Combined H5ND inactivated vaccine protects chickens against challenge by different clades of highly pathogenic avian influenza viruses subtype H5 and virulent Newcastle disease virus

Aim: The aim of the current study was to evaluate the efficacy of a trivalent-inactivated oil-emulsion vaccine against challenge by different clades highly pathogenic avian influenza (HPAI) viruses including HPAI-H5N8 and the virulent genotype VII Newcastle disease virus (NDV) (vNDV). Materials and Methods: The vaccine studied herein is composed of reassortant AI viruses rgA/Chicken/Egypt/ME1010/2016 (clade 2.2.1.1), H5N1 rgA/Chicken/Egypt/RG-173CAL/2017 (clade 2.2.1.2), and “NDV” (LaSota NDV/CK/Egypt/11478AF/11); all used at a concentration of 108 EID50/bird and mixed with Montanide-ISA70 oil adjuvant. Two-week-old specific pathogen free (SPF) chickens were immunized subcutaneously with 0.5 ml of the vaccine, and hemagglutination inhibition (HI) antibody titers were monitored weekly. The intranasal challenge was conducted 4 weeks post-vaccination (PV) using 106 EID50/0.1 ml of the different virulent HPAI-H5N1 viruses representing clades 2.2.1, 2.2.1.1, 2.2.1.2, 2.3.4.4b-H5N8, and the vNDV. Results: The vaccine induced HI antibody titers of >6log2 against both H5N1 and NDV viruses at 2 weeks PV. Clinical protection against all HPAI H5N1 viruses and vNDV was 100%, except for HPAI H5N1 clade-2.2.1 and HPAI H5N8 clade-2.3.4.4b viruses that showed 93.3% protection. Challenged SPF chickens showed significant decreases in the virus shedding titers up to <3log10 compared to challenge control chickens. No virus shedding was detected 6 “days post-challenge” in all vaccinated challenged groups. Conclusion: Our results indicate that the trivalent H5ND vaccine provides significant clinical protection against different clades of the HPAI viruses including the newly emerging H5N8 HPAI virus. Availability of such potent multivalent oil-emulsion vaccine offers an effective tool against HPAI control in endemic countries and promises simpler vaccination programs.


Introduction
Avian influenza (AI) viruses belong to the family Orthomyxoviridae, genus Influenza virus. To date, 18 hemagglutinin (HA) and 11 neuraminidase subtypes have been reported [1]. During the past decade, poultry industry in Egypt was challenged by exposure to different AI virus subtypes, including the highly pathogenic AI (HPAI) H5N1, low pathogenic AI H9N2, and HPAI H5N8 [2][3][4]. Since AI infections emerged in mid-February 2006, HPAI H5N1 showed several mutations and different sub-clades of the virus. The presence of the virus under vaccine immune pressure in vaccinated birds accelerated its mutation rate [5]. Thus [6][7][8].
In the meantime, Newcastle disease (ND) continues to cause serious problems and high economic losses in poultry in Egypt. ND virus (NDV) is an avian paramyxovirus serotype 1 belonging to the genus Avulavirus, subfamily Paramyxovirinae, family Paramyxoviridae [9]. In Egypt, NDV has been reported since 1948 [10] then the country became endemic. Despite adopting vaccination programs that Available at www.veterinaryworld.org/Vol.12/January-2019/14.pdf include both live attenuated and inactivated vaccines, the NDV continues to impact the Egyptian poultry industry [11,12]. The NDV outbreaks are commonly associated with the virulent NDV Genotype VII (vNDV); however, the continuous outbreaks of vNDV were also attributed to poor flock immunity and improper vaccination practices [13,14].
Though over 24 commercial inactivated AI H5 vaccines are licensed for use in poultry in Egypt, the genetic mismatch with poor reactivity of these vaccines to the currently circulating viruses has led to the failure of the HPAI vaccination strategy among poultry in Egypt [3,15]. The spread and co-circulation of different HPAI-H5N1, HPAI-H5N8, and vNDV viruses further complicated the epidemiological situation and control strategies in Egypt with increased economic losses in poultry production. Hence, combined vaccines with matching strains were suggested to facilitate the vaccination programs and minimize the economic losses.

