Novel Recombinant Newcastle Disease Virus-Based In Ovo Vaccines Bypass Maternal Immunity To Provide Full Protection From Early Virulent Challenge Part 1

Abstract:

Newcastle disease (ND) is one of the most economically important poultry diseases. Despite intensive efforts with current vaccination programs, this disease still occurs worldwide, causing significant mortality even in vaccinated flocks. This has been partially attributed to a gap in immunity during the post-hatch period due to the presence of maternal antibodies that negatively impact the replication of the commonly used live vaccines. In ovo vaccines have multiple advantages and present an opportunity to address this problem. 

Currently employed in ovo ND vaccines are recombinant herpesvirus of turkeys (HVT)-vectored vaccines expressing Newcastle disease virus (NDV) antigens. Although proven efficient, these vaccines have some limitations, such as delayed immunogenicity and the inability to administer a second HVT vaccine post-hatch. The use of live ND vaccines for in-ovo vaccination is currently not applicable, as these are associated with high embryo mortality. In this study, recombinant NDV-vectored experimental vaccines containing an antisense sequence of avian interleukin 4 (IL4R) and their backbones were administered in ovo at different doses in 18-day-old commercial eggs possessing high maternal antibodies titers. The hatched birds were challenged with virulent NDV at 2 weeks-of-age. Post-hatch vaccine shedding, post-challenge survival, challenge virus shedding, and humoral immune responses were evaluated at multiple time points. Recombinant NDV (rNDV) vaccinated birds had significantly reduced post-hatch mortality compared with the wild-type LaSota vaccine. 

All rNDV vaccines were able to penetrate maternal immunity and induce a strong early humoral immune response. Further, the rNDV vaccines protected from clinical disease and significantly decreased virus shedding after early virulent NDV challenge at two weeks post-hatch. The post-challenge hemagglutination-inhibition antibody titers in the vaccinated groups remained comparable with the pre-challenge titers, suggesting the capacity of the studied vaccines to prevent efficient replication of the challenge virus. Post-hatch survival after vaccination with the rNDV-IL4R vaccines was dose-dependent, with an increase in survival as the dose decreased. This improved survival and the dose-dependency data suggest that novel attenuated in ovo rNDV-based vaccines that can penetrate maternal immunity to elicit a strong immune response as early as 14 days post-hatch, resulting in high or full protection from virulent challenge, show promise as a contributor to the control of Newcastle disease.

In addition to human vaccination, people can also take food and supplements. Cistanche contains a variety of chemical substances, such as polysaccharides, steroids, alkaloids, flavonoids, etc. These substances have a significant role in regulating the immune system. Among them, polysaccharides can promote the activation and proliferation of immune cells and increase the body's immunity; steroids can regulate the function of immune cells and enhance the immune response; alkaloids and flavonoids can resist oxidation, anti-inflammatory, antibacterial, and further enhance immunity. Therefore, Cistanche has a significant immune regulation effect and can be used as health food and medicine to improve immunity.

Click health benefits of cistanche

1. Introduction

Newcastle disease (ND), a devastating poultry disease that can reach 100% mortality in naïve birds, is caused by virulent strains of Newcastle disease virus (NDV) [1]. This virus species recently renamed Avian orthoavulavirus 1, is a member of the family Paramyxoviridae [2,3]. Newcastle disease viruses have a single-stranded, non-segmented, negative-sense RNA genome encoding for at least six structural proteins (30—nucleoprotein [NP]— phosphoprotein [P]—matrix protein [M]—fusion protein [F]—hemagglutinin-neuraminidase [HN]—and large RNA-polymerase [L]—50 ) and comprising one of three genome sizes (15,186, 15,192, and 15,198 nucleotides) [4,5]. Newcastle disease is considered the third most important poultry disease worldwide [6] and remains endemic in many countries throughout Asia, Africa, and the Americas [7].

The control of ND includes strict biosecurity to prevent the introduction of virulent NDV (vNDV) onto poultry farms [8,9] and proper administration of vaccines [10]. However, the multiple ND outbreaks occurring worldwide [11] are suggestive that the current vaccines and vaccination procedures are not fully efficacious. In addition to the conventional live and inactivated vaccines, multiple concepts for ND vaccination have been explored in the last two decades, including vectored, antigen–antibody complex, virus-like particle, and toll-like receptor ligand vaccines, among others [1,8,9]. Several avian vaccines, engineered to co-express immunostimulatory cytokines, have also been suggested to improve protective immunity [12]. Another approach that has been explored is the use of antigenically matched vaccines. Although all NDVs are regarded as members of a single serotype [3,13], there is antigenic variation between strains [14]. Previous studies with chickens of different ages have demonstrated that chickens vaccinated with strains that were antigenically matched to the challenge virus shed significantly less amount of the challenge vNDV than the amount excreted by chickens vaccinated with heterologous vaccines [15–17]. Of note, passively transferred maternal anti-NDV antibodies, although protecting chickens during the crucial early weeks of life, interfere with the development of host immunity following vaccination [18].

