[Co-expression, purification and bioassay of three avian viral antigens].

To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain, and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine, we co-expressed three avian virus proteins including the fowl adenovirus serotype 4 (FAdV-4) Fiber-2 protein, infectious bursal disease virus (IBDV) VP2 protein and egg-drop syndrome virus (EDSV) Fiber protein in E. coli BL21(DE3) cells after optimization of gene codon, promoter, and tandem expression order. The purified proteins were analyzed by Western blotting and agar gel precipitation (AGP). The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%. Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity. It is the first time to achieve the three different avian virus antigens co-expression and co-purification, which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.

Human respiratory syncytial virus F protein expressed in Pichia pastoris or Escherichia coli induces protective immunity without inducing enhanced respiratory disease in mice.

Human respiratory syncytial virus (hRSV) is the primary cause of severe respiratory tract disease in children and infants as well as in elderly and immunocompromised adults. The fusion protein (F) of hRSV is the major antigen eliciting a neutralizing antibody response and protective immunity in the host, especially those recognizing the prefusion F protein (pre-F). In this study, we made genetic constructs for expression of a recombinant prefusion F protein in Pichia pastoris GS115, called RGF. Using Escherichia coli BL21, we expressed the pre-F and postfusion F protein (Post-F), called RBF and Post-RBF, respectively. RGF and RBF showed high affinity for 5C4, a highly potent monoclonal antibody specific for pre-F. We studied the immunogenicity of RGF and RBF in mice. Compared to mice immunized with formalin-inactivated RSV (FI-RSV), mice immunized with RGF or RBF exhibited superior protective immunity, which was confirmed by serum neutralizing activity and viral clearance after challenge. As judged from the IgG1/IgG2a ratios and numbers of IFN-γ- and IL-4-secreting cells, RGF or RBF with alum adjuvant induced a balanced Th1-biased immune response and produced no signs of enhanced respiratory disease (ERD) upon hRSV challenge. In addition, the immunogenicity and protective efficacy of RGF were superior to those of RBF in mice. Therefore, RGF represents a potential vaccine candidate for the prevention of human infection with hRSV.

Co-expression, purification and bioassay of three avian viral antigens / 生物工程学报

To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain, and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine, we co-expressed three avian virus proteins including the fowl adenovirus serotype 4 (FAdV-4) Fiber-2 protein, infectious bursal disease virus (IBDV) VP2 protein and egg-drop syndrome virus (EDSV) Fiber protein in E. coli BL21(DE3) cells after optimization of gene codon, promoter, and tandem expression order. The purified proteins were analyzed by Western blotting and agar gel precipitation (AGP). The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%. Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity. It is the first time to achieve the three different avian virus antigens co-expression and co-purification, which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.

Establishment of an induced pluripotent cell line from Taiwan black silkie chick embryonic fibroblasts for replication-incompetent virus production.

The objective of this study was to establish a versatile cell line for replication-incompetent virus production and inactivation with formaldehyde to generate a model of cell-based vaccine manufacturing process. To achieve this goal, we took advantage of the easily accessed chick embryonic fibroblasts. Nine-day old chick embryonic fibroblasts were obtained and subjected to be transduced with a set of lentivirus to develop a chick induced pluripotent stem (ciPS) cell line. Morphological features, positive periodic acid-Schiff staining as well as strong immunocytofluorescence of alkaline phosphatase, intestinal (ALPI) and POU class 5 homeobox 1 (POU5F1) proteins suggested that these chick embryonic fibroblasts have been transformed into ciPS cells. Further differentiation and immunocytofluorescence assays confirmed that this ciPS cell line possesses capacities and potentials to form embryoid bodies, differentiate into all three embryonic layers: ectoderm, mesoderm and endoderm with evidence of strongly positive and specific molecular markers. Immunoblot analysis next demonstrated that through recombinant DNA technology and the 2nd generation lentiviral transfer system, the goose hemagglutinin gene (H5) gene was packaged into the replication-incompetent virus and highly expressed in a bladder cancer-derived cell line, T24, after transduction. The titer of ciPS-generated replication-incompetent virus is comparable to that from the Phoenix-AMPHO cell line, which is a commercial and high productive retrovirus producer. Our study successfully established a ciPS cell line which is able to produce replication-incompetent virus, providing a new strategy for cell-based vaccine production after virus inactivation.

