Genetic and Immunological Characterization of Commercial Infectious Bronchitis Virus (IBV) Vaccines Used in Korea

doi:10.21203/rs.3.rs-1387715/v1

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Genetic and Immunological Characterization of Commercial Infectious Bronchitis Virus (IBV) Vaccines Used in Korea

https://doi.org/10.21203/rs.3.rs-1387715/v1

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Infectious bronchitis virus (IBV) is the avian coronavirus that was first isolated eight decades ago and it has remained as a major contagious pathogen in the poultry industry. Although vaccination is considered the most effective preventive strategy, continuously emerging variants prohibit vaccine efficacy and cross protection. In the last two decades, because nephropathogenic strains have been prevalent, the basic structure of numerous live attenuated vaccines was based on them. For a better understanding of their antigenic and immunogenic properties, this study obtained three commercial IBV vaccines commercially available in Korea and analyzed their genetic and immunogenic features. S1 gene sequence comparison with other field strains clustered those vaccine strains into the GI-19 or GI-15 lineage. The genetic variance of vaccine strains from their parental viruses was mostly detected in HVRI. Although each vaccine strain targets both respiratory and nephrotropic IBVs, their amino acid residues were not comparable at multiple locations. Conversely, antigenic stimulation with commercial vaccines and regional IBV variants was not sufficient to modify major immune cell phenotypes. Our study suggests that vaccine selection should be carefully considered according to their structural background because genetic variance can still exist even during the manufacturing process. Further investigation of the precise immune response by each vaccine strain can improve quarantine strategies.

Infectious bronchitis virus (IBV) is the first isolated coronaviruses in the 1930s and has caused enormous economic damage globally. Among its four major structural proteins, the nucleocapsid (N), membrane (M), envelope (E), and spike (S) proteins, the S glycoprotein mediates the core events of infection, such as host invasion, host range, cell tropism, and induction of host immune responses. The S glycoprotein is further cleaved into two subunits. The S1 subunit mediates host cell attachment, which initiates immune responses, and the S2 subunit bridges the viral membrane with the S1 subunit. RNA viruses mutate faster than do DNA viruses and positive-sense single-stranded RNA viruses such as IBV have the highest mutation and substitution rates among all RNA viruses [1, 2, 10, 32]. Specifically, genetic mutation and recombination of IBV occur continuously in hypervariable regions (HVRs) of the S1 glycoprotein because of poor viral polymerase proofreading mechanism. HVRs are divided into three parts: HVR I (amino acid residues 38 ~ 67), HVR II (amino acid residues 91 ~ 141), and HVR III (amino acid residues 274 ~ 387). This results in the continuous emergence of variants, which prohibits vaccine efficacy and cross protection [1, 2, 25, 32]. The IBV variants are clustered into six genetically divergent groups and are further classified into 32 lineages that include genotype I (GI), with 27 lineages, and other five genotypes that are dependent on the S1 glycoprotein sequence [38].

IBV infection prevention can be managed by optimizing the biosecurity of the housing environment. For example, ventilation and light management and the control of flock size can reduce the risk of IBV spread in farms [8]. However, the most effective strategy for protection against this virus is thought to be achieved by vaccination. Compare with other farm animals, the life-span of broilers is much shorter and intensive vaccination is given at a very young age. Because of the heterogenicity of variants, IBV vaccines have been developed based on the antigenicity of regional isolates. The original IBV vaccine was designed from the Mass serotype, which belongs to the GI-1 lineage and had been considered a core strain in the IBV vaccine platform. However, novel variants, such as nephropathogenic strains, were detected that induce a complicated pathogenesis. They initially invade the respiratory epithelium where they begin replicating. Then, the pathogen penetrates deeper epithelial layer and further spreads to remote organs, such as the kidney, liver, and spleen via blood vessels [30]. These novel IBVs originated from the QX strain from China, and QX-like strains were further spread to European and Asian countries. Most of them are clustered into the GI-19 lineage [21, 40, 41]. This prompted a debate regarding the benefit of heterologous vs. homologous vaccination because of possible divergent and unexpected immunity to heterogeneous field strains. Moreover, the risk of vaccine-escape variants exist [11]. Thus, the introduction of the protectotype concept, which combines two heterologous vaccine strains for an additive protection effect has been suggested [15].