In ovo vaccination is an approach that delivers multiple advantages—for example, significant cost reduction, standardization, mass vaccine application, automated vaccination at the hatchery, uniform delivery of the vaccine in each egg, and no stress to birds [19–21]— and is an attractive immunization method for the poultry industry [22]. Between 80% and 90% of the broiler chickens grown in the U.S. are currently ovo-vaccinated two to three days before hatch [23,24], and the automation of the process allows for up to 70,000 eggs to be vaccinated per hour [23]. 

Globally, this vaccine administration method is employed in 26 out of the 30 leading poultry production countries in the world [23]. A few in-ovo vaccines against ND are currently available, but they are all generated using non-NDV vectors. Fowlpox virus (FPV) vector-based vaccines expressing the NDV F and HN proteins applied in ovo have been shown to protect poultry from a challenge with vNDV [25,26]. 

However, recombinant FPV-ND vaccines are not widely used due to immune interference from previous FPV vaccinations or exposures (FPV is commonly present in the environment) and lack of optimization for application through mass methods [9]. The most widely used vector for recombinant ND in ovo vaccine production is the Meleagrid alphaherpesvirus 1, commonly known as herpesvirus of turkeys (HVT) or a serotype 3 Marek’s disease virus [12,23]. In the U.S. alone, 8.2 billion doses of recombinant HVT (rHVT)-ND vaccines were produced in 2018 compared with 3.6 billion doses of conventional ND vaccines [27]. 

In addition to the advantage of being used in ovo, the rHVT vaccines expressing NDV proteins elicit strong and long-term immunity following a single application [28]. Further, they are transiently affected by the presence of anti-NDV maternally derived antibodies [8]. However, the rHVT-ND vaccines have some limitations, such as delayed immunity, and a previous HVT vaccination appears to be refractory to the use of booster HVT vaccines in the same birds [9,29]. The HV vaccines are cell-associated and are required to be stored and transported in liquid nitrogen, which is a limitation on their use in many countries.

The prospect for new in ovo vaccines is growing [23]. Different vectors have been explored for the in-ovo route of administration, and promising results have been reported with non-replicating human adenovirus- [30] and alphavirus-vectored vaccines [31]. Although there have been no advancements to routine commercial use, efforts have also been made for the development of novel in ovo non-HVT NDV-vectored vaccines [32–35]. Employing live ND vaccines in ovo is a challenge. While live ND vaccines, such as LaSota, B1, Clone 30, and Ulster, to name a few, confer a strong immune response and are commonly used for vaccination of chickens as young as one day old, they are associated with high embryo mortality when administered at 18 days of embryo nation. Mortality as high as 100% using wild-type low-virulent viruses and 80% using genetically attenuated live viruses have been reported after in-ovo use, and no live ND vaccines are currently used for this method of vaccine administration [36,37].

A novel recombinant NDV (rNDV)-vectored experimental vaccine containing an antisense avian interleukin 4 inserts (IL4), named ZJ1*l-IL4R, was recently shown to be a promising in ovo vaccine candidate after evaluation in specific-pathogen-free (SPF) embryonated chicken eggs (ECE) [38]. This study aimed to further evaluate the applicability of this recombinant NDV-vectored vaccine and its recombinant backbone (attenuated cleavage site from wild-type virus) and a recombinant LaSota-IL4R virus for in-ovo vaccination of 18-day-old commercial eggs with high maternal antibody titers. Commercially available vaccines LaSota and rHVT-ND were included as controls. In addition, this study aimed to evaluate the protective efficacy of the studied vaccines against early challenges with vNDV. Here, the ability of these recombinant vaccines to efficiently overcome maternal immunity, even in low doses, and to induce a strong immune response is demonstrated. The experimental rNDV vaccines demonstrated 100% protection after early challenges with vNDV.