An Escherichia coli-Expressed Porcine Reproductive and Respiratory Syndrome Virus Chimeric Protein Induces a Specific Immunoglobulin G Response in Immunized Piglets.

Porcine reproductive and respiratory syndrome virus (PRRSV) still poses a threat to the swine industry worldwide. Currently, commercial vaccines against PRRSV, which consist of modified live or inactivated virus, reduce symptoms and viremia in immunized pigs, but efficacy against heterologous strains is variable. This has led to the development of subunit vaccines that contain viral antigens that show the highest variability. In this work, a chimeric protein comprising short amino acid sequences from glycoprotein 3 (GP3), glycoprotein 4 (GP4), glycoprotein 5 (GP5), and M (matrix protein) proteins of PRRSV was designed and expressed in Escherichia coli. This protein, designated as PRRSVchim, was purified by immobilized metal affinity chromatography and evaluated. PRRSVchim was identified by immunoglobulin G (IgG) presence in serum samples from PRRSV-positive pigs. Also, the protein probed to be antigenic in immunized mice and piglets and provided some degree of protection against challenge with a PRRSV field isolate. These results show the potential of PRRSVchim protein for both PRRSV diagnostic and immunoprophylaxis.

Genome based screening of epitope ensemble vaccine candidates against dreadful visceral leishmaniasis using immunoinformatics approach.

Visceral leishmaniasis (VL) is a dreadful protozoan disease caused by Leishmania donovani that severely affects huge populations in tropical and sub-tropical regions. The present study reports an unbiased genome based screening of 4 potent vaccine antigens against 8023 L. donovani proteins by following the criteria of presence of signal peptides, GPI-anchors and ≤1 transmembrane helix using advanced bioinformatics tools viz. SignalP4.0, PredGPI and TMHMM2.0, respectively. They are designated as genome based predicted signal peptide antigens (GBPSPA). The antigenicity/immunogenicity of chosen vaccine antigens (GBPSPA) with 4 randomly selected known leishmanial antigens (RSKLA) was compared by simulation study employing C-ImmSim software for human immune responses. This revealed better immunological responses. These antigens were further evaluated for the presence of B- and T-cell epitopes using immune epitope database (IEDB) based recommended consensus method of MHC class I and II tools. It was found to forecast CD4+ and CD8+ T-cell responses in genetically diverse human population worldwide as well as different endemic regions through IEDB based predicted population coverage (PPC) analysis tool. The worldwide percent PPC value of combined (HLA class I and II) epitope ensemble forecast was found to be 99.98, 99.96 and 50.04, respectively for GBPSPA, RSKLA and experimentally known epitopes (EKE) of L. donovani. Therefore, these potential antigens/epitope ensembles could favor the design of prospective and novel vaccine constructs like self-assembled epitopes as nano vaccine formulations against VL. Overall, the present study will serve as a model framework that might improve the effectiveness of designed vaccine against L. donovani and other related pathogens.

A recombinant vaccine of Riemerella anatipestifer OmpA fused with duck IgY Fc and Schisandra chinensis polysaccharide adjuvant enhance protective immune response.

Riemerella anatipestifer (R. anatipestifer) causes septicemia and infectious serositis in domestic ducks, leading to high mortality and great economic losses worldwide. Vaccination is currently considered the best strategy to prevent R. anatipestifer infection in ducklings. In this study, we fused the duck IgY Fc gene to the outer membrane protein A (ompA) of R. anatipestifer. The eukaryotic expression plasmid carrying the fusion gene was transformed into Pichia pastoris (P. pastoris) to express the recombinant ompA and ompA-Fc proteins. Then, the effects of fused Fc on the vitality and antigen processing efficiency of duck peritoneal macrophages (PMø) were evaluated in vitro, whereas their immunogenicity was evaluated in vivo. Furthermore, Schisandra chinensis polysaccharide (SCP) was used to evaluate its immune-conditioning effects on the activation of PMø. SCP was also used as adjuvant to investigate immunomodulation on immunoresponses induced by the fused ompA-Fc in ducklings. The conventional Freund's incomplete adjuvant served as the control of SCP. Notably, ompA-Fc promoted phagocytosis of PMø and significantly increased serum antibody titers, CD4+ and CD8+ T-lymphocyte counts, lymphocyte transformation rate, and serum levels of IL-2 and IL-4. In addition, ducklings injected with the ompA-Fc vaccine exhibited considerably greater resistance to the R. anatipestifer challenge than those that received vaccines based on standalone ompA. Of note, SCP was demonstrated to boost the secretion of nitric oxide (NO), IL-1ß, IL-6, TNF-α, and IFN-ß by duck macrophages. In addition, the supplementation of SCP adjuvant to the ompA-Fc vaccines led to the further enhancement of immune response and vaccine protection. The dose of 200 µg/mL showed the most pronounced effects. This study provided valuable insights into protective strategies against R. anatipestifer infection.