Ethical approval
Experimental procedures were reviewed and approved by the Animal Care and Use Committee (#171101E001) of the Middle East for Veterinary Vaccines (ME VAC) Company, Egypt.

Vaccine formulation and testing
The vaccine seed viruses were propagated through inoculation of SPF embryonated chicken eggs through allantoic sac route inoculation. Inoculated eggs were incubated at 37°C for 72 h. Harvested allantoic fluids were clarified with a low-speed centrifuger at 2000 rpm for 10 min at 4°C. The viruses were titrated in 10-day SPF embryonated chicken eggs, and then, hemagglutination (HA) titers and the egg infective dose 50 (EID 50 ) were calculated [18,19]. The viruses were inactivated using 0.2% formalin (Sigma-Aldrich, Inc., Germany) and the inactivation was verified by passaging the inactivated antigens into 10-day-old SPF embryonated chicken eggs for three successive passages. The aqueous phase of the vaccine was formulated to contain doses of 10 8 EID 50 /dose from each virus strain and then mixed with Montanide ISA 70 VG adjuvant (SEPPIC® SA, France) at room temperature with a ratio of 70/30 adjuvant/antigen (v/v). Vaccine physicochemical criteria, safety, and sterility were evaluated according to the SEPPIC Montanide ISA 70 VG technical manual and the OIE standards [19].

Chicken experiments
In all experiments, White Leghorn SPF chickens kept in biosafety level III chicken isolators were used.
Hemagglutination inhibition (HI) antibody titers were monitored weekly by HI test. Sera of the vaccinated SPF chickens were tested using a clade 2.2.1.2 HPAI-H5N1 antigen (A/duck/EG/M2583D/2010) and a clade 2.3.4.4b HPAI-H5N8 antigen (A/common coot/EG/CA285/2016/H5N8) according to the OIE manual [19]. Virus challenge was conducted 4 weeks post-vaccination (PV) intranasally using 10 6 EID 50 /0.1 ml of the AI-H5 and vNDV challenge viruses separately. The chosen challenge dose was based on the standard dose being used in Egypt to evaluate all HPAI-H5 and NDV vaccines submitted to the Central Laboratory for Evaluation of Veterinary Biologics, Egypt. Challenged chickens were observed daily for 10 days post-challenge (dpc) for virus shedding and the presence of clinical signs, morbidity, and mortality (Table-1).

Challenge virus shedding detection
Tracheal swabs were collected from all challenged birds in 1 ml of sterile PBS at 3, 6, and 10 dpc to monitor virus shedding titers. Swab samples were vortexed and centrifuged at 2000 rpm for 10 min at 4°C. Supernatants were used for virus titration in 10-day-old SPF embryonated chicken eggs, and EID 50 /ml was calculated [18].
The cloacal samples were collected, but due to the multiple challenges and large data, we presented the data of tracheal swabs only, especially we did not find significant differences in both types of samples.

Statistical analysis
Differences in the virus shedding titers at 3 dpc among different groups were calculated using one-way ANOVA with Tukey's post-test was performed using GraphPad Prism version 5.00 (GraphPad Software, San Diego, California, USA).  (Table-2).