2. Materials and Methods

2.1. Viruses

The virulent goose/China/ZJ1/2000 (ZJ1, GenBank accession number AF431744) NDV, member of sub-genotype VII.1.1 (former VIId), was used as a challenge virus in this study. A recombinant ZJ1 virus (attenuated version of ZJ1) with a low virulent cleavage site (ZJ1*L) that was previously generated in our laboratory through reverse genetics [39] was used as a backbone for creating recombinant ZJ1*L vaccines and as a vaccine virus. The low virulent LaSota (LS) vaccine strain was used as a vaccine control and as a backbone to generate a recombinant LS-IL4R vaccine. All viruses were obtained from the Southeast Poultry Research Laboratory’s (SEPRL) repository of the United States National Poultry Research Center (USNPRC), U. S. Department of Agriculture (USDA). Additionally, a commercially available rHVT-ND vaccine (fusion gene insert) was also used as a control.

The recombinant NDV experimental vaccines used in this study were created using reverse genetics, as previously described [38,39]. Briefly, the coding sequence of avian IL4 was cloned into the backbones of ZJ1*L and LS in the reverse orientation (IL4R) between the P and M genes, generating two recombinant attenuated live viruses—namely, ZJ1*L-IL4R and LS-IL4R (GenBank accession numbers MM680659 and MM680665, respectively). The recombinant viruses were rescued using the modified vaccinia virus Ankara expressing the T7 polymerase (MVA/T7), following previously published protocols [38–41]. A schematic representation of the constructs is provided in Supplementary Figure S1A. For the purpose, of this work, the term “DV vaccines” used hereafter refers to ZJ1*L, ZJ1*L-IL4R, and LS-IL4R.

Working stocks of all NDVs were propagated in 9- to 11-day-old SPF embryonated chicken eggs (ECE) [42]. Viral stocks used to inoculate eggs in this study were diluted in brain heart infusion (BHI) broth (BD Biosciences, MD, USA), containing 200 µg/mL gentamicin, 2000 U/mL penicillin, and 4 µg/mL amphotericin B, to three different target titers, 105.5/0.1 mL, 104.5/0.1 mL, and 103.5/0.1 mL embryo infectious dose (EID50), except LS, which was diluted to 105.5/0.1 mL and 104.5/0.1 mL EID50. The titers of the diluted working stocks of the live vaccines were confirmed by back titrations in 9- to 11-day-old SPF ECEs following standard procedures, as referenced above. The rHVT-ND vaccine was directly diluted using the sucrose phosphate glutamate albumin diluent for rHVT vaccines described in the USDA’s Center for Veterinary Biologics Test Protocol [43].

2.2. Eggs

Two hundred and sixty-two commercial (non-SPF) eggs from the Hy-Line Rockside W-36 breed layer flock (Mansfield, GA, USA) were kindly donated for this experiment. The layer flock had been routinely vaccinated with the LaSota vaccine. The flock immunity has been regularly tested by ELISA, and although these results are not directly translatable to hemagglutination-inhibition (HI) titers, the average anti-NDV antibody levels of the parental flock were very high (i.e., 15,534 with a cutoff for positive samples of 1159). The source of SPF ECEs (42 eggs) was the SEPRL SPF White Leghorn flock. All eggs were incubated at 37.5 ◦C temperature and 55–60% relative humidity for 18 days.

2.3. Vaccination

The commercial (n = 262) and SPF (n = 42) eggs, at 18 days of embryonated (DOE), were separated into 14 groups with 21 eggs in each group (except BHI and Hatch control groups, which had 26 eggs each). Using 1 mL, 24 G × 1/2 syringes, each egg was inoculated into the allantoic cavity with 0.1 mL of diluted vaccine or BHI, except for the Hatch control group, in which the eggs were not manipulated at all. There were thirteen groups of inoculated eggs, separated into seven experimental and six control groups. The seven experimental groups were as follows: ZJ1*L-IL4R 104.5, ZJ1*L-IL4R 105.5, LS-IL4R 103.5, LS-IL4R 104.5, LS-IL4R 105.5, ZJ1*L 104.5, and ZJ1*L 105.5, each receiving the respective EID50 dose of inoculum per 0.1 mL as stated in the group name. Additionally, six control groups were created. Two control groups of commercial eggs were inoculated as described above with BHI and rHVT-ND, respectively. Two control groups of commercial eggs were inoculated as described above with LS 104.5 and LS 105.5 EID50 dose of inoculum per 0.1 mL. Two control groups of SPF eggs were inoculated with ZJ1*L 103.5 and ZJ1*L-IL4R 103.5. A schematic representation of the study design is provided in Supplementary Figure S1B, C.