Development of a multiple-antigen protein fusion vaccine candidate that confers protection against Bacillus anthracis and Yersinia pestis.

Bacillus anthracis and Yersinia pestis are zoonotic bacteria capable of causing severe and sometimes fatal infections in animals and humans. Although considered as diseases of antiquity in industrialized countries due to animal and public health improvements, they remain endemic in vast regions of the world disproportionally affecting the poor. These pathogens also remain a serious threat if deployed in biological warfare. A single vaccine capable of stimulating rapid protection against both pathogens would be an extremely advantageous public health tool. We produced multiple-antigen fusion proteins (MaF1 and MaF2) containing protective regions from B. anthracis protective antigen (PA) and lethal factor (LF), and from Y. pestis V antigen (LcrV) and fraction 1 (F1) capsule. The MaF2 sequence was also expressed from a plasmid construct (pDNA-MaF2). Immunogenicity and protective efficacy were investigated in mice following homologous and heterologous prime-boost immunization. Antibody responses were determined by ELISA and anthrax toxin neutralization assay. Vaccine efficacy was determined against lethal challenge with either anthrax toxin or Y. pestis. Both constructs elicited LcrV and LF-specific serum IgG, and MaF2 elicited toxin-neutralizing antibodies. Immunizations with MaF2 conferred 100% and 88% protection against Y. pestis and anthrax toxin, respectively. In contrast, pDNA-MaF2 conferred only 63% protection against Y. pestis and no protection against anthrax toxin challenge. pDNA-MaF2-prime MaF2-boost induced 75% protection against Y. pestis and 25% protection against anthrax toxin. Protection was increased by the molecular adjuvant CARDif. In conclusion, MaF2 is a promising multi-antigen vaccine candidate against anthrax and plague that warrants further investigation.

Bacterial Factors Required for Transmission of Streptococcus pneumoniae in Mammalian Hosts.

The capacity of Streptococcus pneumoniae to successfully transmit and colonize new human hosts is a critical aspect of pneumococcal population biology and a prerequisite for invasive disease. However, the bacterial mechanisms underlying this process remain largely unknown. To identify bacterial factors required for transmission, we conducted a high-throughput genetic screen with a transposon sequencing (Tn-seq) library of a pneumococcal strain in a ferret transmission model. Key players in both metabolism and transcriptional regulation were identified as required for efficient bacterial transmission. Targeted deletion of the putative C3-degrading protease CppA, iron transporter PiaA, or competence regulatory histidine kinase ComD significantly decreased transmissibility in a mouse model, further validating the screen. Maternal vaccination with recombinant surface-exposed PiaA and CppA alone or in combination blocked transmission in offspring and were more effective than capsule-based vaccines. These data underscore the possibility of targeting pneumococcal transmission as a means of eliminating invasive disease in the population.

Pichia pastoris-expressed Zika virus envelope domain III on a virus-like particle platform: design, production and immunological evaluation.