Discussion
Inadequate biosecurity and relying on vaccination as the only control strategy for the HPAI-H5N1 virus in Egypt led to frequent mutations of the virus and evolution of different subclades, especially with the use of mismatch vaccine strains [3,20]. In late 2016, the epidemiology of AI in Egypt exhibited a substantial change due to the emergence of HP H5N8 in wild birds [4] followed by widespread of the virus in commercial poultry [21,22]. Moreover, other poultry pathogens including vNDV and infectious bronchitis virus became more frequently diagnosed in poultry in Egypt [23].
An immunization strategy depending on using bivalent and multivalent vaccines containing whole inactivated viruses has been advocated before to control several avian pathogens [24]. The objective of the current study was to evaluate the immunogenicity and protective efficacy of a trivalent-inactivated oil-emulsion H5ND vaccine against different clades HPAI subtype H5 and vNDV viruses following a single-dose vaccination regimen. The vaccine contained two reassortant H5N1 viruses representing both 2.2.1.1 and 2.2.1.2 clades registered in Egypt and a LaSota-like NDV strain.
In the vaccination challenge experiments, the vaccine-induced HI antibody titers by 2 weeks post-vaccination against both HPAI-H5N1 and NDV antigens. However, the HI antibody titers against HPAI H5N8 heterologous antigens were only detectable at very low titers 3 weeks PV. By 4 weeks PV, the anti-HPAI H5N8 antibody titers were ≥5.0 log2. These relatively low antibody titers were rather expected due to the genetic and antigenic differences in the HA between HPAI H5N1 and H5N8 viruses isolated in Egypt [25,26].
The protective efficacy of the developed H5ND vaccine was evaluated by challenge of vaccinated birds with 10 6 EID 50 /100 µl of the different clades HPAI AI viruses or vNDV at 4 weeks post-vaccination (PV). All non-vaccinated chickens showed severe clinical signs and 100% mortality by 3 and 4 dpc in all HPAI and vNDV virus challenge groups, respectively. Although few reports indicated that HPAI H5N8 viruses produce asymptomatic disease in geese and ducks with prolonged virus shedding [27], increased virus adaptation to chickens was observed within the HPAI of 2.3.4.4 clade viruses [28,29]. This was supported by the finding that members of the HPAI H5N8 challenge group showed typical AI signs and 100% mortality rate.
There was a significant (p<0.01) reduction in both virus shedding titers and the number of active virus shedders in all challenged groups, including those receiving HPAI H5-2.3.4.4b, despite the low HI antibody titers against HPAI-H5N8-2.3.4.4 clade virus. In contrast, Yuk et al. [32] showed that while commercial clade 2.3.2 H5 vaccines protected chickens against HPAI-H5N8 virus challenge, they failed to prevent virus shedding. It is worthwhile to note that the seed virus of clade 2.3.2 viruses showed 84.6-87.7% amino acid identities with the HPAI H5N8 challenge virus, compared to 89. .4% in the current study.
In another study, the efficacy of commercial vaccines available in Egypt was studied. Most of the  I  I  I  I  I  I  119 K T  T  T  T  T  T  198  I  I  I  I  I  I [33][34][35][36][37]. HPAI=Highly pathogenic avian influenza, EG=Egypt, CK=Chicken, DU=Duck, PBS=Phosphate buffered saline  [25,38,39]. Moreover, previous reports indicated that changes in HA amino acids may not correspond to both clade and subclade grouping and the protective efficacy of vaccine preparations [35].
Although the protection rate (≥90%) against HPAI-H5N8-2.3.4.4b is acceptable for a combined vaccine and the vaccine could be regarded as effective, this finding cannot be extrapolated to the field conditions considering the first 3 weeks gap observed until a relatively high cross antibody titers are detected. The complicated poultry field situation and inadequate biosecurity measures may reduce the efficacy   [25].

Conclusion
The trivalent H5ND vaccine was found to be immunogenic, and it provides protection in SPF chickens against HPAI H5 AI and virulent ND infections. The current study also demonstrates that the multivalent oil-emulsion vaccines could be a useful strategy to simplify the vaccination programs for controlling multiple poultry viruses, especially in endemic countries. ----------10 ----------PBS negative control

All
Not detected 1 dpc=Days post-challenge-additional days in challenge control groups are the days at which deaths of infected birds occur; 2 Virus shedding titers at the same column at 3 dpc and followed by different superscript small letters indicate significant differences (p≤0.05); 3 NT=Not tested, 4 All birds died by 3-4 dpc, HPAI=Highly pathogenic avian influenza, PBS=Phosphate buffered saline