Progress 07/01/18 to 06/30/23
Outputs
Target Audience:Presentation: Newcastle disease virus (NDV) vectored vaccine candidates against the respiratory syncytial virus (RSV). Conference of Research Workers in Animal Diseases (CRWAD), 2021 Veterinary and Biotech community: Manuscripts in preparations. Changes/Problems:The COVID-19 pandemic has significantly impacted the progress and achievements of research. It has become more difficult to recruit highly qualified postdoc scientists with a background in virology and immunology, as well as experience in performing animal studies. Additionally, the rising costs of research materials and animals have led to a tight budget, which is affecting the accomplishment of testing vaccine efficacy against bovine respiratory syncytial virus in calves. We are currently seeking more funding and resources to achieve our final goal, and we will continue to acknowledge the support of this NIFA grant in our publications. What opportunities for training and professional development has the project provided?Edris Shirvani, postdoc, now works at a biotech company Sunny Khattar, Assistant Research Professor, now works at a biotech company How have the results been disseminated to communities of interest?Presentation - Newcastle disease virus (NDV) vectored vaccine candidates against the respiratory syncytial virus (RSV), Conference of Research Workers in Animal Diseases (CRWAD), 2021. What do you plan to do during the next reporting period to accomplish the goals?Prepare and submit the manuscript: Construction of aNewcastledisease Virus (NDV) vector expressingbovine respiratory syncytial virus perfusion F protein Mucosal delivery ofperfusion F protein againstrespiratory syncytial virus infection Efficient mucosal vaccination with tandem RBD antigensagainst infection and transmissionof sarbecovirus
Impacts
What was accomplished under these goals?
Construction, recovery, and characterization of recombinant NDVs containing the F gene of BRSV. The recovered viruses have been characterized in vitro for their ability to replicate and their expression levels of BRSV F protein and BRSV pre- F protein. rNDVs expressing BRSV proteins were passaged in eggs. The allantoic fluids containing high titers of each recombinant NDV virus were aliquoted in vials and stored at -70°C. The titer of each virus was determined by plaque forming unit (PFU) assay on DF1 cells in the presence of Dulbecco's minimal essential medium (DMEM) containing 0.8% methylcellulose and 10% fresh allantoic fluid overlay. All rNDVs replicated in eggs efficiently with titers of 28 HAU/50µl. All rNDVs developed almost similar plaque sizes in monolayers of DF1 cells, suggesting the insertion of BRSV F protein does not affect the replication capacity of recombinant NDV. The expression of BRSV F and pre-F proteins was detected in DF1 cells by Western blot analysis using an F-specific antibody. Motavizumab monoclonal antibody (mAb) recognizes human RSV F and pre-F proteins and 8G12 mAb interacts with BRSV F and pre-F proteins. In denaturing condition, the motavizumab mAb detected approximately 64 kDa, approximately 50 kDa, and approximately 20 kDa bands which respectively represent an uncleaved F0, and cleaved F1 and F2 subunits of F protein, expressed by rNDV/BRSV-F and rNDV/BRSV-F+NDV F-TMCT. However, a slightly higher level of F protein expressed by rNDV/BRSV-F+NDV F-TMCT was detected than that of rNDV/BRSV-F. The motavizumab mAb also detected approximately 64 kDa and approximately 50 kDa which respectively represent an uncleaved F0, and cleaved F1 subunit of pre-F protein, expressed by rNDV/BRSV-pre-F and rNDV/BRSV-pre-F+NDV F-TMCT. There was no approximately 20 kDa band detected for the F2 subunit. Similarly, a higher level of F protein expressed by rNDV/BRSV-pre-F+NDV F-TMCT was detected than that of rNDV/BRSV-pre-F. In non-denaturing conditions, an approximately 180 kDa band was detected for F protein expressed by rNDV/BRSV-F and rNDV/BRSV-F+NDV F-TMCT, but not for pre-F protein expressed by rNDV/BRSVpre-F and rNDV/BRSV-preF+NDV-FTMCT using motavizumab or 8G12 mAb. An approximately 70 kDa band was also detected