Zika virus (ZIKV) is an arbovirus which shares antigenic similarity and the mosquito vector with dengue viruses (DENVs). ZIKV is a neurotropic virus capable of causing congenital neurodevelopmental birth defects. As ZIKV antibodies (Abs) can potentially enhance infection by DENVs, a preventive ZIKV vaccine must be designed to eliminate antibody dependent enhancement of infection. We developed a Zika Subunit Vaccine (ZSV) consisting of two proteins, ZS and S, in a genetically pre-determined ratio of 1:4, using the methylotrophic yeast Pichia pastoris. ZS is an in-frame fusion of ZIKV envelope domain III with the Hepatitis B virus (HBV) surface antigen, and S is the un-fused HBV surface antigen. Using specific monoclonal Abs we showed the presence of ZS and S in the co-purified material which were found to co-assemble into virus-like particles (VLPs), based on dynamic light scattering and electron microscopic analyses. These VLPs were immunogenic in BALB/c mice, eliciting Abs capable of neutralizing ZIKV reporter virus particles. Further, the VLP-induced Abs did not enhance a sub-lethal DENV-2 challenge in AG129 mice. This important safety feature, coupled to the well-documented advantage of P. pastoris expression system, warrants further exploration of ZSV VLP as a possible vaccine candidate.

Reverse vaccinology approach to design a novel multi-epitope subunit vaccine against avian influenza A (H7N9) virus.

H7N9, a novel strain of avian origin influenza was the first recorded incidence where a human was transited by a N9 type influenza virus. Effective vaccination against influenza A (H7N9) is a major concern, since it has emerged as a life threatening viral pathogen. Here, an in silico reverse vaccinology strategy was adopted to design a unique chimeric subunit vaccine against avian influenza A (H7N9). Induction of humoral and cell-mediated immunity is the prime concerned characteristics for a peptide vaccine candidate, hence both T cell and B cell immunity of viral proteins were screened. Antigenicity testing, transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis and molecular docking approach were adopted to generate the most antigenic epitopes of avian influenza A (H7N9) proteome. Further, a novel subunit vaccine was designed by the combination of highly immunogenic epitopes along with suitable adjuvant and linkers. Physicochemical properties and secondary structure of the designed vaccine were assessed to ensure its thermostability, h ydrophilicity, theoretical PI and structural behavior. Homology modeling, refinement and validation of the designed vaccine allowed to construct a three dimensional structure of the predicted vaccine, further employed to molecular docking analysis with different MHC molecules and human immune TLR8 receptor present on lymphocyte cells. Moreover, disulfide engineering was employed to lessen the high mobility region of the designed vaccine in order to extend its stability. Furthermore, we investigated the molecular dynamic simulation of the modeled subunit vaccine and TLR8 complexed molecule to strengthen our prediction. Finally, the suggested vaccine was reverse transcribed and adapted for E. coli strain K12 prior to insertion within pET28a(+) vector for checking translational potency and microbial expression.

A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E. coli as a host.

Chemical synthesis of conjugate vaccines, consisting of a polysaccharide linked to a protein, can be technically challenging, and in vivo bacterial conjugations (bioconjugations) have emerged as manufacturing alternatives. Bioconjugation relies upon an oligosaccharyltransferase to attach polysaccharides to proteins, but currently employed enzymes are not suitable for the generation of conjugate vaccines when the polysaccharides contain glucose at the reducing end, which is the case for ~75% of Streptococcus pneumoniae capsules. Here, we use an O-linking oligosaccharyltransferase to generate a polyvalent pneumococcal bioconjugate vaccine with polysaccharides containing glucose at their reducing end. In addition, we show that different vaccine carrier proteins can be glycosylated using this system. Pneumococcal bioconjugates are immunogenic, protective and rapidly produced within E. coli using recombinant techniques. These proof-of-principle experiments establish a platform to overcome limitations of other conjugating enzymes enabling the development of bioconjugate vaccines for many important human and animal pathogens.

Development of a novel multiepitope chimeric vaccine against anthrax.