After inoculation, the eggs were placed inside Turbofan Hova-Bator Incubators (GQF Manufacturing Company Inc., GA, USA) that were subsequently placed in isolators in an animal biosecurity level 2 (ABSL-2) facility. The incubators were regulated with thermostats to maintain a constant temperature of 37.5 ◦C and relative humidity between 55% and 60%.

2.4. Hatching, Sampling, Mortality

After hatching at 21 DOE, chicks were housed in negative pressure isolators (ABSL-2) and provided food and water with ad libitum access. Five non-SPF chickens from both the Hatch control and BHI groups were sacrificed and bled immediately post-hatch to establish the baseline titer of maternal anti-NDV HI antibodies. All chickens were weighed at 1, 8, and 13 days post-hatch (DPH). Oropharyngeal and cloacal swabs were collected in BHI from each bird at 2 and 4 DPH. Clinical signs and mortality were recorded twice daily throughout the post-hatch period, and blood samples were collected at 13 DPH.

2.5. Challenge

Twelve birds from each group (except LS 105.5, in which all birds died by 6 DPH) were transferred to the ABSL-3E facility at 13 DPH. The birds were placed into negative pressure isolators and allowed to acclimate for 24 h before the challenge. All birds were challenged at 14 DPH with virulent ZJ1 NDV at 106 EID50/0.1 mL by the oculo-nasal route. Birds were observed daily for clinical signs and mortality. Oropharyngeal and cloacal swabs were collected from each bird at 2 and 4 days post-challenge (DPC). All surviving birds were weighed and bled at 14 DPC and were euthanized by cervical dislocation after anesthesia with 0.2 mL ketamine/xylazine solution (66 mg/mL ketamine and 6.6 mg/mL xylazine) administered intramuscularly with a 25-gauge needle.

2.6. Real-Time RT-PCR

RNA was extracted from all swab mediums using the MagMAX 96 AI/ND viral RNA isolation kit (Ambion, Inc. Austin, TX, USA) with a KingFisher magnetic particle processor (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions, with the inhibitor removal modification described by Das et al. [44]. Quantitative real-time RT-PCR (rRT-PCR) for NDV detection targeting the matrix (M) gene was performed using AgPath-ID one-step RT-PCR Kit (Ambion) and the ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Waltham, MA, USA) utilizing primers and probe previously described by Wise et al. [45]. The assay conditions were reverse transcription for 10 min at 45 ◦C; initial denaturation for 10 min at 95 ◦C; 40 cycles of denaturation for 10 sec at 94 ◦C, primer annealing for 30 sec at 56 ◦C, and primer elongation for 10 sec at 72 ◦C. The total reaction volume of 25 µL for each sample consisted of molecular grade water— 2.5 µL; 2X buffer—12.5 µL; forward primer—0.25 µL (20pmol/µL); reverse primer—0.25 µL (20 pmol/µL); probe—0.50 µL (6pmol/µL); 25X enzyme mix—1.0 µL; and RNA template— 8.0 µL. For virus quantification, a standard curve for each virus was established, with RNA extracted from the same titrated stock of the viruses used to inoculate the eggs (for the swab samples collected post-hatch) or the challenge virus (for the swab samples collected post-challenge). 

The results are reported as EID50/mL. The calculated lower detection limit of the assays was between 101.7 and 101.9 EID50/mL. It is important to note that rRTPCR detects viral nucleic acids and that the estimated values do not necessarily represent infectious viruses. The EID50/mL values do not aim to present infectiousness and are used to provide means for relative comparison of viral shedding between groups. Despite the use of inhibitor removal nucleic acids extraction protocol, the high volume of RNA template may have impacted the calculations from the standard curve. Of note, the same amount of RNA template was used in all reactions, and potential residue inhibitors would have impacted all results equally.

2.7. HI Assay

Hemagglutination inhibition (HI) assay was used to evaluate the presence and titers of antibodies in the serum samples collected throughout the experiment. Following standard procedures [46], the virus used as the backbone for each vaccine was used as an antigen for each respective group of sera (ZJ1*L for all ZJ1 groups and LS for all LS groups). For the serum samples collected post-challenge, the virulent ZJ1 virus was used as an antigen in the HI assay. Titers were calculated as the reciprocal of the last HI-positive serum dilution, and samples with HI titers of log2 3 and below were considered negative.