for the HN protein of rNDVs using a mAb against the HN protein of NDV. The expressions of BRSV F and pre-F proteins in embryonated chicken eggs were assessed by Western blot analysis using motavizumab mAb. In denaturing condition, the motavizumab mAb detected an approximately 50 kDa band for cleaved F1 of F protein expressed by rNDV/BRSV-F, rNDV/BRSV-F+NDV F-TMCT, rNDV/BRSV-pre-F and rNDV/BRSV-pre-F+NDV FTMCT. However, a higher level of F protein expressed by rNDV/BRSV-F+NDV F-TMCT was detected than that of rNDV/BRSV-F. Similarly, the level of F protein expressed by rNDV/BRSV-pre-F+NDV F-TMCT was higher than that of rNDV/BRSV-pre-F. An approximately 70 kDa band was also detected for the HN protein of rNDVs using a mAb against the HN protein of NDV. Therefore, the Western blot analysis showed that rNDVs expressed F and pre-F proteins of BRSV. However, rNDV/BRSV-F+NDV F-TMCT and rNDV/BRSV-F+NDV pre-F-TMCT expressed higher levels of F and pre-F proteins, respectively. The pattern and the level of expression detected for F and pre-F protein showed some differences: i) uncleaved F0 and cleaved F1 and F2 were detected for F protein, whereas uncleaved F0 and cleaved F1 were only detected for pre-F protein. The F2 subunit was not detected for pre-F protein, ii) F protein was detected in an oligomeric form by motavizumab and 8G12 mAbs, while an oligomer form was not found in pre-F protein and iii) F protein was detected by both motavizumab and 8G12 mAbs, but pre-F protein was detected by motavizumab mAb and was not detected or detected at very low level by 8G12 mAb. BRSV F protein naturally exists as a trimeric form in the virus envelope. We further determined if the F proteins expressed by the NDV vector maintain their trimeric conformation. First, BS3, a hydrophilic, 11 ångström cross-linker that covalently links proteins, can stabilize trimeric proteins. Thus, we cross-linked the pre-F or F proteins with BS3, and the treated proteins were subjected to SDS-PAGE analysis under a reducing and denaturing condition. As shown, the treated pre-F or F proteins migrated to a position at approximately 175 kDa in comparison with the untreated F protein that migrated at 57 kDa position, suggesting the Pre-f or F protein exists as a trimer. The interaction of BRSV F and pre-F proteins expressed by rNDVs with antibodies specific for fusion and pre-fusion forms of HRSV F protein was also analyzed by ELISA using motavizumab anti-F protein and pre-F protein of human RSV mAb or AM14 mAb anti pre-F protein of human RSV and a secondary anti-human HRP conjugated antibody. The plates were analyzed using an ELISA reader after developing with substrate peroxidase ABTS. The ELISA result showed that motavizumab mAb interacted with F and pre-F proteins expressed by rNDV/BRSV-F, rNDV/BRSV-F+NDV F-TMCT, rNDV/BRSV-pre-F and rNDV/BRSV-pre-F+NDV F-TMCT. However, compared to pre-F protein expressed by rNDV/BRSVpreF, F protein expressed by rNDV/BRSV-F interacted at a higher level. Similarly, compared to pre-F protein provided by rNDV/BRSV-preF+NDV F-TMCT, F protein provided by rNDV/BRSV-F+NDV F-TMCT was detected at a higher level. Compared to pre-F protein expressed by rNDV/BRSV-preF, pre-F protein from the rNDV/BRSV-pre-F+NDV F-TMCT interacted at a higherlevel. Compared to F protein in rNDV/BRSV-F, F protein expressed by rNDV/BRSV-F+NDV F-TMCT was detected at a higher level. AM14 mAb specific for human RSV pre-fusion antibody only detected pre-F protein provided by rNDV/BRSV-preF+NDV F-TMCT. In conclusion, the recombinant NDVs expressing F and pre-F proteins of BRSV were successfully generated. They replicated efficiently and provided high titers in embryonated chicken eggs. They formed similarly sized plaques in chicken DF1 cells. The expression of F or pre-F protein was detected by Western blot and ELISA analysis. Western-blot analysis did not confirm the stabilizing pre-fusion form of the pre-F protein. However, the pre-F protein was detected by a pre-fusion-specific mAb using ELISA. Among the constructs, rNDV/BRSV-F+NDV F-TMCT and rNDV/BRSV-pre-F+NDV F-TMCT will be selected for animal studies in the next year as described below.
