with IgY against HPS virus at different formulations. Vaccination was carried out in chicks of 1 week age, so that it can produce immunity by the time birds become susceptible to HPS virus infection at the age of 3-5 weeks. Whitfill et al. (1995) in their studies administered IBD immune complex vaccine subcutaneously into nape of day old chicks and also in ovo into 18 day old embryos. Gupta et al. (2005) reported vaccination with inactivated cell culture vaccine to 10 day old chicks, so as to protect birds from Hydropericardium syndrome when they reached market age.

Experimental evaluation of HPS-Icx Vaccine
Single dose of 80 units of HPS IgY complexed with 100 TCID 50 /50μl of HPS virus demonstrated protective immunity against challenge virus at 14 day post vaccination. There was 66.7% protection (Group II) compared to 53.4% of protection offered by 40 units of IgY/50μl (group I birds). Our findings were in correlation with Whitfill et al. (1995) studies on Immune complex vaccine against IBD reported that IBDV immune complex vaccine prepared by 100 EID 50 /50µl mixed 20-80 units of bursal disease antibody (BDA) showed increase antibody response 3 days post challenge with IBDV (31 st day post vaccination). In our study peak antibody response at 1 week post challenge in groups I and II, demonstrated protective immunity against the challenge virus. Kaur et al. (1997) showed that birds vaccinated with 1000 TCID 50 /0.2 ml live adenovirus inoculated at one week of age at dose rate of 1ml per chick showed elevated neutralizing antibody titre 1 week post challenge (4 th week after vaccination). These findings show the immunogenic response of live virus as a candidate for vaccine development against IBH-HPS.

Clinical signs and mortality
All the birds in vaccinated and unvaccinated groups were active and healthy up to 14 days post vaccination, showing no signs of illness. Following challenge, birds did not show any apparent clinical signs till 24 hours. After that few birds in vaccinated group and most of the birds in unvaccinated challenge group developed greenishyellow diarrhea, by 36 hours some of the birds in both vaccinated group and unvaccinated challenge group were dull, depressed, debilitated. Some birds exhibited ruffled feathers, pale skin and attained a crouching posture by resting their chest and beak on ground with closed eyelids. Some birds struggled vigorously and died. Clinical signs were more prominent and extensive in birds died on 3 rd and 4 th day post challenge (d.p.c.). Hydropericardium was not observed in the dead group I and II birds on 3 rd d.p.c.
However, 4 out of 8 dead group III birds showed presence of 3-5 ml straw coloured fluid in pericardial sac. Similarly, 5 out of 7 dead group IV and 6 out of 08 group V dead birds showed presence of 6-8 ml pericardial fluid. Birds died on 4 th and 5 th d.p.c. in groups I-V showed accumulation of nearly 8-10 ml straw coloured fluid having thick consistency in pericardial sac. It was also observed that amount of pericardial fluid was less in vaccinated groups compared to unvaccinated challenge group. In most of the cases epicardium showed congestion and engorgement of blood vessels. There were no clinical signs in birds survived in both vaccinated groups. All survived birds in unchallenged control groups did not show clinical signs and were healthy throughout the experiment. Livers of all dead birds were friable, swollen with rounded ends and mottled with multifocal necrotic areas. Some birds revealed severe congestion and haemorrhages, while others exhibited pale livers with large areas of necrotic foci. Kidneys in most of dead birds were swollen, congested and hemorrhagic. Intestine in most of the dead birds were congested and haemorrhagic. Spleen in most of the birds showed congestion, necrotic foci and enlargement. Bursa of fabricius was enlarged in most of the birds died; however, no abnormality could be detected on cut surface as there was no evidence of edema or hemorrhages.
Peak mortality was noticed on 3 rd d.p.c. and mortality continued up to day 6 post challenge. Mortality rates in group I, II, III, IV and V were 46.6%, 33.3%, 93%, 100% and 93%, respectively. No mortality was recorded in group VI throughout the experiment (Table 1). Hydropericardium syndrome is highly infectious and devastating disease of broiler birds causing high mortality in the age group of 3-5 weeks, resulting in heavy economic losses to poultry industry (Kataria et al., 2013, Asthana et al., 2013. High mortality in groups III and IV might be because of high amount of virus leading to improper immune complex formation leaving most of the virus in free State. This free virus may be readily metabolized by body so little exposure of antigen to immune system resulting low antibody formation. Immune complex formation to elicit immune response the amount of virus and antibody should be in proper proportion as showed by Pokric et al. (1993) that immune complexes generating effective immune stimulation were prepared at slight antigen excess or at equivalence. High mortality in groups III, IV and V, may be attributed to the high virulence of challenge virus, absence of protection due to low or no antibody titre. Some strains of broiler birds and route of inoculation also affect the pathogenesis of disease. Roy et al. (2004) reported that some B1 white strains of broilers were highly susceptible and recorded mortality rates of 97%, 92.3%, where as in colored B2 broilers, mortality rate was low, ranging from 15.7% to 23.3% in 3 rd and 5 th week broilers. Route that we used was intramuscular which produced high mortality and reduces Incubation period and duration of disease (Abdul-Aziz et al., 1995). Clinical signs, gross lesions and incubation period were similar to that reported by other investigators (Gowda and Satyanaryana, 1994;Roy et al., 2004, Asthana et al., 2013 in experimental and natural HPSV infection. Incubation period of 48-72 hours was reported by Aleiv et al. (1997) and duration of disease of 7 day was observed in our study which was in agreement with findings of Rani et al. (2012).