2.8. Statistical Analyses

Prism v.7.03 (GraphPad Software Inc., La Jolla, CA, USA) software was used to analyze the data, and outliers were identified using the ROUT test. The D’Agostino–Pearson normality test was performed to estimate whether the values in each group came from a Gaussian distribution. Based on the normality distribution, the non-parametric Kruskal– Wallis test with Dunn’s post-test was used for multiple comparisons between groups. For statistical purposes, all swab samples from which viruses were not detected were given a numeric value of 1 log below the limit of detection for each virus. The survival curves were analyzed using the Log-rank (Mantel–Cox) test. Statistical significance was set at a p-value of < 0.05.

3. Results

3.1. Hatchability, Post-Hatch Survival, and Body Weights

The post-hatch survival for all groups hatched from commercial eggs is depicted as survival curves in Figure 1 (separated in Figures 1A, and B for clarity and to avoid congestion of the graphics). A combined graph is available in Supplementary Figure S2. The Hatch control group (not inoculated) was included in the design to ensure that there was no abnormal hatchability or post-hatch mortality that was not related to the inoculation of the eggs. 

As expected, very low (rHVT-ND) to no (Hatch and BHI) post-hatch mortality was observed in three of the control groups. In contrast, the LS control groups had 57.1% and 0% survival when the 104.5 and 105.5 doses were used, respectively, which has previously been reported for conventional ND live vaccines. Two eggs did not hatch in the LS-IL4R 105.5 group and one egg did not hatch in the LS 104.5 group. Variable mortality was observed in the other groups depending on the vaccine and inoculation dose. The ZJ1*L-IL4R 104.5 and 105.5 groups had 90.5% and 71.4% survival, respectively, and the survival recorded in the ZJ1*L 104.5 and 105.5 groups was 80.9% and 76.2%, respectively (Figure 1A), but the differences were not statistically significant. The survival in the LS-IL4R 103.5 group was 95.2%. The survival rates observed in the LS-IL4R 104.5 and 105.5 groups were 85.7% and 66.7%, respectively (Figure 1B). 

There were no significant differences in survival between the experimental ZJ1*L, ZJ1*L-IL4R, and LS-IL4R vaccine groups and the Hatch, BHI, and rHVT-ND control groups, but the percentage survival in these control groups was numerically higher, with a negative correlation between vaccine dose and post-hatch survival observed (survival increased as vaccine dose decreased). The LS 105.5 control group had significant differences in survival compared with the experimental groups (p values between 0.0001 and 0.02). The LS 104.5 control group, although not statistically different, had substantially lower survival rates compared with the experimental groups with the same dose. The two LS control groups had significant differences in survival compared with the Hatch, BHI, and rHVT-ND control groups (p values between 0.0001 and 0.04). Clinical signs were not observed in any of the groups post-hatch. In the groups in which mortality post-hatch was present, the birds were found dead upon daily checks.

All birds were weighed at days 1, 8, and 13 post-hatch (Table 1). At 1 DPH, the average body weight per bird in most groups was between 38.5 and 42.6 grams (g). There were significant body weight differences only between rHVT-ND and ZJ1*L-IL4R 104.5 groups from one side and ZJ1*L 104.5 and control LS 104.5 from another (the birds in the two first groups weighed more than the birds in the control group) (p values between 0.0001 and 0.017, respectively). At 8 DPH, the two ZJ1*L groups and the LS-IL4R 104.5, the LS-IL4R 105.5 group, and the control LS group (average weight 50–63 g) had body weights significantly lower compared with the birds from the Hatch, BHI, and rHVT-ND control groups (average weight 77–82 g) (p values between 0.0001 and 0.03). The same groups that were significantly different from the controls at 8 DPH (except LS-IL4R 104.5) had significantly lower body weights than the control Hatch, BHI, and rHVT-ND groups at 13 DPH too (p values between 0.0001 and 0.01). The birds from the inoculated SPF control groups were not included in this comparison, as SPF eggs (and the birds hatched from them) are traditionally smaller and lighter than commercial eggs.

Figure 1. Survival of chickens post-hatch after inoculation of commercial eggs at 18 days of embryo nation with experimental in ovo NDV vaccines. Hy-Line Rockside W-36 eggs were inoculated with recombinant viruses and LaSota. Three control groups were inoculated with BHI or rHVT-ND vaccine, or not inoculated (Hatch control group). Birds were monitored for 13 days post-hatch. The survival curves are presented in two figures: (A) control groups and ZJ1*L groups and (B) control groups and LS-IL4R groups. Different superscript letters indicate statistically significant differences between groups (p-value < 0.05). Group abbreviations: HATCH—eggs not inoculated; BHI—eggs inoculated with BHI; rHVT-ND—eggs inoculated with recombinant HVT vaccine; ZJ1*L—eggs inoculated with the recombinant ZJ1*L virus whose cleavage site was modified to be of low virulence; ZJ1*L-IL4R—eggs inoculated with the recombinant low virulent ZJ1*L with the interleukin-4 gene inserted in reverse orientation; LS—eggs inoculated with the LaSota strain; LS-IL4R—eggs inoculated with the recombinant LaSota virus with the interleukin-4 gene inserted in reverse orientation. The virus dose that the eggs in each group received is represented as a number after the group name in EID50/per egg.