Publications
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Progress 07/01/21 to 06/30/22
Outputs
Target Audience:Presentaton Newcastle disease virus (NDV) vectored vaccine candidates against respiratory syncytial virus (RSV). Conference of Research Workers in Animal Diseases (CRWAD), 2021 Changes/Problems:The pandemic continues to affect research progress. It is difficult to recruit highly qualified postdoc scientists with a background in molecular virology and with experience in performing animal studies. What opportunities for training and professional development has the project provided?Edris Shirvani, postdoc, now works at a biotech company Sunny Khattar, Assistant Research Professor, now works at a biotech company How have the results been disseminated to communities of interest?Presentation - Newcastle disease virus (NDV) vectored vaccine candidates against respiratory syncytial virus (RSV), Conference of Research Workers in Animal Diseases (CRWAD), 2021. What do you plan to do during the next reporting period to accomplish the goals?Test immunogenicity and protective efficacy of rNDV vectored BRSV vaccine candidates in guinea pig and/or cotton rat. Test immunogenicity and protective efficacy of rNDV vectored BRSV vaccine candidates in calves.
Impacts
What was accomplished under these goals?
Construction, recovery, and characterization of recombinant NDVs containing the F gene of BRSV. The recovered viruses have been characterized in vitro for their ability to replicate and their expression levels of BRSV F protein and BRSV pre- F protein. rNDVs expressing BRSV proteins were passaged in eggs. The allantoic fluids containing high titers of each recombinant NDV virus were aliquoted in vials and stored at -70°C. The titer of each virus was determined by plaque forming unit (PFU) assay on DF1 cells in presence of Dulbecco's minimal essential medium (DMEM) containing 0.8% methylcellulose and 10% fresh allantoic fluid over layer. All rNDVs replicated in eggs efficiently with titers of 28 HAU/50µl. All rNDVs developed almost similar plaques sizes in monolayers of DF1 cells, suggesting insertion of BRSV F protein does not affect the replication capacity of recombinant NDV. The expression of BRSV F and pre-F proteins was detected in DF1 cells by Western blot analysis using F-specific antibody. Motavizumab monoclonal antibody (mAb) recognizes human RSV F and pre-F proteins and 8G12 mAb interacts with BRSV F and pre-F proteins. In denaturing condition, the motavizumab mAb detected an approximately 64 kDa, an approximately 50 kDa and an approximately 20 kDa bands which respectively represents an uncleaved F0, and cleaved F1 and F2 subunits of F protein, expressed by rNDV/BRSV-F and rNDV/BRSV-F+NDV F-TMCT. However, a slightly higher level of F protein expressed by rNDV/BRSV-F+NDV F-TMCT was detected than that of rNDV/BRSV-F. The motavizumab mAb also detected an approximately 64 kDa, an approximately 50 kDa which respectively represents an uncleaved F0, and cleaved F1 subunit of pre-F protein, expressed by rNDV/BRSV-pre-F and rNDV/BRSV-pre-F+NDV F-TMCT. There was not approximately 20 kDa band detected for F2 subunit. Similarly, a higher level of F protein expressed by rNDV/BRSV-pre-F+NDV F-TMCT was detected than that of rNDV/BRSV-pre-F. In non-denaturing conditions, an approximately 180 kDa band was detected for F protein expressed by rNDV/BRSV-F and rNDV/BRSV-F+NDV F-TMCT, but not for pre-F protein expressed by rNDV/BRSVpre-F and rNDV/BRSV-preF+NDV-FTMCT using motavizumab or 8G12 mAb. An approximately 70 kDa band was also detected for HN protein of rNDVs using a mAb against HN protein of NDV. The expressions of BRSV F and pre-F proteins in embryonated chicken eggs were assessed by Western blot analysis using motavizumab mAb. In denaturing condition, the motavizumab mAb detected an approximately 50 