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. 2024 Jun 18;103(9):103988. doi: 10.1016/j.psj.2024.103988

An avian-origin internal backbone effectively increases the H5 subtype avian influenza vaccine candidate yield in both chicken embryonated eggs and MDCK cells

Fan Yang *, Xinyu Zhao *, Chenzhi Huo *, Xinyu Miao *,†,‡,§, Tao Qin *,†,‡,§, Sujuan Chen *,†,‡,§, Daxin Peng *,†,‡,§,1, Xiufan Liu *,†,§
PMCID: PMC11269899  PMID: 38970848

Abstract

Inactivated vaccines play an important role in preventing and controlling the epidemic caused by the H5 subtype avian influenza virus. The vaccine strains are updated in response to alterations in surface protein antigens, while an avian-derived vaccine internal backbone with a high replicative capacity in chicken embryonated eggs and MDCK cells is essential for vaccine development. In this study, we constructed recombinant viruses using the clade 2.3.4.4d A/chicken/Jiangsu/GY5/2017(H5N6, CkG) strain as the surface protein donor and the clade 2.3.4.4b A/duck/Jiangsu/84512/2017(H5N6, Dk8) strain with high replicative ability as an internal donor. After optimization, the integration of the M gene from the CkG into the internal genes from Dk8 (8GM) was selected as the high-yield vaccine internal backbone, as the combination improved the hemagglutinin1/nucleoprotein (HA1/NP) ratio in recombinant viruses. The r8GMΔG with attenuated hemagglutinin and neuraminidase from the CkG exhibited high-growth capacity in both chicken embryos and MDCK cell cultures. The inactivated r8GMΔG vaccine candidate also induced a higher hemagglutination inhibition antibody titer and microneutralization titer than the vaccine strain using PR8 as the internal backbone. Further, the inactivated r8GMΔG vaccine candidate provided complete protection against wild-type strain challenge. Therefore, our study provides a high-yield, easy-to-cultivate candidate donor as an internal gene backbone for vaccine development.

Key words: Avian influenza virus, vaccine, internal backbone, yield, MDCK cell

INTRODUCTION

Avian influenza (AI) is a zoonotic disease caused by the avian influenza virus (AIV), a member of the family Orthomyxoviridae. Due to the antigenic differences between hemagglutinin (HA) and neuraminidase (NA), AIVs are classified into 16 HA and 9 NA subtypes, all of which have been isolated from wild birds (Webster, et al., 1992; Fouchier, et al., 2005). The H5 and H7 subtypes of highly pathogenic AIVs pose a constant global threat to the poultry industry and public health and have attracted increasing attention (WHO, 2023). Since A/Goose/Guangdong/1/1996 (H5N1, Gs/GD/1996) was first identified in China in 1996 (Peiris, et al., 2001), the HA gene of the H5 subtype AIV has undergone continuous evolution, ultimately leading to the emergence of distinct clades and subclades (clades 0–9). In 2014, clade 2.3.4.4 H5 subtype AIVs spread globally and evolved into subclades known as 2.3.4.4a to 2.3.4.4h (Cui, et al., 2022; Lee, et al., 2017a; WHO/OIE/FAO H5N1 Evolution Working Group, et al.,2008). In addition to culling all poultry in outbreak areas, vaccination plays a crucial role in preventing highly pathogenic avian influenza (Ping, et al., 2015). Efforts have focused on developing avian influenza vaccines to protect high-risk individuals.

Inactivated whole-virus vaccines are widely used against highly pathogenic avian influenza in poultry in certain countries and regions (Sims, et al., 2012; Peiris, et al., 2016). Chicken embryonated eggs are the most commonly used primary material for producing inactivated avian influenza vaccines. However, there are problems with the inadequate supply and unstable quality of chicken embryos (Barr, et al., 2018). Therefore, cell culture possesses the potential to enhance the existing situation and solve the problem of large-scale rapid production (Robertson, et al., 1995). The HA protein, which mediates receptor binding, membrane fusion, pathogenicity, serves as the predominant antigenic glycoprotein on the surface of influenza virus particles (Skehel, et al., 2000; Wu, et al., 2020). The NA protein, as the second most abundant surface glycoprotein, primarily functions in cleaving sialic acid on the cell surface to facilitate the release and dissemination of virus particles (Suzuki, et al., 2005). It also serves as a crucial target for vaccine development. Through reverse genetic technology (Hoffmann, et al., 2000), the surface proteins HA and NA of an epidemic strain can be recombined with a high-yield backbone to rescue the vaccine strain. PR8, also known as A/Puerto Rico/8/1934(H1N1), is a common high-yield vaccine backbone(Chen, et al., 2009) for seasonal and pandemic influenza vaccine development; however, it may be incompatible with the HA and NA genes of H5 subtype AIV (Horimoto, et al., 2007). Therefore, the development of an avian-origin backbone for avian influenza vaccine strains that can replicate effectively in both chicken embryos and MDCK cells is crucial.