The major IBV vaccine type is live attenuated, which is constituted according to emerging variants. Therefore, a critical step in vaccine development is the identification of field strains, antigenicity of which determines vaccine structure. Both inappropriate vaccine selection and a poor application strategy in the field tend to increase the risk of generation of unexpected variants. For instance, massive-delivery methods lower vaccine efficiency which can lead to the mutation and recombination of field viruses and vaccine strains. Although a proper vaccine strain is chosen, its possible reversion to virulence can accelerate novel virus derivation [12, 13]. Consequently, frequent vaccine alterations and lowered cross protection can render protection difficult. After the initial IBV outbreak of 1986, live attenuated vaccines derived from the Mass strain (H120 and Ma5) were widely used in Korea up to the late 1990s. The subsequent emergence of nephrotropic strains prompted the development of the K2 vaccine strain, which is based on a Korean native strain, KM91. In the 2000s, a novel respiratory strain (KI strain) was detected in Korea and other new variants were reported thereafter [7, 34]. We also analyzed the genetic feature of the novel IBV strains, K046-12 and K047-12, which were correlated with other Korean isolates and QX-like strains, respectively [24].

Despite the development of numerous vaccines for long-lasting immunity and cross protection against multiple viral strains, the repeated emergence of variants has increased the demand for restored vaccine development. For better control of IBV and execution of vaccine strategies, evaluation of the genetic stability and subpopulations of commercial vaccines is necessary. To this end, we analyzed the genetic sequence of the S1 glycoprotein in three commercial vaccine strains. Using a phylogenetic analysis, we examined the genetic closeness between vaccine strains and their parental strains and regional variants. The genetic stability of vaccine strains was evaluated by comparing their amino acid sequences. Moreover, the immune responses elicited by antigens from vaccine strains and regional variants were examined.

Vaccines and viruses

Three commercial live-attenuated IBV vaccines produced by different manufacturers were collected. These vaccines included the K40/09, the Kr/D85/06, and a recombination of the KM91 and QX IBV strains. Here, they were coded as A, B and C, respectively, and their features and GenBank accession numbers are shown in Table 1. As previously reported, the lyophilized vaccines were reconstituted in PBS for the sequence analysis [39]. In addition, Vaccine B was propagated in 9 ~ 11-day-old specific pathogen free (SPF) embryonated chicken eggs (Sungmin Farm, Korea) and a single passage of Vaccine B was performed.

Table 1

List of the commercial vaccine strains selected for this study. The parental strains, target, and GenBank accession numbers of each vaccine are indicated.
Name	Parental strain	Target	GenBank ID
Vaccine A	K40/09	Respiratory and renal variants	OM685193
Vaccine B	D85/06	Respiratory and renal variants	OM685194
Vaccine B_single passage	D85/06	Respiratory and renal variants	OM685195
Vaccine C	KM91 & QX-like	Respiratory and renal variants	OM685196

PCR and cloning of the S1 gene

Total RNA of each vaccine was isolated using the TRIzol® Reagent (Invitrogen™) and then reverse transcribed into cDNA with AccuPower® RT PreMix (BIONEER) using an S1000™ Thermal Cycler (Bio-Rad) according to the manufacturer’s instructions. Each reaction was carried out in a final volume of 20 µl and the resultant cDNA was immediately used for conventional PCR or stored at -70°C for later use. The S1 gene of the vaccines was amplified by conventional PCR using AccuPower® PCR PreMix (Bioneer). The primer sets (forward: 5′-TAG TGA CCC TTT TGT GTG CAC TAT-3′ and reverse: 5′-GTT TGT ATG TAC TCA TCT GTA AC-3′) were used as reported previously [23]. PCR was performed using a PCR Thermal Cycler Dice™ Touch (TaKaRa Bio Inc., Australia) and the following conditions: predenaturation at 94°C for 5 min; followed by 35 cycles of denaturation (94°C, 90 s), annealing (45°C, 30 s), and polymerization (72°C, 90 s); and a postpolymerization step was at 72°C for 3 min. The amplified sequences were analyzed by 1% agarose gel electrophoresis. PCR products from the three vaccines and the single-passaged Vaccine B were purified using a the Gel Extraction kit (QIAGEN), ligated into the TOPO TA pCR®2.1 vector and transformed into One Shot®TOP10 competent E.coli (Invitrogen), as recommended by the manufacturer. Transformants were grown in Luria-Bertani (LB) medium supplemented with kanamycin (50 ng/µl). Plasmid purification was performed using a DNA-spin Plasmid DNA purification kit (iNtRON) and the isolates were sent to Macrogen Inc. (Seoul, South Korea) for sequencing using the universal primers M13F (5′-GTA AAA CGA CGG CCA GT-3′) and M13R-pUC (5′-CAG GAA ACA GCT ATG AC-3′).