Bacillus anthracis (BA), the etiological agent of anthrax, secretes protective antigen (PA), lethal factor (LF), and edema factor (EF) as major virulence mediators. Amongst these, PA-based vaccines are most effective for providing immunity against BA, but their low shelf life limits their usage. Previous studies showed that B-cell epitopes, ID II and ID III present in PA domain IV possess higher toxin neutralization activity and elicit higher antibody titer than ID I. Moreover, N-terminal region of both LF and EF harbors PA-binding sites which share 100% identity with each other. Here, in this study, we have developed an epitope-based chimeric vaccine (ID-LFn) comprising ID II-ID III region of PA and N-terminal region of LF. We have also evaluated its protective efficacy as well as stability and found it to be more stable than PA-based vaccine. Binding reactivities of ID-LFn with anti-PA/LF/EF antibodies were determined by ELISA. The stability of chimeric vaccine was assessed using circular dichroism spectroscopy. ID-LFn response was characterized by toxin neutralization, lymphocyte proliferation isotyping and cytokine profiling. The protective efficacy was analyzed by challenging ID-LFn-immunized mice with B. anthracis (pXO1+ and pXO2+). ID-LFn was found to be significantly stable as compared to PA. Anti-ID-LFn antibodies recognized PA, LF as well as EF. The T-cell response and the protective efficacy of ID-LFn were found to be almost similar to PA. ID-LFn exhibits equal protective efficacy in mice and possesses more stability as compared to PA along with the capability of recognizing PA, LF and EF at the same time. Thus, it can be considered as an improved vaccine against anthrax with better shelf life. ID-LFn, a novel multiepitope chimeric anthrax vaccine: ID-LFn comprises of immunodominant epitopes of domain 4 of PA and N-terminal homologous stretch of LF and EF. The administration of this protein as a vaccine provides protection against anthrax.

The rational simplification of a recombinant cocktail vaccine to control the parasitic nematode Teladorsagia circumcincta.

Using data from five independent vaccine trials, which employed a subunit cocktail vaccine containing eight recombinant proteins to protect sheep against Teladorsagia circumcincta, a strategy was developed to simplify antigen complexity of the vaccine. A meta-analysis of data from these five trials demonstrated statistically significant reductions in cumulative faecal egg count and worm burden in vaccinated sheep when compared with those which had received adjuvant only (P = 0.009 and P < 0.0001, respectively). Relationships between antigen-specific antibody levels, antibody avidity and parasitological parameters of efficacy were analysed for each of the eight proteins in these trials. Of these, the strongest correlations between percentage reduction in cumulative faecal egg count and avidity were obtained for the vaccine antigen T. circumcincta apyrase-1 (Tci-APY-1) in relation to either total antigen-specific IgG or IgG1 in sera (P = 0.019 and P = 0.030, respectively). In addition, IgG and IgA within the serum and abomasal mucus of control (parasite challenged) lambs strongly recognised Tci-APY-1 and T. circumcincta metalloproteinase-1 (Tci-MEP-1) but only weakly bound the other six antigens, indicating Tci-APY-1 and Tci-MEP-1 are most effectively recognised by the parasite-induced antibody response. On the basis of these findings, a two-protein vaccine comprising Tci-APY-1 and Tci-MEP-1 was tested in a direct comparison with the original eight-component vaccine. A further group was immunised with Tci-MEP-1 in combination with a mutated form of Tci-APY-1 (mTci-APY-1), which had no enzymatic activity. Across the trial, the mean faecal egg count levels of the eight-antigen recipients were lower than those of the adjuvant only control group (P = 0.013) and the mean FEC of the mTci-APY-1 and Tci-MEP-1 recipients was lower, although not statistically significantly, than that of the adjuvant-only control group (P = 0.093). Mean cumulative faecal egg count levels were reduced by 43% in lambs immunised with mTci-APY-1 plus Tci-MEP-1 compared with the controls (P = 0.079).

Assessment of the impact of manufacturing changes on the physicochemical properties of the recombinant vaccine carrier ExoProtein A.

Efforts to develop a vaccine for the elimination of malaria include the use of carrier proteins to assemble monomeric antigens into nanoparticles to maximize immunogenicity. Recombinant ExoProtein A (EPA) is a detoxified form of Pseudomonas aeruginosa Exotoxin A which has been used as a carrier in the conjugate vaccine field. A pilot-scale process developed for purification of EPA yielded product that consistently approached a preset upper limit for host cell protein (HCP) content per human dose. To minimize the risk of bulk material exceeding the specification, the purification process was redeveloped using mixed-mode chromatography resins. Purified EPA derived from the primary and redeveloped processes were comparable following full biochemical and biophysical characterization. However, using a process specific immunoassay, the HCP content was shown to decrease from a range of 0.14-0.24% w/w of total protein to below the level of detection with the revised process. The improved process reproducibly yields EPA with highly similar quality characteristics as the original process but with an improved profile for the HCP content.

Efficient production of porcine circovirus virus-like particles using the nonconventional yeast Kluyveromyces marxianus.