In this study, we chose the clade 2.3.4.4d H5 subtype AIV CkG strain as the donor for surface proteins and screened the AIV Dk8 strain with a high yield in both chicken embryos and MDCK cells as the vaccine backbone. After optimization, the vaccine candidates were rescued and evaluated for their biological characteristics, immunogenicity, and protective efficacy.

MATERIALS AND METHODS

Facility and Ethics Statements

Animal experiments were conducted under the guidelines for experimental animal welfare and ethics. All animal studies followed the protocols of the Jiangsu Province Administrative Committee for Laboratory Animals (approval number: SYXKSU-2021-0027). All infection experiments using highly pathogenic avian influenza viruses were conducted at the Animal Biosafety Level 3 Laboratory of Yangzhou University.

Cell Culture and Virus Infection

Human embryonic kidney (HEK) 293T cells (ATCC; CRL-11268) were cultured in Dulbecco's modified Eagle's medium (DMEM) (HyClone, Logan, Utah, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand island, New York, USA). MDCK cells (ATCC CCL-34) were cultured in minimum essential medium (MEM) (HyClone, Logan, Utah, USA) supplemented with 10% FBS. Suspension MDCK cells were cultured in a serum-free medium (Yishengke, China). Chicken embryo fibroblast (CEF) cells were prepared from 10-day-old specific pathogen-free (SPF) embryos and cultured in M199 medium (Thermo Fisher Scientific, Waltham, Massachusetts, USA) with 4% FBS. Cells were routinely cultured at 37°C with 5% CO2. Three highly pathogenic AIVs, A/chicken/Jiangsu/GY5/2017 (H5N6, CkG), A/duck/Jiangsu/84512/2017 (H5N6, Dk8), and A/chicken/Yangzhou/YZC08101/2023 (H5N1, CkY), were isolated from birds in live poultry markets and plaque-purified (Naguib, et al., 2017) in MDCK cells for 3 consecutive generations before passaging in 10-day-old SPF chicken embryonated eggs.

Phylogenetic Analysis

Reference sequences (Bi, et al., 2016) and H5N6 subtype sequences from 2010 to 2023 worldwide were downloaded from GISAID (http://www.gisaid.org) and filtered using BioAider (v. 1.334) to remove sequences with > 99% similarity. Phylogenetic analysis of different genes of the H5N6 subtype of the avian influenza virus was conducted in PhyloSuite v1.2.2(Bi, et al., 2016; Xu, et al., 2022b) using IQ-TREE (v.1.6.8). ITOL (v.6.7) (https://itol.embl.de/) was used to annotate the branches of the selected strains and enhance the aesthetics of the evolutionary tree.

Construction of Plasmids for Expressing Influenza Virus Genes and Virus Rescue

H5N6 AIV CkG strain and Dk8 strain were selected as parental viruses. Eight fragments including polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), polymerase acidic protein (PA), HA, NP, NA, matrix protein (M), and nonstructural protein (NS) of CkG strain, Dk8 strain, and a weakened HA fragment of CkG strain, Dk8 strain, and CkY strain were constructed by removing the multibasic amino acid motif (Senne, et al., 1996; Li, et al., 1999). These fragments were cloned into the pHW2000 bidirectional expression vector (Hoffmann, et al., 2000) to rescue the recombinant viruses. The plasmids of PR8 were stored in our laboratory (Yang, et al., 2022). The genomes of the fifth-passage recombinant viruses were sequenced to confirm the correction of the rescued viruses (Shi, et al., 2016). All rescued viruses were titrated for HA titer, 50% embryo infective dose (EID50) and 50% tissue culture infective dose (TCID50).

Replication Kinetics

CEF and MDCK cells were inoculated with the first-generation viruses at the multiplicity of infection (MOI) of 0.001. The supernatants were harvested at 12, 24, 36, 48, 60, and 72 hours postinfection (hpi). The TCID50 was calculated as previously described by Reed and Muench (1938).