Sequence comparisons and phylogenetic analysis

The sequences received from Macrogen were analyzed using the Lasergene package version 10 (DNASTAR, Inc., USA) and deposited in GenBank as follows: Vaccine A, OM685193; Vaccine B, OM685194; Vaccine B_single passage, OM685195; Vaccine C, OM685196. The nucleotide sequences were aligned via the Clustal W method using the DNASTAR 2.0 MegAlign program and the phylogenetic tree was constructed via the neighbor-joining method with 1,000 bootstrap replicates using MEGA X software (version 10.2.2). The nucleotide sequences of the S glycoproteins, for comparison, were downloaded from the NCBI GenBank database as follows: Conn48725 (FJ899692.1), Beaudette (M95169.1), M41 (AY561711.1), H120 (FJ888351.1), K046-12 (MK618758.1), BP-CaKII (MF924724.1), QXIBV (AF193423.1), K40/09 (HM486957.1), K047-12 (MK618759.1), ArkDPI (AF006624.1), GX2-98 (AY251816.1), K210-02 (AY257068.1), and Kr/D85/06 (EF621400.1). The chromatograms were obtained and reviewed manually using BioEdit version 7.2.5 to detect the presence of viral subpopulations in the vaccines.

Immune cell preparation and culture

Total splenocytes were collected from 4 ~ 5-week-old SPF chickens (Sungmin Farm, Korea). After RBS lysis, cells were seeded into 96-well U-bottom plates (5 x 10⁵ cells/well) and cultured for 4 ~ 5 days with Concanavalin A (5 µg/ml) (Sigma Aldrich, MO) and chicken IL-2 (10 ng/ml) (Kingfisher Biotech, MN, USA) in RPMI-1640 supplemented with 10% FBS. For antigenic stimulation, vaccines and IBV variants suspension (K046-12 and K047-12, 10 µL/mL) were added to the culture. The animal work was approved by the Institutional Animal Care and Use Committee (IACUC) of Kangwon National University (No: KW-211021-6).

Cell staining and flow cytometry

Cultured splenocytes were harvested and stained with the LIVE/DEAD Fixable Aqua Dead Cell Stain (L3457; Thermo Fisher) and antibodies specific for CD4 (CT-4, SouthernBiotech), CD8α (CT-8, SouthernBiotech), CD44 (AV6, SouthernBiotech), Monocyte/Macrophage (KUK01, SouthernBiotech), MHCII (2G11, SouthernBiotech), Bu-1 (AV20, SouthernBiotech). Stained cells were analyzed using flow cytometry (Cytoflex, Beckman Coulter).

Statistics

Dunnett’s test with ANOVA was used to assess the statistical significance of the results obtained from the viral-antigen-stimulated groups compared with the untreated group. Comparisons among the stimulated groups were analyzed by one-way ANOVA. Significance was set as P < 0.05.

Cloning of the S1 glycoprotein gene from commercial vaccine strains

The PCR products of the S1 glycoprotein gene from three different commercial vaccine strains and the single-passaged commercial vaccine strain were inserted into the pCR®2.1 vector, which contains a kanamycin resistance ORF region (Fig. 1A). The inserted PCR product (~ 1,400 bp) was verified by digestion of cloned plasmids with EcoRI, followed by gel electrophoresis (Fig. 1B). This procedure confirmed that the suitable preparation for the DNA sequencing of the S1 gene.

Classification of vaccine strains and IBV variants based on S1 glycoprotein gene

Using the determined sequences of the full-length S1 gene in commercial vaccines (Fig. S1), we compared genetic relatedness to their parental strains and ten different field strains, which were included in five distinct lineages. Moreover, two Korean isolates (K046-12 and K047-12; the genetic features of which were reported by us in 2019) [24] were added in the clustering. The S1 sequences of Vaccines A and C were grouped in the GI-19 lineage, which includes nephrotropic strains such as the QX-strain and its regional variant, K047-12. As expected, Vaccine A was genetically close to its parental strain, K40/09, and its passaged strain, K40/09 CE50. In contrast, Vaccine C was genetically more related to the K40/09 strain compared with the KM91 and QX-like strains. Vaccine B and its single-passaged form were grouped in the GI-15 lineage together with their parental strain, D85/06. Otherwise, none of the commercial vaccine strains were allotted to the conventional respiratory GI-1 lineage, which comprises the Connecticut, Beaudette, M41, and H120 strains (Fig. 2). Although most of the recent vaccines target nephrotropic strains that induce a complicated pathology [30], the recent variant K046-12 was genetically remote from the three commercial vaccine strains. This phylogenetic analysis indicated that genetic features of the S1 gene of IBV in commercial vaccines are clustered according to their parental strains, but do not completely cover emerging variants.