Porcine circovirus type 2 (PCV2) is a ubiquitous virus with high pathogenicity closely associated with the postweaning multisystemic wasting syndrome (PMWS) and porcine circovirus diseases (PCVDs), which caused significant economic losses in the swine industry worldwide every year. The PCV2 virus-like particles (VLPs) are a powerful subunit vaccine that can elicit high immune response due to its native PCV2 virus morphology. The baculovirus expression system is the widely used platform for producing commercial PCV2 VLP vaccines, but its yield and cost limited the development of low-cost vaccines for veterinary applications. Here, we applied a nonconventional yeast Kluyveromyces marxianus to enhance the production of PCV2 VLPs. After codon optimization, the PCV2 Cap protein was expressed in K. marxianus and assemble spontaneously into VLPs. Using a chemically defined medium, we achieved approximately 1.91 g/L of PCV2 VLP antigen in a 5-L bioreactor after high cell density fermentation for 72 h. That yield greatly exceeded to recently reported PCV2 VLPs obtained by baculovirus-insect cell, Escherichia coli and Pichia pastoris. By the means of two-step chromatography, 652.8 mg of PCV2 VLP antigen was obtained from 1 L of the recombinant K. marxianus cell culture. The PCV2 VLPs induced high level of anti-PCV2 IgG antibody in mice serums and decreased the virus titers in both livers and spleens of the challenged mice. These results illustrated that K. marxianus is a powerful yeast for cost-effective production of PCV2 VLP vaccines.

Ligand binding properties of two Brugia malayi fatty acid and retinol (FAR) binding proteins and their vaccine efficacies against challenge infection in gerbils.

Parasitic nematodes produce an unusual class of fatty acid and retinol (FAR)-binding proteins that may scavenge host fatty acids and retinoids. Two FARs from Brugia malayi (Bm-FAR-1 and Bm-FAR-2) were expressed as recombinant proteins, and their ligand binding, structural characteristics, and immunogenicities examined. Circular dichroism showed that rBm-FAR-1 and rBm-FAR-2 are similarly rich in α-helix structure. Unexpectedly, however, their lipid binding activities were found to be readily differentiated. Both FARs bound retinol and cis-parinaric acid similarly, but, while rBm-FAR-1 induced a dramatic increase in fluorescence emission and blue shift in peak emission by the fluorophore-tagged fatty acid (dansyl-undecanoic acid), rBm-FAR-2 did not. Recombinant forms of the related proteins from Onchocerca volvulus, rOv-FAR-1 and rOv-FAR-2, were found to be similarly distinguishable. This is the first FAR-2 protein from parasitic nematodes that is being characterized. The relative protein abundance of Bm-FAR-1 was higher than Bm-FAR-2 in the lysates of different developmental stages of B. malayi. Both FAR proteins were targets of strong IgG1, IgG3 and IgE antibody in infected individuals and individuals who were classified as endemic normal or putatively immune. In a B. malayi infection model in gerbils, immunization with rBm-FAR-1 and rBm-FAR-2 formulated in a water-in-oil-emulsion (®Montanide-720) or alum elicited high titers of antigen-specific IgG, but only gerbils immunized with rBm-FAR-1 formulated with the former produced a statistically significant reduction in adult worms (68%) following challenge with B. malayi infective larvae. These results suggest that FAR proteins may play important roles in the survival of filarial nematodes in the host, and represent potential candidates for vaccine development against lymphatic filariasis and related filarial infections.

Recombinant infectious bronchitis viruses expressing heterologous S1 subunits: potential for a new generation of vaccines that replicate in Vero cells.

The spike glycoprotein (S) of infectious bronchitis virus (IBV) comprises two subunits, S1 and S2. We have previously demonstrated that the S2 subunit of the avirulent Beau-R strain is responsible for its extended cellular tropism for Vero cells. Two recombinant infectious bronchitis viruses (rIBVs) have been generated; the immunogenic S1 subunit is derived from the IBV vaccine strain, H120, or the virulent field strain, QX, within the genetic background of Beau-R. The rIBVs BeauR-H120(S1) and BeauR-QX(S1) are capable of replicating in primary chicken kidney cell cultures and in Vero cells. These results demonstrate that rIBVs are able to express S1 subunits from genetically diverse strains of IBV, which will enable the rational design of a future generation of IBV vaccines that may be grown in Vero cells.