kDa band for cleaved F1 of F protein expressed by rNDV/BRSV-F, rNDV/BRSV-F+NDV F-TMCT, rNDV/BRSV-pre-F and rNDV/BRSV-pre-F+NDV FTMCT. However, a higher level of F protein expressed by rNDV/BRSV-F+NDV F-TMCT was detected than that of rNDV/BRSV-F. Similarly, the level of F protein expressed by rNDV/BRSV-pre-F+NDV F-TMCT was higher than that of rNDV/BRSV-pre-F. An approximately 70 kDa band was also detected for HN protein of rNDVs using a mAb against HN protein of NDV. Therefore, the Western blot analysis showed that rNDVs expressed F and pre-F proteins of BRSV. However, rNDV/BRSV-F+NDV F-TMCT and rNDV/BRSV-F+NDV pre-F-TMCT expressed higher levels of F and pre-F proteins, respectively. The pattern and the level of expression detected for F and pre-F protein showed some difference: i) uncleaved F0 and cleaved F1 and F2 were detected for F protein, whereas uncleaved F0 and cleaved F1 were only detected for pre-F protein. The F2 subunit was not detected for pre-F protein, ii) F protein was detected at an oligomeric form by motavizumab and 8G12 mAbs, while an oligomer form was not found in pre-F protein and iii) F protein was detected by both motavizumab and 8G12 mAbs, but pre-F protein was detected by motavizumab mAb and was not detected or detected at very low level by 8G12 mAb. BRSV F protein naturally exists as a trimeric form in the virus envelop. We further determined if the F proteins expressed by NDV vector maintains its trimeric conformation. First, the BS3, a hydrophilic, 11 ångström cross-linker that covalently links proteins, can stabilize trimeric protein. Thus, we cross-linked the pre-F or F proteins with BS3 and the treated proteins were subjected to SDS-PAGE analysis under a reducing and denaturing condition. As shown, the treated pre-F or F proteins migrated to a position at an approximately 175 kDa in comparison with the untreated F protein that migrated at 57 kDa position, suggesting the Pre-f or F protein exists as a trimer. The interaction of BRSV F and pre-F proteins expressed by rNDVs with antibody specific for fusion and pre-fusion forms of HRSV F protein was also analyzed by ELISA using motavizumab anti F protein and pre-F protein of human RSV mAb or AM14 mAb anti pre-F protein of human RSV and a secondary anti-human HRP conjugated antibody. The plates were analyzed using an ELISA reader after developing with substrate peroxidase ABTS. The ELISA result showed that motavizumab mAb interacted with F and pre-F proteins expressed by rNDV/BRSV-F, rNDV/BRSV-F+NDV F-TMCT, rNDV/BRSV-pre-F and rNDV/BRSV-pre-F+NDV F-TMCT. However, compared to pre-F protein expressed by rNDV/BRSVpre-F, F protein expressed by rNDV/BRSV-F interacted at higher level. Similarly, compared to pre-F protein provided by rNDV/BRSV-preF+NDV F-TMCT, F protein provided by rNDV/BRSV-F+NDV F-TMCT detected at a higher level. Compared to pre-F protein expressed by rNDV/BRSV-preF, pre-F protein from the rNDV/BRSV-pre-F+NDV F-TMCT interacted at higher level. Compared to F protein in rNDV/BRSV-F, F protein expressed by rNDV/BRSV-F+NDV F-TMCT was detected at higher level. AM14 mAb specific for human RSV pre-fusion antibody only detected pre-F protein provided by rNDV/BRSV-preF+NDV F-TMCT. In conclusion, the recombinant NDVs expressing F and pre-F proteins of BRSV were successfully generated. They replicated efficiently and provided high titers in embryonated chicken eggs. They formed similarly sized plaques in chicken DF1 cells. The expression of F or pre-F protein was detected by Western-blot and ELISA analysis. Western-blot analysis did not confirm the stabilizing pre-fusion form of the pre-F protein. However, pre-F protein was detected by a pre-fusion specific mAb using ELISA. Among the constructs, rNDV/BRSV-F+NDV F-TMCT and rNDV/BRSV-pre-F+NDV F-TMCT will be selected for animal studies in the next year as described below.