Virus Particle Purification

Viruses were grown in 10-day-old eggs, and the allantoic fluid was harvested and clarified by centrifugation at 18,000 × g for 30 min using a centrifuge (Eppendorf, Hamburg, Germany). The clarified supernatant was concentrated using a 30% sucrose cushion (Hutchinson, et al., 2014), and the virions were pelleted by ultracentrifugation in an SW32 rotor (Beckman, Brea, California, USA) at 4°C for 90 min at 112,000 × g. They were then resuspended, and the sediment was placed into tubes with 60%, 40%, and 20% sucrose solutions and subjected to ultracentrifugation in an SW41 rotor at 4°C at 209,000 × g for 150 min. The virion band was transferred into a new tube containing chilled NTC buffer (100 mM NaCl, 20 mM Tris-HCl, and 5 mM CaCl2), and virions were pelleted by ultracentrifugation in an SW32 rotor at 4°C for 60 min. The supernatant was aspirated, and the pellets were resuspended in NTC buffer. The resuspended virions were stored at -80°C in small aliquots.

Deglycosylation of Virion Proteins and Quantification

Purified viruses were quantified using a BCA protein assay kit (Thermo Fisher,Waltham, Massachusetts, USA), and 20 µg of total protein was treated with PNGase F (Harvey, et al., 2011; Hussain, et al., 2019) (New England Biolabs,Beverly, Massachusetts, USA) following the manufacturer's instructions. Briefly, 20 µg of purified viruses were mixed with glycoprotein denaturing buffer and denatured by incubating at 100°C for 10 min. The denatured glycoproteins were cooled on ice and briefly centrifuged (Eppendorf). Glyco Buffer 2, 10% NP-40, and PNGase F were added, and the samples were incubated at 37°C for 1 h. Samples were analyzed by labeling on a 10% SDS-PAGE gel (Bio-Rad, Hercules, California, USA). The gel was subjected to Coomassie staining for visualization and immunostaining by western blotting. Viral protein concentrations were determined by WB, and the bands were analyzed using ImageJ software. The HA1/NP was calculated (Ridenour, et al., 2015).

The cRNA, mRNA, and vRNA Expression Levels of NP Genes

The first generation of recombinant virus was inoculated into MDCK cells at MOI=0.1, and cell samples were collected at 1, 3, 4.5, and 6 hpi. Primers (Table S2) for different types of RNA were used to amplify the cRNA, mRNA, and vRNA of NP gene, and the relative expression levels were determined by quantitative RT-PCR.

Vaccine Preparation

The vaccine strains with an HA titer of 8Log2 were treated with 0.1% formaldehyde at 4°C for 24 h. After inactivation, the virus was inoculated into SPF chicken embryos to verify complete inactivation. The aqueous phase of the vaccine was mixed with Tween 80 and white oil adjuvant in a 1:3 ratio and was then emulsified to form an emulsion characterized by oil droplets in a water-like fluid.

Chicken Experiments

Six-week-old SPF chickens (10 chickens in each group) were inoculated intravenously with diluted allantoic fluid (0.1 mL) containing each virus to assess viral pathogenicity. Experimental SPF chickens were monitored daily for clinical signs of disease for 10 d, and intravenous pathogenicity indices (IVPI) were calculated following the recommendations of WOAH (WOAH, 2021). The 50% chicken lethal dose (CLD50) of the challenge CkG strain was determined in 4-wk-old SPF chickens after intranasal inoculation. The chickens were monitored for survival for 2 wk.

For the protection experiments, 3-wk-old SPF chickens were randomly divided into eight groups of 10 chickens each group. Chickens were immunized subcutaneously with 0.2 mL candidates or PBS. Serum samples were collected postvaccination on d 7, 14, and 21 to determine HI and MNT titers. In the third week after immunization, vaccinated chickens were intranasally challenged with the H5N6 AIV CkG strain at 100 CLD50/0.1 mL doses. Throat and cloacal swabs were collected at 3, 5, and 7 d postchallenge and inoculated into SPF chicken embryos. The protection rate was recorded after 14 d of continuous observation. Viral shedding in swabs was periodically monitored after challenge (Zhang, et al., 2015b).

HI Assays and Microneutralization Assays

Antisera that were serially diluted 2-fold were mixed with 4 HA units of virus (25 μL each) and incubated for 15 min at 37°C. Subsequently, 25 μL of 0.5% chicken red blood cells were added to each well and incubated for 15 min at 37°C. The hemagglutination inhibition (HI) titer had the highest dilution of antiserum, which completely inhibited hemagglutination. Microneutralization (MNT) titers were determined as previously described (Lessler, et al., 2012). Sera were serially diluted 2-fold and mixed with 200 TCID50 viruses to perform the microneutralization assays. After incubation at 37°C for 1 h, the mixture was inoculated into MDCK cells. Neutralization titers were defined as the reciprocal of the highest serum dilution.

Graphs and Statistical Analyses

All statistical analyses were performed using GraphPad Prism software V8.0.1. Data are presented as the means ±SD unless otherwise indicated. A t-test or 1-way analysis was used to determine the differences between the experimental and control groups. For all tests, differences between groups were considered significant when the P value was >0.05 (ns); P < 0.05(*), P < 0.01(**); For letters, identical letter cases denote no statistical differences, while distinct lowercase letters indicate P <0.05.