Amino acid variance in S1 glycoprotein among vaccine and parental strains

The S1 glycoprotein consists of 520 amino acid residues and induces a neutralizing antibody response. Because of frequent genetic modification in the HVRs, viral clearance in the host is prohibited. The major HVRs of the S1 glycoprotein have multiple locations, i.e., at amino acid residues 38 ~ 67, 91 ~ 141, and 274 ~ 387 [3, 25]. To evaluate the genetic modification of vaccines from their parental strains, we compared the amino acid arrangement in HVRs and HVR-adjacent regions. We found that Vaccine A, which originated from a nephrotropic virus, showed polymorphism within HVRs compared with the parental strain K40/09 and its passaged strain, K40/09 CE50. The amino acid at position 83 was an asparagine (Asn) in the K40/09 strains, whereas it was an aspartic acid (Asp) in the Vaccine A strain. Similarly, the amino acids at positions 97 and 365 were serine (Ser) and isoleucine (Ile) in the K40/09 strains, whereas they were phenylalanine (Phe) and valine (Val) in the Vaccine A strain. Conversely in the HVRIII-adjacent area (412 amino acid residues), all three vaccine strains carried a glutamic acid (Glu), with a point mutation detected in Vaccine A exclusively. This indicates that the difference from K40/09 was more obvious in Vaccine A than changes occurring after 50 passages (Fig. 3A). Amino acid variance in Vaccine B was mostly observed adjacent to HVRI at position 8, 11, and 13, compared with its parental strain, D85/06. AVR1/08 is an attenuated vaccine strain that was passaged 89 times [7], and its genetic similarity in the upstream of S1 glycoprotein was higher with D85/06 than it was with Vaccine B. The amino acids encoded were valine (Val), isoleucine (Ile), and phenylalanine (Phe) for D85/06 and AVR1/08, whereas the Vaccine B strains carried leucines (Leu) or cysteine (Cys). Moreover, at the 56th position, D85/06 encoded serine (Ser) but other strains encoded tyrosine (Tyr). In addition, a single passage of Vaccine B yielded a change from glutamic acid (Glu) to glycine (Gly) at HVRII (amino acid residue number 96) (Fig. 3B). Despite the overall similarity in the whole S1 genomic sequence, the vaccine strains under study were composed of multiple polymorphisms exclusively among HVRs and the upstream of S1 glycoprotein.

Genetic variance of the S1 glycoprotein among commercial vaccines and new variants

Next, we addressed the genetic similarity of the S1 gene among the three commercial vaccine strains. Dissimilarity was detected before HVRI and II at the 83rd, 257th, and 262nd amino acid positions. Although Vaccines A and C were genetically closer in terms of the whole S1 protein sequence (Fig. 2), Vaccines A and B exhibited an identical amino acid sequence near HVRI and II (Fig. 4A). Furthermore, at the 315th amino acid position, Vaccine C showed a second peak that encoded serine (Ser), whereas the major peak of each of three strains encoded cysteine (Cys). This suggests the existence of two viral subpopulations in a vaccine strain (Fig. 4B). Because the three vaccines are designed to deal with regional variants, we examined whether these vaccines are genetically close to emerging Korean variants, the genetic features of which were previously analyzed by us [24]. The amino acid sequences of HVRI (up to the 130th position) from K046-12 and K047-12 were aligned with those of the three commercial vaccine strains. Heterogeneity was most frequently observed around the 30 ~ 50th and 120 ~ 130th amino acid positions (Fig. 5A). To further analyze the genetic proximity regarding HVRI, we drew another phylogenetic tree based on the amino acid sequences of this region. We found that Vaccines A and C were grouped with the nephrotropic variant, K047-12, whereas Vaccine B was less associated. Moreover, none of the vaccine strains showed genetic closeness to the respiratory variant, K046-12 (Fig. 5B). The result of this analysis agrees with the phylogenetic tree of whole S1 gene sequences, which implies that HVRI variation is a dominant factor that attributes genetic characteristics to both field and vaccine strains.

Immune responses by commercial vaccines and regional variants

We recently reported the action of the nephrotropic variant K047-12 on the innate immune response and identified key immunological factors that can govern the acute response in chicken embryonic kidney (CEK) cells [20]. The avian spleen, similar to that of mammals, is a major secondary lymphoid tissue in which various immune cells, such as lymphocytes and myeloid cells regulate adaptive immunity. In the spleen, dendritic cells and macrophages recognize antigens and present them to lymphocytes, i.e., T and B cells. Activated lymphocytes differentiate into effector cells, thus initiating both cellular and humoral immune responses [14, 36]. We examined the manner in which viral antigens in vaccine strains and regional variants affect the immune cell phenotype. Total splenocytes from 4 ~ 5 week-old SPF chickens were stimulated with Concanavalin A and chicken IL-2 for 4 ~ 5 days in the presence of the three vaccine strains or the regional variants (K046-12 and K047-12). To examine the memory T cell population, which potentially interacts with viral antigens, we stained the surface of CD4⁺ and CD8⁺ T cells with an anti-CD44 antibody. None of the treatments changed the frequencies of memory CD4⁺ and CD8⁺ T cells (Fig. 6A). To assess the antigen-presenting capacity of macrophages, monocytes were stained with an antibody to MHCII molecule. Although IBVs induced a bit more activated macrophages to a slightly greater extent, significant differences between the variant and vaccine strains were hardly observed. A similar trend was observed for B cell frequency (Fig. 6B and C). These data illustrate the contention that different antigenic stimulations in vitro are not sufficient to change the surface phenotype of major immune cells. Further functional analyses, such as those of intracellular molecules and cytokine regulation within each cell type, are warranted to evaluate these issues.