Publications
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Progress 07/01/20 to 06/30/21
Outputs
Target Audience:1. Veterinarians 2. Bovine practitioners 3. Cattle farmers 4. Scientific community, especially vaccine development on human respiratory syncytial virus. 5. Researchers working on animal diseases 6. Researches working on bovine respiratory diseases Changes/Problems:The COVID-19 crisis has affected our research plan dramatically; particularly calve immunization and infection study. In 2020, our research capacity was limited to the essential employees who only performed COVID-19 related research projects; then transition to 50% employees, and currently 75% employee capacity starting on April 6, 2021. Therefore, we have to adjust our research strategies and objectives according to pandemic situation. Our objectives in this research will be: 1. Construction and characterization of recombinant NDVs containing the F gene of BRSV. F protein exhibits a trimeric pre-fusion and post-fusion structure. We will first make mutations to stabilize the pre-fusion form of the F protein of BRSV strain A51908 by different modifications. The wild type and stabilized pre-fusion F proteins will be expressed in NDV vector infected cells and further evaluated using NDV vector. The specific antibodies recognizing pre-fusion form of human RSV F protein are used to verify the pre-fusion form of BRSV F protein. Biochemical approach is employed to demonstrate the trimeric nature of F proteins. We will select the recombinant NDV expressing high level of F proteins for testing in animal models. 2. Evaluation of immunogenicity and protective efficacy of recombinant NDVs expressing BRSV F protein in guinea pigs, cotton rats, and calves. Based on the current pandemic situation, we elect to use small animal models to test immunogenicity and the protective efficacy of rNDV vectored BRSV vaccine candidates in guinea pigs and cotton rat that are excellent animal modles for testing efficacy and safety of respiratory sycyticial vaccines. This test would optimize our rNDV vaccine candidates. The protective efficacies of BRSV pre-fusion and fusion protein expressed by NDV vector will be further evaluated by intranasally immunizing two to four-week-old, BRSV-seronegative calves. Calves will be challenged four weeks after vaccination by a virulent BRSV strain. Animals will be euthanized seven days after challenge to determine virus replication in lungs and to evaluate pulmonary pathology. What opportunities for training and professional development has the project provided?1. This study will support and train graduate student and post-doctoral scientists 2. This study will initiate collaboration with researchers at other universities How have the results been disseminated to communities of interest?The results were presented in Conference of Research Workers in Animal Diseases (CRWAD). What do you plan to do during the next reporting period to accomplish the goals?Test immunogenicity and protective efficacy of rNDV vectored BRSV vaccine candidates in guinea pig and/or cotton rat. Cotton rat and guinea pig are well recognized small animal modles for testing efficacy and safety of respiratory sycytical vaccines. The protective efficacy of rNDV expressing F or Pre-F gene of BRSV against virulent strains of BRSV, respectively, will be evaluated using six groups of guinea pigs or cotton rats. Each experiment includes two groups of 10 guinea pigs or 10 cotton rats inoculated with rNDV/BRSV vaccine candidates (expressing F or PreF genes of BRSV). Experimental animals will include: a group of 10 guinea pigs or 10 cotton rats inoculated with rNDV as an empty vector control, a group of 10 guinea pigs or 10 cotton rats inoculated with PBS, a group of 10 guinea pigs or 10 cotton rats immunized with a commercial BRSV, and a non-vaccinated group of 10 guinea pigs or 10 cotton rats inoculated with PBS which will not be infected with a virulent BRSV virus. Guinea pigs or cotton rats will be anesthetized and intranasally (i.n.) inoculated with 106 PFU (100 ul each nostril) of empty vector, rNDV/BRSVs or rNDV/RSVs. Three