RESULTS

The rCkG Provides the Surface Protein, While the rDk8 Serves as the Backbone

After screening for H5 subtype AIVs, the clade 2.3.4.4d strain A/chicken/Jiangsu/GY5/2017 (H5N6, CkG) was selected as the surface protein donor to meet the antigen requirements for prevailing strains. In contrast, the clade 2.3.4.4b strain A/duck/Jiangsu/84512/2017(H5N6, Dk8) with high replication in embryonated eggs and MDCK cells was chosen as the backbone donor (Figure S1). Recombinant viruses rCkG strain and rDk8 strain were constructed using the reverse genetics method and passaged in chicken embryos and MDCK cells. As expected, the rDk8 strain exhibited higher HA titers, TCID50, and EID50 than rCkG strain (Table 1).

Table 1.

Biological characteristics of 2 H5N6 subtype avian influenza viruses.

Viruses HA titer(Log2) Titer (Log10TCID50/mL)
 Titer (Log10EID50/mL)
CEF MDCK
rDk8 9.667±1.506a* 8.796±0.602a 7.964±0.465a 9.277±0.449a
rCkG 7.000±1.225b 7.982±0.615b 6.963±0.439b 8.526±0.654b
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Different letters indicate significant differences, P <0.05.

Integration of the M Gene From the CkG Improves the Internal Backbone of the rDk8

To clarify the genetic background of the 2 parental strains, their internal genes were subjected to phylogenetic analysis, as presented in Figure S1. The PB2, PB1, PA, M, and NS genes of the Dk8 strain originated from low-pathogenicity avian influenza viruses (LPAIV), and the HA, NP, and NA genes originated from H5 viruses. The PB2 and PB1 genes of the CkG strain were from LPAIV, and the PA, HA, NP, NA, M, and NS genes were from H5 viruses (Figure S1). Indicating that the internal genes of the Dk8 strain predominantly originate from LPAIVs and those of the CkG strain are mainly from H5 subtype AIVs.

The growth curves of rDk8 and rCkG were similar on CEF but differed on MDCK (Figure S2). To investigate the effect of internal genes on replication ability in MDCK, recombinant viruses were individually rescued through single-segment reciprocal replacement using either the CkG strain or the Dk8 strain as the backbone (Figure 1A), named rDk8+CkG-PB2/PB1/PA/NP/M/NS or rCkG+Dk8-PB2/PB1/PA/NP/M/NS. The growth curves of the recombinant viruses in MDCK cells were assessed (Figures 1B and 1C).

Figure 1.

Figure 1

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The model diagram and growth curves of H5N6 avian influenza virus with single-segment replacement. (A) The eight genes from 2 H5N6 AIV strains were subjected to genetic evolution analysis, and different lineages were labelled in different colours. (B) Growth curves in MDCK cells for recombinant viruses using the Dk8 strain as the backbone and substitution with a single segment from the CkG strain. (C) Growth curves in MDCK cells for recombinant viruses using the CkG strain as the backbone and substitution with a single segment from the Dk8 strain. Data are the means ± standard deviations (SD) from 3 independent experiments (*P < 0.05, **P < 0.01).

The rDk8+CkG-M with substitution of the M gene from the CkG strain exhibited significantly increased titers at 12, 24, and 36 hpi when compared to those of the wild-type rDk8 strain, while rDk8+CkG-PB2, rDk8+CkG-PA, and rDk8+CkG-NS exhibited significantly increased titers only at 12hpi. In contrast, all recombinant CkG strains substituting a single gene from Dk8 strain exhibited increased considerably titers. These data indicated that integrating the M gene from the CkG strain into the backbone of the Dk8 strain may optimize the internal donor of the vaccine, which can be named 8GM and used as a vaccine donor.

The Recombinant Attenuated Viruses With Optimized Backbone Exhibit Higher Titers and Growth Performance

To determine the effect of the optimized backbone on virus replication, the recombinant viruses with HA (modified cleavage site from RERRRKR↓GLF to RET—R↓GLF) and NA genes from the CkG strain (ΔG) and the internal backbone from the PR8, Dk8, or 8GM strains were rescued, and the IVPIs of the recombinant viruses were determined. As expected, all the recombinants exhibited low pathogenicity with a score of 0.

To assess the titer and gene stability of the recombinant virus postpassage, we propagated it through 5 generations on embryonated eggs and cells. The strains cultured on chicken embryos were labeled as rΔG, rPΔG, rDk8ΔG, r8GMΔG, while those on cells were labeled as C-ΔG, C-rPΔG, C-rDk8ΔG, C-r8GMΔG. HA titers of the recombinant viruses from the first generation to the fifth generation are shown in Table S1. The biological characteristics of the recombinant viruses in chicken embryos, CEF, and MDCK cells were determined (Table 2).