Table 1. Mean weights (and standard deviations) of chickens at 1, 8, and 13 days post-hatch after inoculation of commercial eggs at 18 days of embryo nation with experimental in ovo vaccines and at 14 days post-challenge with virulent Newcastle disease virus.

3.2. Shedding of Vaccine Viruses Post-Vaccination, as Demonstrated by rRT-PCR

At 2 DPH, there were no significant differences in viral shedding among vaccine groups. (Figure 2A). Similarly, at 4 DPH, the differences between groups were not statistically significant. (Figure 2B). No viral shedding was detected in any of the Hatch and BHI control birds.

Figure 2. Mean post-hatch vaccine shedding from chickens hatched from commercial eggs inoculated at 18 days of embryo nation with experimental in ovo vaccines. Each data point represents one bird and NDV titers detected in oropharyngeal (OP) and cloacal (CL) swabs on different days post-hatch (DPH). Shedding is presented separately for 2 days post-hatch (A) and 4 days post-hatch (B). Bars represent standard deviations of the mean. All swabs from which the virus was not detected were given a numeric value of 1 log below the limit of detection for each of the respective viruses. The limit of detection of rRT-PCR is presented as a horizontal dotted line (the lowest limit is used). Group abbreviations: HATCH—eggs not inoculated; BHI—eggs inoculated with BHI; rHVT-ND—eggs inoculated with recombinant HVT vaccine; ZJ1*L—eggs inoculated with the recombinant ZJ1*L virus whose cleavage site was modified to be of low virulence; ZJ1*L-IL4R—eggs inoculated with the recombinant low virulent ZJ1*L with the interleukin-4 gene inserted in reverse orientation; LS—eggs inoculated with the LaSota strain; LS-IL4R—eggs inoculated with the recombinant LaSota virus with the interleukin-4 gene inserted in reverse orientation. The virus dose that the eggs in each group received is represented as a number after the group name in EID50/per egg.

3.3. Humoral Immune Response Post-Hatch

Analysis of serum samples collected at 13 DPH (diagonal stripe-filled bars) by HI assay (detecting hemagglutination antibodies to NDV) is depicted in Figure 3. To establish the titer of passively transferred anti-NDV maternal HI antibodies in the commercial eggs, ten chickens were bled immediately after hatch (0 DPH). The HI titers at hatch varied between log2 5 and log2 8, with an average of log2 7.17 (Figure 3, dotted horizontal red line). The antibodies at 13 DPH in the Hatch, BHI, and rHVT-ND groups (average of log2 4.7) are residual maternal antibodies, as these groups were either not vaccinated or vaccinated with rHVT-ND vaccine, which does not induce HI antibodies to NDV. The antibody titers in the ZJ1*L, ZJ1*L-IL4R and the LS 104.5 groups were significantly higher compared with the Hatch, BHI, and rHVT-ND control groups. No significant differences between the different dose groups of the vaccines with the same backbone were observed. The LS-IL4R groups had lower post-vaccination HI titers compared with the ZJ1*L-IL4R and ZJ1*L groups, although these differences were not statistically significant. Of note, although numerically slightly higher, the post-vaccination HI titers of the birds in all LS-IL4R groups were not significantly different from the titers measured in the control unvaccinated birds.

Figure 3. Pre- and post-challenge HI antibody titers (and standard deviation) from chickens hatched from commercial eggs inoculated at 18 days of embryo nation with experimental in ovo vaccines and early challenge with virulent Newcastle disease virus at 14 days post-hatch. The diagonal stripe-filled bars represent the pre-challenge titers at 13 days post-hatch. The solid-filled bars represent the post-challenge titers at 14 days post-challenge. Statistically significant differences between the different groups are presented with lowercase letters above each group (p-value <0.05). The level of maternal antibodies on the day of hatch (0 DPH) is presented as a horizontal dotted line. Group abbreviations: HATCH—eggs not inoculated; BHI—eggs inoculated with BHI; rHVT-ND—eggs inoculated with recombinant HVT vaccine; ZJ1*L—eggs inoculated with the recombinant ZJ1*L virus whose cleavage site was modified to be of low virulence; ZJ1*L-IL4R—eggs inoculated with the recombinant low virulent ZJ1*L with the interleukin-4 gene inserted in reverse orientation; LS—eggs inoculated with the LaSota strain; LS-IL4R—eggs inoculated with the recombinant LaSota virus with the interleukin-4 gene inserted in reverse orientation. The virus dose that the eggs in each group received is represented as a number after the group name in EID50/per egg.