weeks after immunization, blood samples will be collected from all guinea pigs or cotton rats for analysis of antibody responses. All groups except unvaccinated/uninfected groups, will be challenged i.n. with 106 PFU (50-100 ul each nostril) of BRSV strain. Some additional animals will be observed daily after challenge for signs of disease until day 4 at which time animals will be euthanized and bronco-tracheal lavage, nasal turbinate and lung tissues will be collected for virus isolation and/or virus genome quantification and histopathology. The spleen, lungs, and draining lymph nodes will be collected for measuring cellular immune response. We expect small animal models will provide valuable information to test this recombinant vaccine. It also helps to overcome the experimental obstacles caused by COVID-19 pandemic. Test immunogenicity and protective efficacy of rNDV vectored BRSV vaccine candidates in calves. Thirty BRSV- seronegative calves (2-4 weeks old) will be obtained from a local animal supplier. After arrival of animals, during the week of acclimatization, all the calves will be treated with procaine benzyl penicillin. The calves will be randomized into four groups (six calves in each group) so that the mean body weight is similar in each group. This study will be performed in a blind manner. All calves in Group I will be vaccinated with a commercial MLV BRSV vaccine according to the manufacturer's instructions. Each animal in groups II, III and IV will be inoculated either with 2 ml of allantoic fluid containing 106 PFU of rNDV by IN route. Each animal in Group II will be immunized with rNDV expressing pre-fusion F protein. Group III will be inoculated with control rNDV expressing F protein. Group IV will act as mock vaccinated control and will be inoculated with PBS. 3-4 weeks after the immunization, each calf will be challenged with a virulent BRSV (104 PFU/ml) by aerosolization with a nebulizer. This BRSV strain and the challenge procedure has been shown to produce moderate to severe disease in calves. Clinical signs will be evaluated and scored daily using the system previously developed by Gershwin et al. The observed clinical signs will include temperature, respiratory rate, anorexia, conjunctivitis, ocular discharge, nasal discharge, dyspnea, mouth breathing, cough and auscultation. Usually, infected calves develop a fever on day 4-5 after infection with cough beginning as early as day 3. Abnormal lung sounds are auscultated from day 4 through day 10. In severe cases, open mouth breathing and dyspnea on day 5-6. Virus is usually shed in nasal secretion from day 3 through day 7. Heparinized and unmodified blood samples will be collected from all calves. Peripheral blood mononuclear cells (PBMC) and serum will be extracted from heparinized and unmodified blood, respectively. PBMC will be used in lymphocyte proliferation assay. After challenge, nasal secretions will be collected daily using sterilized cotton swabs (NS) and tampons (NT). On day 7 post challenge the calves will be euthanized humanely. BAL samples will be collected post-mortem from lung of all challenged calves. The upper and lower respiratory tracts will be removed and examined for gross lesions. Lungs will be photographed. The percentage area of lesioned lung will be determined by computer software. Histopathology of each lung lobe at a pneumonic area will be conducted in a blind manner. BRSV RNA coding for N protein in nasal secretions and in BAL cells will be quantified by RT-PCR to determine virus load. Isolation of BRSV present in nasal secretion and in BAL cells will be attempted in bovine turbinate cells. BRSV-specific IgG1 and IgA and NDV-specific IgG and IgA will be determined by commercial ELISA. Neutralizing antibody titer to BRSV and NDV will be determined by plaque reduction assay. Cellular responses will be determined by BRSV-specific lymphocyte proliferation assay and flow cytometric analysis of BRSV-specific IFN producing lymphocytes. Bovine IL-4 and interferon gamma will be detected in supernatant in re-stimulated lymphocytes.
Impacts
What was accomplished under these goals?