Table 2.

Biological characteristics of H5 subtype avian influenza vaccine candidates.

Viruses HA titer(Log2) Titer (Log10TCID50/mL)
 Titer (Log10EID50/mL)
CEF MDCK
rΔG 7.556±1.130b* 7.518±0.035a 5.563±1.394b 8.404±0.754b
rPΔG 9.400±1.517a 7.666±0.274a 6.652±0.943a 8.533±0.141ab
rDk8ΔG 9.143±1.069a 7.956±0.722a 6.622±1.355a 9.189±0.500a
r8GMΔG 9.714±1.113a 8.375±1.041a 7.372±0.831a 9.211±0.594a
C-rΔG 7.500±0.707a 6.750±0.354a 6.000±1.414a 8.701±0.059ab
C-rPΔG 7.000±1.732b 7.222±0.481a 6.902±0.830a 8.7561±0.084ab
C-rDk8ΔG 7.00±1.00b 7.61±0.35a 6.72±0.26a 7.556±0.096b
C-r8GMΔG 8.667±0.577a 8.334±0.577a 7.675±0.743a 9.056±0.096a
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⁎

Different letters indicate significant differences, P <0.05.

The HA titers (Log2) of rΔG, rPΔG, rDk8ΔG and r8GMΔG were 7.56, 9.40, 9.14, and 9.71, respectively. All recombinant viruses significantly increased HA titers compared to the wild-type strain rΔG. The EID50 titers (Log10) of rΔG, rPΔG, rDk8ΔG and r8GMΔG were 8.40, 8.53, 9.19, and 9.21, respectively. When the titers of recombinant viruses were determined in CEF or MDCK cells, the r8GMΔG exhibited the highest TCID50 titer. There was no significant difference between r8GMΔG and rDk8ΔG. After passage in the suspension MDCK cultivation for five generations, the HA titers (Log2) of C-rΔG, C-rPΔG, C-rDk8ΔG, and C-r8GMΔG were 7.50, 7.00, 7.00, and 8.67, respectively. The HA titer of C-r8GMΔG is significantly higher than that of C-rDk8ΔG and C-rPΔG. The C-r8GMΔG also exhibited the highest EID50 and TCID50 compared to other recombinant viruses. The EID50 of C-r8GMΔG is considerably higher than that of C-rDk8ΔG.

Considering vaccine production using chicken embryos or suspended MDCK cells, we determined the growth curves on CEF and MDCK cells. In CEF cells, the r8GMΔG or C-r8GMΔG exhibited a significant increase in titers during 24 to 72 hpi when compared to that of the parent strains rΔG or C-rΔG (Figures 2A and 2B). C-r8GMΔG also exhibited a significant increase in titers during 12 to 72 hpi compared to C-rPΔG with the internal backbone from PR8 strain. There was no significant difference between r8GMΔG or C-r8GMΔG and rDk8ΔG or C-rDk8ΔG. In MDCK cells, the r8GMΔG or C-r8GMΔG exhibited a significant increase in titers during 24 to 72 hpi compared to the parent strains (Figures 2C and 2D). Additionally, the r8GMΔG exhibited a considerable increase in titers during 24 to 60 hpi when compared to rDk8ΔG, and C-r8GMΔG exhibited a considerable increase in titers during 36 to 72 hpi when compared to the C-rPΔG or C-rDk8ΔG. These findings suggest that integrating the M gene from the CkG strain into the internal backbone of Dk8 strain resulted in faster replication and higher titers in the early stage. These data indicated that the 8GM internal backbone could improve the replication ability of recombinant viruses in either chicken embryos or MDCK cells.

Figure 2.

Figure 2

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Growth curves of H5 avian influenza vaccine candidates in CEF (A and B) and MDCK cells (C and D). Data are the means ±SD from 3 independent experiments, and different letters indicate significant differences; P < 0.05.