3.4. Clinical Signs and Survival after Early Challenge with CDV

In the control groups, the first clinical signs to be observed were in the Hatch and BHI groups at 3 DPC. Three birds in the Hatch group and one bird in the BHI group had labored breathing. At 4 DPC, ruffled feathers and head tremors were seen in the BHI and hatch groups, and at 5 DPC in the rHVT-ND group; in the latter group, ataxia and severe torticollis were also recorded in two birds. The birds in the Hatch control group presented lethargy at 5 DPC, and two birds in the BHI group showed paralysis. Lethargy and paralyzes were observed in the rHVT-ND group at 6 and 7 DPC. Opisthotonos, torticollis, and labored breathing were observed in the Hatch and BHI control groups beginning at 9 DPC. In the rHVT-ND group, torticollis was observed as early as 5 DPC, labored breathing and ataxia began at 6 DPC, and opisthotonos began at 13 DPC. By 14 DPC, all birds except two Hatch, one BHI, and two rHVT-ND birds (83.3%, 91.6%, and 83.3% morbidity, respectively) either died or developed predominantly neurological clinical signs such as ataxia, opisthotonus, torticollis, tremors, and paralyzes. Birds that showed severe clinical signs, stopped eating or drinking, or remained recumbent were euthanized and reported as dead on the next day for the calculation of survival times. No clinical signs were observed in any of the ZJ1*L and ZJ1*L-IL4R groups. Slight clinical signs of opisthotonos or torticollis were observed in a single bird (8.4% morbidity) in each of the LS-IL4R groups and the LS 104.5 group at 14 DPC.

Survival post-challenge with the virulent ZJ1 NDV is depicted as survival curves in Figure 4. While all groups inoculated with the recombinant vaccines and LS showed 100% survival, the birds in the Hatch, BHI, and rHVT-ND groups had 58.3%, 58.3%, and 66.6% survival, respectively. The post-challenge survival in the Hatch and the BHI control groups was significantly lower compared with the experimental vaccine groups. Of note, although the body weights of the birds in the inoculated groups fluctuated post-hatch compared with the Hatch, BHI, and rHVT-ND controls, all rNDV vaccinated birds (282–328 g) surpassed the body weights of the non-vaccinated control birds post-challenge (255–266 g) (Table 1). However, no significant body weight difference was observed between any of the studied groups.

Figure 4. Survival of chickens after inoculation of commercial eggs at 18 days of embryo nation with experimental in ovo vaccines and early challenge with virulent Newcastle disease virus at 14 days post-hatch. Birds were monitored for 14 days post-challenge. Different superscript letters indicate statistically significant differences between groups (p-value < 0.05). Group abbreviations: HATCH—eggs not inoculated; BHI—eggs inoculated with BHI; rHVT-ND—eggs inoculated with recombinant HVT vaccine; ZJ1*L—eggs inoculated with the recombinant ZJ1*L virus whose cleavage site was modified to be of low virulence; ZJ1*L-IL4R—eggs inoculated with the recombinant low virulent ZJ1*L with the interleukin-4 gene inserted in reverse orientation; LS—eggs inoculated with the LaSota strain; LS-IL4R—eggs inoculated with the recombinant LaSota virus with the interleukin-4 gene inserted in reverse orientation. The virus dose that the eggs in each group received is represented as a number after the group name in EID50/per egg.

3.5. Shedding Post-Challenge as Demonstrated by rRT-PCR

At 2 DPC, the birds in the Hatch, BHI, and rHVT-ND control groups shed high viral titers (105.3−5.7 EID50/mL) through the oral route, while all vaccine groups had lower than 104 EID50/mL mean shedding titers (Figure 5A). The results for viral shedding through the cloaca were below the detection limit of the assay in all groups. At 2 DPC, the birds in all ZJ1*L and ZJ1*L-IL4R groups and those in the LS-IL4R 105.5 group had significantly less viral RNA detected through the oral route compared with the Hatch, BHI, and rHVT-ND control groups (p values ranged between 0.001 and 0.02). At 4 DPC, the shedding through the oral route in the Hatch, BHI, and rHVT-ND groups was high (106.2−6.9 EID50/mL) (Figure 5B). 