The first IBV vaccine was developed based on the Mass serotype and was as the only licensed vaccine for many years. Since the isolation of novel IBV serotypes from the GI-19 lineage, vaccine construction has been updated depending on emerging variants. In addition, two or more serotypes are combined in vaccine programs. Currently, numerous IBV vaccines are available and the selection of a proper vaccine strain against regional variants is critical [16]. To better evaluate vaccine stability and safety, it is necessary to identify genetic variation from the parental strains as well as the immune response elicited by vaccination. Thus, we obtained three vaccines that are commercially available in Korea and assessed their genetic sequence encoding the S1 protein together with that of field strains. Subsequently, we investigated the genetic variance of the three vaccine strains compared with the parental strains using S1 gene sequences. The vaccine strains exhibited genetic modification primarily in HVRI and its adjacent region. Near HVRII, genetic variance was also detected. However, compared with the regional variants, antigenic stimulation with the vaccine strains did not significantly change the phenotype of major immune cells.

Vaccines A and C were clustered into the GI-19 lineage, whereas Vaccine B and its passaged strain were included in the GI-15 lineage. Because the majority of emerging IBVs are classified as GI-19, we observed genetic distance between Vaccines A and C and other field strains. The parental strain of Vaccine A, K40/09, was developed from a QX-like strain that was reported in 2011 and is thought to be generated by recombination with a KM91-like strain [22]. When chickens are immunized with K40/09, cross protection against both respiratory and renal IBV strains was shown [17]. Based on our phylogenetic analysis, Vaccine A maintained genetic stability compared with its parental strains. Vaccine A was also close to the new regional variant, K047-12, in terms of the S1 gene sequence. HVRI includes the receptor-binding domain, which determines the interaction between the virus and respiratory tissues. Hence, genetic modification of amino acid residues in this region can generate novel variants [29, 32]. The HVRI amino acid sequences differed between K40/09 and Vaccine A to a certain extent, although genetic variation of HVRI was less observed after 50 passages. Therefore, whether Vaccine A is sufficient to provide broad immunity to novel variants warrants further exploration. Moreover, the potential risk of recombination with field viruses should be evaluated.

KM91 is a native Korean variant that was first isolated in 1991, and KM-like variants have been isolated that exhibit recombination with QX-like strains [6, 20]. According to the manufacturer, Vaccine C is a recombinant of the QX and KM91 strains that targets both respiratory and nephropathogenic IBVs; however it is difficult to determine the parental strain in this case. Our analysis showed that Vaccine C also belonged to the GI-19 lineage, which confirmed that this vaccine stems from the KM91 and QX-like strains. Considering the genetic analysis of the S1 glycoprotein and the information provided by the manufacturer, we suggest that Vaccine C shares a genetic background with Vaccine A. However, the efficacy of the vaccines to new regional respiratory variants, such as K046-12, which belongs to the GI-1 lineage, remains to be examined.

The D85/06 strain was isolated from Korea in 2006 and clustered into the K-I type together with other respiratory IBV strains. The AVR1/08 strain was developed from the D85/06 strain via 89 passages. A point mutation was observed at the 56th amino acid position of AVR1/08 after 47 passages, which attenuated the virulence of the strain [5, 19]. Vaccine B was introduced from AVR1/08 and showed genetic modification at the upstream of HVRI. We performed a single passage of Vaccine B to evaluate its genetic stability and detected a point mutation in HVRI after this procedure. According to a previous report, AVR1/08 yielded broad protection against both regional respiratory and renal IBVs [7]. However, our phylogenetic analysis revealed that Vaccine B was grouped into the GI-15 lineage, which is genetically separated from the major nephropathogenic strains from the GI-19 lineage. Moreover, Vaccine B was genetically distant from respiratory strains, such as M41 or Beaudette and the regional isolate, K046-12. The investigation of the genetic variance among the three vaccine strains revealed a genetic difference was only observed outside HVRs with the exception of the second peak at the 315th amino acid position in Vaccine C. To estimate the protective efficacy of vaccines against novel IBVs, we aligned the HVRI amino acid sequences of the three commercial vaccines with those of the regional isolates K046-12 and K047-12. A major modification was observed around upstream of the HVRI. Of note, the phylogenetic distance of HVRI was comparable with phylogenetic results obtained for the whole S1 gene sequence. To some extent, our results suggest that protectotype conception is applicable to the three vaccine strains, whereas an alternative vaccine for respiratory strain is necessary to provide broader protection against emerging variants.