Integration of the M Gene From the CkG in the Backbone Improves HA Expression of Recombinant Virus

To compare the HA expression levels of the recombinant viruses, the HA1 expression of purified virus particles was evaluated using Coomassie brilliant blue staining and western blotting, both with and without the addition of PNGase (Figures 3A and 3B). Without the addition of PNGase, r8GMΔG exhibited a substantial increase in HA1/NP rate (127.94%) when compared to those of rΔG (set as baseline, 100%), rPΔG (113.77%), and rDk8ΔG (100.56%) (Figure 3C). C-r8GMΔG exhibited a significant increase in HA1/NP rate (208.11%) compared to that of C-rΔG (set as baseline, 100%), whereas C-rDk8ΔG and C-rPΔG exhibited values of 164.11% and 93.24%, respectively (Figure 3F). With the addition of PNGase, r8GMΔG exhibited a significantly higher HA1/NP rate of 187.05% compared to that of rΔG, while rPΔG and rDk8ΔG yielded 169.46% and 175.49%, respectively (Figure 3D). C-r8GMΔG exhibited a substantial increase in HA1/NP rate at 239.39%, whereas C-rDk8ΔG and C-rPΔG exhibited values of 201.11% and 177.70% for HA1/NP rate, respectively (Figure 3F). These data indicate that r8GMΔG with a selected internal backbone can effectively improve HA expression. The morphology and RNA expression were determined to further compare the strains rDk8ΔG and r8GMΔG. Electron microscopy revealed no differences in the shape and diameter of either virus particle (Figures 4A and 4B). The expressions of cRNA, mRNA, and vRNA from the r8GMΔG strain in MDCKs were significantly higher than that from the rDk8ΔG strain (Figure 4C). These findings suggest that integrating the M gene from the CkG strain into the internal backbone of Dk8 strain resulted in enhanced replication ability in chicken embryonated eggs or MDCK cells.

Figure 3.

Figure 3

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Yield analyses of H5 avian influenza vaccine candidates. The virus particles cultured in chicken embryonated eggs (A) and suspended MDCK cells (B) were purified by ultracentrifugation, and a total of 10 μg of protein for each virus was treated with (+) or without (-) prior treatment with PNGase and loaded into a 10% SDS-PAGE gel. Protein bands were visualized by Coomassie blue staining (upper panel) or Western blotting (lower panel). Molecular mass (kDa) markers and specific proteins were labelled. (C) and (D) represent grey values of glycosylated HA1/NP (%) for Western blotting (n = 2), while (E) and (F) represent grey values of deglycosylated HA1/NP (%). Data are the means ±SD from 3 independent experiments, and different letters indicate significant differences; P < 0.05.

Figure 4.

Figure 4

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Morphology observation and NP gene expression of rDk8ΔG and r8GMΔG. A B, Morphology of rDk8ΔG (A) and r8GMΔG (B) was observed by transmission electron microscopy. C, The cRNA, mRNA, and vRNA expression levels of NP genes in AIVs-infected MDCK cells were estimated by quantitative RT-PCR. All samples were normalized to rDk8ΔG at 1 hpi. Data are the means ±SD from 3 independent experiments (*P < 0.05).

The Recombinant Virus With Optimum Backbone Induces a Higher Antibody Response and Immune Protection

To evaluate their immunogenicity and protection efficacy, the recombinant viruses rΔG, rPΔG, r8GMΔG, C-rΔG, C-rPΔG, and C-r8GMΔG were selected. Viruses with identical HA titers of 8Log2 were inactivated and emulsified with white oil adjuvants for chicken vaccination. Serum samples were collected to determine HI and MNT titers (Figures 5A and 5B). In the first week after immunization, the groups exhibited no significant differences in HI titers. In the second week, the HI titers induced by the r8GMΔG group as well as the C-r8GMΔG group were significantly higher than that induced by rPΔG or C-rPΔG and were similar to those induced by rΔG or C-rΔG. There were no significant differences between the chicken embryo cultured r8GMΔG group and the suspension cell cultured C-r8GMΔG group. The HI titer trends in the third week were similar to that in the second week; however, the HI titer induced by the C-rΔG group was lower than that of the C-r8GMΔG group. The MNT titers in the third-week postvaccination were positively correlated with HI titers (Figure 5B). The rΔG, r8GMΔG, and C-r8GMΔG groups induced similar MNT titers that were higher than those of the C-rΔG and C-rPΔG groups.

Figure 5.

Figure 5

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Hemagglutination inhibition (A) and microneutralization (B) titers of chicken vaccination sera. HI titers of sera were determined at each week postvaccination for 3 wk. MNT titers of sera were determined in the third-week postvaccination. Different letters indicate significant differences; P < 0.05.

After the challenge with H5N6 virus CkG strain, all nine chickens in the mock group died within 2 d, with the remaining chicken succumbed by d 4, whereas the chickens in all vaccine groups exhibited a 100% survival rate (Table 3). Viral shedding was not detected in any of the egg-based vaccine groups. However, 30% viral shedding was observed in the chickens vaccinated with the C-rPΔG strain.

Table 3.

Protective efficacy induced by inactivated vaccine candidates in SPF chickens.