Humoral immune response
Humoral immune response of vaccinated birds was determined by indirect ELISA. In group I, chicks showed an immune response with low antibody titre at 7 days post vaccination (d.p.v.), which increased by day 14 and attained peak by 21 d.p.v., a week following challenge. Later, the titre decreased to half of the peak titre on 28 d.p.v. By 35 th d.p.v. it reduced to about 20% of the peak titre. In group II, antibody titre showed similar pattern but was slightly lower than that of group I (Fig. 1). Antibody titres in group I chicks were slightly higher throughout the experiment when compared to group II.
There was an increase in antibody titre post vaccination and peak titre was attained a week following challenge infection, which acts as booster to elicit secondary immune response. Gupta et al. (2005) reported that peak antibody titre was attained 14 days following post challenge in birds vaccinated with HPS inactivated cell culture vaccine. In comparison with findings of Gupta et al. (2005) the immune response in this experiment was attained early and high antibody titre was attained within a week post challenge. These findings suggested assumption that early surge in peak antibody titre following challenge virus may be attributed to presence of B-memory cells and formation of immune complexes by preformed antibodies with challenge virus which help in early selection and maturation of B cells to antibody secreting plasma cells. Marusic et al. (1991) suggested that antigen binding into the Immune complex could play an important role in enhancing specific cellular immune responses during the secondary contact with small amount of antigen. Antibodies already present in the circulation of primed animal would rapidly form immune complexes with antigen when it enters the body. This would allow for faster and more efficient binding of antigen to the Antigen presenting cells and consequently stronger stimulation of specific T cells when limiting amounts of antigen enter the body. These findings suggest that there is need for a booster dose required to boost the immune response before challenging with virus. In the same context Klaus (1978) demonstrated the adjuvant effect of antibody in Immune complex is mainly in priming B memory cells and therefore, triggering of memory cells to antibody production would require a further dose of antigen. Klaus and Humphrey (1986) opined that immune complexes generate memory more rapidly than antigen alone and antibody in the immune complex involves in the generation of B memory cells in germinal centers which are the birth places of B memory cells. Klaus (1978) concluded that In vitro preformed Icx have been shown to be 100 times more efficient in inducing humoral immune responses in vivo than the native protein antigen. They also demonstrated the adjuvant effect of antibody is manifested priming B memory cells and suggested that triggering of memory cells to antibody production could require a further dose of antigen. From various investigations and this study it is clear that immune complexes induce B memory cells and triggering of which require a booster dose, which enhances secondary immune response to protect birds from high level of infection.

Detection of viral antigens in fecal samples
All the fecal samples collected from birds of group I and II were screened for presence of viral antigens by sandwich ELISA.
O The route of transmission of HPS virus in poultry birds is feco-oral (Akhtar, 1995) and the fecal samples tested from vaccinated Group I & II by sandwich ELISA were negative for presence of HPS virus, which is important from point of view of horizontal transmission of virus. The absence of virus in fecal sample might be due to development of humoral immune response in vaccinated birds of group I and II. Whereas, unvaccinated birds from group V excretes virus in fecal sample from 3 rd day post challenge when peak mortality was noticed. Rani et al. (2012) noticed similar findings in experimental passive immune therapy using purified IgY against HPS in broilers, detected HPS virus in feces on 3 rd day post infection. Kaleta et al. (1980) showed that development of neutralizing antibodies coincides with cessation of virus excretion in Egg Drop Syndrome disease. Hwang et al. (1980) correlates the cessation of virus excretion with development of humoral and local immunity by appearance of neutralizing antibodies in adenoviral infection.

CONCLUSION
This is the first report that describes preparation of an immune complex vaccine against hydropericardium syndrome in domestic fowl. Present study also showed that egg derived antibodies (IgY) can be used as substitute for serum IgG. Immune complex vaccine significantly reduced the mortality and stopped the excretion of the virus in feces. However, additional studies using maternal antibody-positive chicks in combination with in-ovo vaccination along with methods to increase the protection level will be needed to determine if immune complex vaccines will be useful to protect commercial chickens.