The induction of adaptive immunity is accelerated when an IBV-specific epitope stimulates the cytotoxic T lymphocyte (CTL) response. An S1 glycoprotein-associated peptide can work as a CTL epitope. When chicken splenocytes were activated with a peptide epitope, IFN-γ production and CD8⁺ T cell proliferation were facilitated. Furthermore, administration of this epitope induced a DNA vaccine-like effect and protected the host from the IBV challenge [33, 37]. The major purpose of vaccination is to induce specific immune responses against an invading pathogen. However, IBV variants are likely to escape host immune surveillance by modifying their antigenicity because genetic modification of the S1 glycoprotein alters the affinity of the virus for host cell receptors. As shown previously, protection can be achieved by adoptive transfer of memory CD8⁺ T cells. The key protective action was mediated by memory T cells that had experienced the IBV antigen [27]. IBV variants with dissimilar amino acid residues in the S1 glycoprotein are less likely to express a coherent epitope. However, we found that the S1 antigenic variance was not sufficient to modify the immune cell phenotype. When splenocytes were stimulated with Concanavalin A, memory CD8⁺ (CD8⁺CD44⁺) T cells occupied more than 50% of the total CD8⁺ T cell; however, the proportion of memory CD4⁺ (CD4⁺CD44⁺) T cells was < 20% of the total CD4⁺ T cell. Moreover, the frequencies of each subset of T cells were not changed by antigenic stimulation with IBV variants or vaccine strains. This implies that an immune-boosting strategy needs to be considered during vaccine development. Although phenotypic change was not observed here, during infection, inhibitory receptors (IRs) are expressed on surface of T cells and regulates T cell activation together with T cell receptor (TCR) signal. IR activation can be mediated by macrophage-originated cytokine, such as IL-27. Consequently, the T cell phenotype can be shaped expression of IRs, such as PD-1, CTLA-4, TIGIT, and LAG-3 [4, 9]. Although studies of chicken IRs have yet to be well established, and detection antibodies are not currently available, a recent study reported an analysis of chicken IRs such as chPD-1 and chPD-L1. This is useful for identifying unknown features of T cells during IBV infection [31]. Antigen presentation and cytokine production by macrophages are required for the induction of pathogen-specific T cell activity. For example, IFN-γ production in CTLs is mediated by M1 macrophage generation during intracellular pathogen infection [26]. The role of macrophages in IBV infection is also crucial. Upon infection with the M41 variant, the viability and phagocytic function of macrophages are inhibited. Conversely, overall innate immunity was enhanced such as antimicrobial activity, toll like receptor (TLR) activation, and type I IFN or pro-inflammatory cytokine induction [35]. We detected a minimal number of activated macrophages that expressed MHCII molecules on their surface, probably because of the optimal inducing condition for myeloid cells was not provided. Therefore, a comprehensive analysis of viral antigen-induced macrophage activity needs to be carried out using bone marrow-derived myeloid cells and stimulating cytokines. Moreover, revised IBV vaccines that boost MHCII expression on myeloid cells for enhanced antigen presentation can be applied [18]. This will provide improved antigen-specific T cell proliferation and afford immunity geared toward novel variants. Together with cellular immunity, humoral response by B cells is required. IBV-specific antibody-secreting cells (ASCs) are maximally activated around 10 days after IBV infection. However, during in vitro culture, splenic ASCs are transiently observed only when restimulated with the viral antigen [28]. In our splenocyte culture system, viral antigens enhanced B cell frequency but the level of the response did not rely on antigens. Therefore, kinetic observations and modified culture conditions need to be further considered.

The inconsistent antigenic phenotype of IBV variants is a major hurdle for IB prevention, despite the continuous vaccine development of vaccines. The majority of IBV vaccines are of the live-attenuated type, which requires stability during the manufacturing process. In the host, vaccines should be safe and provide long-lasting immunity to multiple variants. Throughout this study, we identified the genetic features and stability of IBV vaccines that are currently available in Korea. In addition, the possible host immune response was examined after the stimulation of host cells with the viral antigen. Our study provides guidance for the evaluation of commercial vaccines and suggestions for the new IBV vaccine development.

Acknowledgments

This study was supported by funding from a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (no. 2022R1C1C1002793).