Group Shedding rate (No. of shedding/No. of total)
 Protection rate (%)
3 d
 5 d
 7 d
Cloacal Oropharyngeal Cloacal Oropharyngeal Cloacal Oropharyngeal
rΔG 0/10 0/10 0/10 0/10 0/10 0/10 100
rPΔG 0/10 0/10 0/10 0/10 0/10 0/10 100
r8GMΔG 0/10 0/10 0/10 0/10 0/10 0/10 100
C-ΔG 0/10 0/10 0/10 0/10 0/10 0/10 100
C-rPΔG 0/10 2/10 0/10 3/10 0/10 0/10 70
C-r8GMΔG 0/10 0/10 0/10 0/10 0/10 0/10 100
Mock 1/11 1/1 -2 - - - 0
Control 0/10 0/10 0/10 0/10 0/10 0/10 /
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1Only one chicken survived on 3 d postchallenge.

2

All chickens died within 4 d postchallenge.

The Optimized Backbone Exhibits a High Degree of Adaptability for the Updating of Surface Antigens

To confirm if the backbone 8GM is suitable for developing other AIV vaccine candidates, we rescued rΔ8, rPΔ8, and r8GMΔ8 with a modification of the basic cleavage site of HA gene from the Dk8 strain(Δ8). The modified HA segment from the latest epidemic clade 2.3.4.4b H5 subtype AIV CkY strain(ΔY) was used as a donor for surface proteins. The 8GM-based vaccine candidates exhibited higher HA, TCID50, and EID50 titers than PR8-based (Table 4).

Table 4.

Viral titers of the recombined vaccine candidates with surface proteins from clade 2.3.4.4b H5 AIV strains.

Viruses HA titer(Log2) Titer (Log10TCID50/mL)
 Titer (Log10EID50/mL)
CEF MDCK
rΔ8 9.500±0.500 8.722±1.110 6.666±0.665 8.417±0.589
rPΔ8 8.667±2.082 6.5±0.000 6.584±0.118 8.584±0.118
r8GMΔ8 9.833±1.169 9.04±0.698 7.087±0.714 9.22±0.631
rPΔY 7.667±0.577 7.750±0.354 7.415±0.120 7.750±0.354
r8GMΔY 8.330±0.577 8.084±0.589 7.750±0.354 9.667±0.235
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DISCUSSION

In China, although a diverse range of vaccine types, including attenuated live vaccines (Sun, et al., 2023), recombinant vaccines (Qiao, et al., 2009; Zhao, et al., 2024), virus-like particles (Park, et al., 2023), DNA vaccines (Xu, et al., 2022a), have been developed, inactivated vaccines are the predominant strategy for preventing and controlling H5 subtype influenza. In clinical applications, developing high-titer vaccines is crucial for cost control while considering factors such as the inherent specificity of a given strain (Ping, et al., 2015), matrix elements (Kistner, et al., 1998; Pau, et al., 2001; Tree, et al., 2001), culture, and infection (Frensing, et al., 2014; Klenk et al., 1975). Site mutations or modifications of surface proteins are beneficial for improving titers. HA K119N (Nicolson, et al., 2012), N133D (Chen, et al., 2014), N149D, G196E, and L217Q (Ridenour, et al., 2015) have all been observed to increase the HA protein yield. Long NA stalks in recombinant H5N1 vaccine candidates increase the viral yield in eggs and MDCK cells (Zhang, et al., 2011). As the HA and NA genes of the prevailing influenza viruses are gene donors for vaccine candidates, more efforts have been focused on optimizing the backbone to improve replication efficiency. Y360 of PR8 PB2 is responsible for viral enhancement of polymerase activity and viral growth in MDCK cells but not in eggs (Murakami, et al., 2008). PB1 and HA from the same source in the PR8 internal backbone enhanced the replication efficiency of the H1N1 2009pdm vaccine candidate in embryonated eggs and cell culture (Mostafa, et al., 2016; Almeida, et al., 2022). Additionally, the PA E31K mutant of the PR8 internal backbone in the avian influenza vaccine exhibited enhanced replication in Vero cells (Lee, et al., 2017b). The NS2 K86R mutation in the PR8 internal backbone also promoted the growth of the PR8 vaccine strain in Vero cells (Zhang, et al., 2015a).

In this study, we attempted to identify an avian-origin backbone for avian influenza vaccine development. After screening, the H5 subtype AIV Dk8 strain was observed to efficiently replicate in both embryonated eggs and MDCK cell cultures. The majority of internal genes from LPAIV Dk8 strain confer higher replicative capacity than those predominantly from H5 CkG strain. Using a single-segment substitution strategy, substituting the M gene from the H5 subtype AIV CkG strain into the internal backbone of virus Dk8 strain significantly increased viral titers in MDCK cells.