Construction and characterization of recombinant NDVs containing the F gene of BRSV. We have made four cDNAs, including BRSV pre-F, BRSV F, ectodomain of BRSV pre-F or F gene that were fused with NDV F protein transmembrane and cytoplasmic tail. These cDNAs all contained the NDV gene-start and gene-end sequences. The F protein transcriptional units have been inserted at the unique Pme I site between P and M genes of the full-length cDNA of a chimeric NDV strain Beaudette C (BC) backbone with the ectodomains of F and HN proteins in NDV LaSota strain, which has been constructed using a pBR322 plasmid. Infectious NDV recombinants have been generated using reverse genetics technique. The rNDVs expressing BRSV-pre-F, BRSV F, BRSV pre-F ectodomain fused with NDV F protein transmembrane and cytoplasmic tail and BRSV F ectodomain fused with NDV F protein transmembrane and cytoplasmic tail have been designated as rNDV/BRSV-preF, rNDV/BRSV-F, rNDV/BRSV-preF+NDV F-TMCT and rNDV/BRSV-F+NDV FTMCT, respectively. The presence of transcription cassettes containing BRSV genes in rNDVs genomes was confirmed by RT-PCR. Briefly, a primer pair set: a) NDV P gene 2841 forward primer and b) NDV M gene 3322 reverse primer was used to amplify the transcription cassettes containing BRSV genes from cDNA synthesized from extracted rNDVs genome. The PCR was used to amplify the fragments. The correct sequence of BRSV genes were confirmed by sequence analysis using a set of primers for fully sequencing the F and Pre-F genes of BRSV. We also successfully generated three other recombinant viruses as described above. The recovered viruses have been characterized in vitro for their ability to replicate and their expression levels of BRSV F protein and BRSV pre- F protein. rNDVs expressing BRSV proteins were passaged in eggs. The allantoic fluids containing high titers of each recombinant NDV virus were aliquoted in vials and stored at -70°C. The titer of each virus was determined by plaque forming unit (PFU) assay on DF1 cells in presence of DMEM containing 0.8% methylcellulose and 10% fresh allantoic fluid over layer. All rNDVs replicated in eggs efficiently with titers of 28 HAU/50µl. All rNDVs developed almost similar plaques sizes in monolayers of DF1 cells, suggesting insertion of BRSV F protein does not affect the replication capacity of recombinant NDV. The expression of BRSV F and pre-F proteins was detected in DF1 cells by Western blot analysis using F-specific antibody. Motavizumab monoclonal antibody (mAb) recognizes human RSV F and pre-F proteins and 8G12 mAb interacts with BRSV F and pre-F proteins. In denaturing condition, the motavizumab mAb detected an approximately 64 kDa, an approximately 50 kDa and a an approximately 20 kDa bands which respectively represents a uncleaved F0, and cleaved F1 and F2 subunits of F protein, expressed by rNDV/BRSV-F and rNDV/BRSV-F+NDV F-TMCT. However, a slightly higher level of F protein expressed by rNDV/BRSV-F+NDV F-TMCT was detected than that of rNDV/BRSV-F. The motavizumab mAb also detected an approximately 64 kDa, an approximately 50 kDa which respectively represents a uncleared F0, and cleaved F1 subunit of pre-F protein, expressed by rNDV/BRSV-pre-F and rNDV/BRSV-pre-F+NDV F-TMCT. There was not approximately 20 kDa band detected for F2 subunit. Similarly, a higher level of F protein expressed by rNDV/BRSV-pre-F+NDV F-TMCT was detected than that of rNDV/BRSV-pre-F. In non-denaturing conditions, an approximately 180 kDa band was detected for F protein expressed by rNDV/BRSV-F and rNDV/BRSV-F+NDV F-TMCT, but not for pre-F protein expressed by rNDV/BRSVpre-F and rNDV/BRSV-preF+NDV-FTMCT using motavizumab or 8G12 mAb. An approximately 70 kDa band was also detected for HN protein of rNDVs using a mAb against HN protein of NDV. The expressions of BRSV F and pre-F