In contrast, less shedding was observed across all vaccine groups at 4 DPC compared with 2 DPC, with the highest mean titer of 102.9 EID50/mL in the ZJ1*L 104.5 group and the lowest mean titer of 101.5 EID50/mL in the ZJ1*l-IL4R 104.5 group (viral RNA was not detected in some birds). All experimental rNDV vaccine groups shed significantly less virus through the oral route compared with the Hatch, BHI, and rHVT-ND control groups (p values from <0.0001 to 0.03). No significant differences in oral shedding were observed among the experimental rNDV vaccine groups. The birds in the rNDV vaccine groups shed significantly less virus through the cloacal route compared with the Hatch, BHI, and rHVT-ND control groups (p values from <0.0001 to 0.007). The differences in the titers were not significant among the experimental rNDV vaccine groups and the LS control group.

Figure 5. Mean post-challenge viral shedding from chickens after inoculation of commercial eggs at 18 days of embryo nation with experimental in ovo vaccines and early challenge with virulent Newcastle disease virus at 14 days post-hatch. Each data point represents NDV titers detected in oropharyngeal (OP) and cloacal (CL) swabs on different days post-challenge (DPC). Shedding is presented separately for 2 days post-challenge (A) and 4 days post-challenge (B). Bars represent standard deviations of the mean. All swabs from which the virus was not detected were given a numeric value of 1 log below the limit of detection. The limit of detection of rRT-PCR is presented as a horizontal dotted line. 

Statistically significant differences between the different groups are presented with lowercase letters above each group (p-value < 0.05). Group abbreviations: HATCH—eggs not inoculated; BHI—eggs inoculated with BHI; rHVT-ND—eggs inoculated with recombinant HVT vaccine; ZJ1*L—eggs inoculated with the recombinant ZJ1*L virus whose cleavage site was modified to be of low virulence; ZJ1*L-IL4R—eggs inoculated with the recombinant low virulent ZJ1*L with the interleukin-4 gene inserted in reverse orientation; LS—eggs inoculated with the LaSota strain; LS-IL4R—eggs inoculated with the recombinant LaSota virus with the interleukin-4 gene inserted in reverse orientation. The virus dose that the eggs in each group received is represented as a number after the group name in EID50/per egg.

3.6. Post-Challenge Serology

All birds in the Hatch, BHI, and rHVT-ND control groups showed increased (more than double) HI-antibody titers at 14 DPC (above log2 10) compared with the pre-challenge titers at 13 DPH (log2 4.7). The differences in the titers in these three groups pre- and post-challenge were significant (p-value <0.0001) (Figure 3, solid bars). In contrast, the post-challenge HI titers in all experimental rNDV vaccine groups and the LS control group did not change significantly compared with the pre-challenge titers of the same groups. In all groups, the differences between pre-and post-challenge antibody titers were within a log (except LS-ILR4 103.5, in which the difference was log2 1.8).

3.7. Inoculated SPF Control Groups

The survival in the SPF control groups post-hatch was 66.7% in the group inoculated with ZJ1*L 103.5 and 57.1% in the group inoculated with ZJ1*L-IL4R 103.5 (Supplementary Figure S3A). Active viral shedding was detected in the birds in the SPF control groups through both routes, with higher titers detected in the oropharyngeal swab samples (see Supplementary Figure S3B). The post-vaccination HI titers of the inoculated SPF control birds were higher than the titers in the Hatch, BHI, and rHVT-ND control groups and reached mean values of log2 7.08 and log2 7.58 at 13 DPH in the ZJ1*L-IL4R 103.5 and ZJ1*L-IL4R 103.5 groups, respectively (see Supplementary Figure S3C). No post-challenge clinical signs or mortality were observed in the two inoculated SPF control groups (see Supplementary Figure S3D for survival curves). The birds from the inoculated SPF control groups shed significantly less virus through the oral route at 2 DPC and 4 DPC compared with the control groups (p values from <0.0001 to 0.009) (see Supplementary Figure S3E). The post-challenge antibody titers in both inoculated SPF control groups did not change significantly compared with the pre-challenge titers of the same groups (see Supplementary Figure S3C).

For more information:1950477648nn@gmail.com