Vaccine candidates possessing the optimized donor 8GM exhibited higher growth titers compared to Dk8 in cell-cultured from 24 to 72 hpi, and significantly higher titers in egg-cultured from 24 to 60 hpi. It means that 8GM exhibited enhanced viral yields in both MDCK cell cultures and chicken embryonated eggs, thus indicating that the M gene from the H5 subtype AIV CkG strain contributes to the high replication ability of 8GM-based vaccine candidates. The finding is consistent with the previous study, which suggests that incorporating the M gene from H5 into the H9N2 backbone can enhance viral replication (Arai, et al., 2019).

Genetic analysis revealed that the M segment of the CkG strain was derived from H5 viruses, whereas the M segment of Dk8 strain was classified as LPAIV (Okazaki, et al., 2000; Duan, et al., 2007; Bi, et al., 2016) (Figure. S1). Li et al reported that the matrix protein 1 (M1) derived from the H9N2 virus contributes to the enhanced replication capacity of the H5N6 virus in mammalian cells (Li, et al., 2021). Mutation at the M1 site results in the morphological transformation of viral particles (Elleman, et al., 2004), thus influencing the titration of chicken embryos (Kilbourne, et al., 1960). In this study, we observed that alterations in the M segment did not affect the morphology of virus particles. r8GMΔG exhibited higher expression of cRNA, mRNA, and vRNA levels during the initial replication round compared to those of rDk8ΔG (Figure 4C). Further, the HA1/NP rate of 8GMΔG was higher than that of rDk8ΔG. Our findings suggest that the M gene of viral CkG strain is pivotal for enhancing vaccine production, possibly by influencing gene expression.

Previous studies have demonstrated that the same vaccine strain induces higher HI and IgG titers in cell-based candidates than in egg-based candidates (Shin, et al., 2015). In this study, although the HA titers of vaccine candidates were standardized to 8Log2, the HA titers of rPΔG and C-rPΔG following the inactivation decreased to 6Log2, while the HA titers of the remaining groups maintained at 8Log2. This indicated that the vaccine candidates with the PR8 internal backbone are not stable during the inactivation of formalin. Consistent with the HA titer, both r8GMΔG and C-8GMΔG induced higher HI and MNT titers than rPΔG and C-rPΔG. However, there were no differences in HI and MNT titers between r8GMΔG and C-8GMΔG. Animal experiments revealed that all vaccine candidates provided complete survival protection after H5 subtype AIV challenge. However, protection against shedding varied among the vaccination groups. Parallel analysis showed that the immunized chickens with an HI titer < 5Log2 were more likely to shed, while no virus shedding occurred in the immunized chickens with an HI titer of ≥5Log2.

The PR8 backbone is extensively utilized, and the surface protein of the current H5 subtype vaccine in China has been updated to Re-13 and Re-14 (Zeng, et al., 2022). In some cases, the H5 subtype recombinant virus with the PR8 backbone exhibits suboptimal growth in eggs and/ or cell culture (Stephenson, et al., 2006). Therefore, a universal avian-origin vaccine backbone can effectively match the prevailing surface proteins, facilitating the efficient production of recombinant viruses during an update to the H5 epidemic strain.

In summary, we developed an avian-origin backbone with high replication ability in chicken embryonated eggs and MDCK cells. Substitution of the M gene from CkG in Dk8 can improve viral replication in MDCK cells. The 8GM-based vaccine candidates exhibited good immunogenicity and immune protection, thus providing a promising alternative to the traditional PR8-based avian influenza vaccines.

DISCLOSURES

The authors declare no conflicts of interest.

ACKNOWLEDGMENTS

This work was supported by the National Key R&D Project (2022YFD1801001-4), National Natural Science Foundation of China (32373042, 32172942), the Yangzhou University “Jie Bang Gua Shuai” Project (YZUXK202316), the Agricultural Science and Technology Independent Innovation Fund of Jiangsu Province (SCX[22]3547), the Jiangsu Province University Outstanding Science and Technology Innovation Team Project (([2021] NO.1), 111 Project D18007), and the Priority Academic Program Development of Jiangsu Higher Education (PAPD).

Author Contributions: P. and F.Y. conceived and designed the study. F. Y., X. Z., C. H., and X. M. performed the experiments. D. P., F. Y., S. C., and T. Q. analyzed the data. D.P. and X.L. provided critical resources. D. P. and F. Y. wrote the manuscript. All the authors contributed to the review and approval of the manuscript.

Footnotes

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.103988.

Appendix. Supplementary materials

mmc1.docx (1.6MB, docx)
mmc2.docx (74.9KB, docx)
mmc3.docx (16.4KB, docx)
mmc4.docx (16.3KB, docx)

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