Belouzard S, Millet JK, Licitra BN, Whittaker GR (2012) Mechanisms of coronavirus cell entry mediated by the viral spike protein. Viruses 4:1011-1033
Cavanagh D (1983) Coronavirus IBV Giycopolypeptides: Size of Their Polypeptide Moieties and Nature of Their Oligosaccharides. J gen Virol 64:1187-1191
Cavanagh D, Davis PJ, Mockett AA (1988) Amino acids within hypervariable region 1 of avian coronavirus IBV (Massachusetts serotype) spike glycoprotein are associated with neutralization epitopes. Virus research 11:141-150
Chen DY, Wolski D, Aneja J, Matsubara L, Robilotti B, Hauck G, de Sousa PSF, Subudhi S, Fernandes CA, Hoogeveen RC, Kim AY, Lewis-Ximenez L, Lauer GM (2020) Hepatitis C virus-specific CD4+ T cell phenotype and function in different infection outcomes. The Journal of clinical investigation 130:768-773
Choi K-S, Jeon W-J, Lee E-K, Kye S-J, Park M-J, Kwon J-H (2011) Development of an attenuated vaccine strain from a korean respiratory type infectious bronchitis virus. Korean Journal of Veterinary Research 51:193-201
Choi KS, Lee EK, Jeon WJ, Park MJ, Kim JW, Kwon JH (2009) Pathogenicity and antigenicity of a new variant of Korean nephropathogenic infectious bronchitis virus. J Vet Sci 10:357-359
Choi KS, et al. (2011) Development of an attenuated vaccine strain from a korean respiratory type infectious bronchitis virus. Korean J Vet Res 51:193-201
De Wit J, Swart W, Fabri T (2010) Efficacy of infectious bronchitis virus vaccinations in the field: association between the α-IBV IgM response, protection and vaccine application parameters. Avian Pathology 39:123-131
DeLong JH, O’Hara Hall A, Rausch M, Moodley D, Perry J, Park J, Phan AT, Beiting DP, Kedl RM, Hill JA, Hunter CA (2019) IL-27 and TCR Stimulation Promote T Cell Expression of Multiple Inhibitory Receptors. ImmunoHorizons 3:13-25
Duffy S, Shackelton LA, Holmes EC (2008) Rates of evolutionary change in viruses: patterns and determinants. Nature Reviews Genetics 9:267-276
Franzo G, Legnardi M, Tucciarone CM, Drigo M, Martini M, Cecchinato M (2019) Evolution of infectious bronchitis virus in the field after homologous vaccination introduction. Vet Res 50:92
Gallardo RA (2021) Infectious bronchitis virus variants in chickens: evolution, surveillance, control and prevention. Austral journal of veterinary sciences 53:55-62
Guzmán M, Hidalgo H (2020) Live Attenuated Infectious Bronchitis Virus Vaccines in Poultry: Modifying Local Viral Populations Dynamics. Animals 10:2058
Hirakawa R, Nurjanah S, Furukawa K, Murai A, Kikusato M, Nochi T, Toyomizu M (2020) Heat Stress Causes Immune Abnormalities via Massive Damage to Effect Proliferation and Differentiation of Lymphocytes in Broiler Chickens. Frontiers in Veterinary Science 7
Jackwood MW, Jordan BJ, Roh HJ, Hilt DA, Williams SM (2015) Evaluating Protection Against Infectious Bronchitis Virus by Clinical Signs, Ciliostasis, Challenge Virus Detection, and Histopathology. Avian Dis 59:368-374
Jordan B (2017) Vaccination against infectious bronchitis virus: A continuous challenge. Vet Microbiol 206:137-143
Kim BY, Lee DH, Jang JH, Lim TH, Choi SW, Youn HN, Park JK, Lee JB, Park SY, Choi IS, Song CS (2013) Cross-protective immune responses elicited by a Korean variant of infectious bronchitis virus. Avian Dis 57:667-670
Larsen FT, Guldbrandtsen B, Christensen D, Pitcovski J, Kjærup RB, Dalgaard TS (2020) Pustulan activates chicken bone marrow-derived dendritic cells in vitro and promotes ex vivo CD4+ T cell recall response to infectious bronchitis virus. Vaccines 8:226
Lee E-K, Jeon W-J, Lee Y-J, Jeong O-M, Choi J-G, Kwon J-H, Choi K-S (2008) Genetic diversity of avian infectious bronchitis virus isolates in Korea between 2003 and 2006. Avian diseases 52:332-337
Lee HC, Jeong S, Cho AY, Kim KJ, Kim JY, Park DH, Kim HJ, Kwon JH, Song CS (2021) Genomic Analysis of Avian Infectious Bronchitis Viruses Recently Isolated in South Korea Reveals Multiple Introductions of GI-19 Lineage (QX Genotype). Viruses 13
Lee HJ, Youn HN, Kwon JS, Lee YJ, Kim JH, Lee JB, Park SY, Choi IS, Song CS (2010) Characterization of a novel live attenuated infectious bronchitis virus vaccine candidate derived from a Korean nephropathogenic strain. Vaccine 28:2887-2894
Lim TH, Lee HJ, Lee DH, Lee YN, Park JK, Youn HN, Kim MS, Lee JB, Park SY, Choi IS, Song CS (2011) An emerging recombinant cluster of nephropathogenic strains of avian infectious bronchitis virus in Korea. Infect Genet Evol 11:678-685
Lim TH, Kim MS, Jang JH, Lee DH, Park JK, Youn HN, Lee JB, Park SY, Choi IS, Song CS (2012) Live attenuated nephropathogenic infectious bronchitis virus vaccine provides broad cross protection against new variant strains. Poultry Science 91:89-94
Moon H-W, Sung HW, Kwon HM (2019) Genomic characteristics of natural recombinant infectious bronchitis viruses isolated in Korea. Korean Journal of Veterinary Research 59:123-132
Moore KM, Jackwood MW, Hilt DA (1997) Identification of amino acids involved in a serotype and neutralization specific epitope within the s1 subunit of avian infectious bronchitis virus. Arch Virol 142:2249-2256
Park J, Hunter CA (2020) The role of macrophages in protective and pathological responses to Toxoplasma gondii. Parasite immunology 42:e12712
Pei J, Briles WE, Collisson EW (2003) Memory T cells protect chicks from acute infectious bronchitis virus infection. Virology 306:376-384
Pei J, Collisson EW (2005) Specific antibody secreting cells from chickens can be detected by three days and memory B cells by three weeks post-infection with the avian respiratory coronavirus. Developmental and comparative immunology 29:153-160
Promkuntod N, van Eijndhoven RE, de Vrieze G, Gröne A, Verheije MH (2014) Mapping of the receptor-binding domain and amino acids critical for attachment in the spike protein of avian coronavirus infectious bronchitis virus. Virology 448:26-32
Reddy VR, Trus I, Desmarets LM, Li Y, Theuns S, Nauwynck HJ (2016) Productive replication of nephropathogenic infectious bronchitis virus in peripheral blood monocytic cells, a strategy for viral dissemination and kidney infection in chickens. Veterinary research 47:1-19
Reddy VRAP, Mwangi W, Sadigh Y, Nair V (2019) In vitro Interactions of Chicken Programmed Cell Death 1 (PD-1) and PD-1 Ligand-1 (PD-L1). Frontiers in Cellular and Infection Microbiology 9
Rohaim MA, El Naggar RF, Abdelsabour MA, Mohamed MHA, El-Sabagh IM, Munir M (2020) Evolutionary Analysis of Infectious Bronchitis Virus Reveals Marked Genetic Diversity and Recombination Events. Genes 11:605
Seo SH, Wang L, Smith R, Collisson EW (1997) The carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic T lymphocytes and protects chickens from acute infection. Journal of virology 71:7889-7894
Song CS (2017) An Attenuated Infectious bronchitis virus and An Infectious bronchitis vaccine using the same.
Sun X, Wang Z, Shao C, Yu J, Liu H, Chen H, Li L, Wang X, Ren Y, Huang X (2021) Analysis of chicken macrophage functions and gene expressions following infectious bronchitis virus M41 infection. Veterinary research 52:1-15
Sutton KM, Morris KM, Borowska D, Sang H, Kaiser P, Balic A, Vervelde L (2021) Characterization of Conventional Dendritic Cells and Macrophages in the Spleen Using the CSF1R-Reporter Transgenic Chickens. Frontiers in Immunology 12
Tan L, Liao Y, Fan J, Zhang Y, Mao X, Sun Y, Song C, Qiu X, Meng C, Ding C (2016) Prediction and identification of novel IBV S1 protein derived CTL epitopes in chicken. Vaccine 34:380-386
Valastro V, Holmes EC, Britton P, Fusaro A, Jackwood MW, Cattoli G, Monne I (2016) S1 gene-based phylogeny of infectious bronchitis virus: An attempt to harmonize virus classification. Infect Genet Evol 39:349-364
van Santen VL, Toro H (2008) Rapid selection in chickens of subpopulations within ArkDPI-derived infectious bronchitis virus vaccines. Avian Pathol 37:293-306
Wang Y, Wang Y, Zhang Z, Fan G, Jiang Y, Liu X, Ding J, Wang S (1998) Isolation and identification of glandular stomach type IBV (QX IBV) in chickens. Chinese Journal of Animal Quarantine 15:1-3
Worthington KJ, Currie R, Jones RC (2008) A reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in Western Europe from 2002 to 2006. Avian pathology 37:247-257

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Genetic and Immunological Characterization of Commercial Infectious Bronchitis Virus (IBV) Vaccines Used in Korea

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