proteins in chicken eggs were assessed by Western blot using motavizumab mAb. In denaturing condition, the motavizumab mAb detected an approximately 50 kDa band for cleaved F1 of F protein expressed by rNDV/BRSV-F, rNDV/BRSV-F+NDV F-TMCT, rNDV/BRSV-pre-F and rNDV/BRSV-pre-F+NDV FTMCT. However, a higher level of F protein expressed by rNDV/BRSV-F+NDV F-TMCT was detected than that of rNDV/BRSV-F. Similarly, the level of F protein expressed by rNDV/BRSV-pre-F+NDV F-TMCT was higher than that of rNDV/BRSV-pre-F. An approximately 70 kDa band was also detected for HN protein of rNDVs using a mAb against HN protein of NDV. Therefore, the Western blot analysis showed that rNDVs expressed F and pre-F proteins of BRSV. However, rNDV/BRSV-F+NDV F-TMCT and rNDV/BRSV-F+NDV pre-F-TMCT expressed higher levels of F and pre-F proteins, respectively. The pattern and the level of expression detected for F and pre-F protein showed some difference: i) uncleaved F0 and cleaved F1 and F2 were detected for F protein, whereas uncleaved F0 and cleaved F1 were only detected for pre-F protein. The F2 subunit was not detected for pre-F protein, ii) F protein was detected at an oligomeric form by motavizumab and 8G12 mAbs, while an oligomer form was not found in pre-F protein and iii) F protein was detected by both motavizumab and 8G12 mAbs, but pre-F protein was detected by motavizumab mAb and was not detected or detected at very low level by 8G12 mAb. BRSV F protein naturally exists as a trimeric form in the virus envelop. We determined if the F proteins expressed by NDV vector maintains its trimeric conformation. First, the BS3, a hydrophilic, 11 ångström cross-linker that covalently links proteins, can stabilize trimeric protein. Thus, we cross-linked the pre-F or F proteins with BS3 and the treated proteins were subjected to SDS-PAGE analysis under a reducing and denaturing condition. As shown, the treated pre-F or F proteins migrated to a position at an approximately 175 kDa in comparison with the untreated F protein that migrated at 57 kDa position, suggesting the Pre-f or F protein exists as a trimer. The interaction of BRSV F and pre-F proteins expressed by rNDVs with antibody specific for fusion and pre-fusion forms of HRSV F protein was also analyzed by ELISA using motavizumab anti F protein and pre-F protein of human RSV mAb or AM14 mAb anti pre-F protein of human RSV and a secondary anti-human HRP conjugated antibody. The plates were analyzed using an ELISA reader after developing with substrate peroxidase ABTS. The ELISA result showed that motavizumab mAb interacted with F and pre-F proteins expressed by rNDV/BRSV-F, rNDV/BRSV-F+NDV F-TMCT, rNDV/BRSV-pre-F and rNDV/BRSV-pre-F+NDV F-TMCT. However, compared to pre-F protein expressed by rNDV/BRSVpre-F, F protein expressed by rNDV/BRSV-F interacted at higher level. Similarly, compared to pre-F protein provided by rNDV/BRSV-preF+NDV F-TMCT, F protein provided by rNDV/BRSV-F+NDV F-TMCT detected at a higher level. Compared to pre-F protein expressed by rNDV/BRSV-preF, pre-F protein from the rNDV/BRSV-pre-F+NDV F-TMCT interacted at higher level. Compared to F protein in rNDV/BRSV-F, F protein expressed by rNDV/BRSV-F+NDV F-TMCT was detected at higher level. AM14 mAb specific for human RSV pre-fusion antibody only detected pre-F protein provided by rNDV/BRSV-preF+NDV F-TMCT. In conclusion, the recombinant NDVs expressing F and pre-F proteins of BRSV were successfully generated. They replicated efficiently and provided high titers in embryonated chicken eggs. They formed similarly sized plaques in chicken DF1 cells. The expression of F or pre-F protein was detected by Western-blot and ELISA analysis. Western-blot analysis did not confirm the stabilizing pre-fusion form of the pre-F protein. However, pre-F protein was detected by a pre-fusion specific mAb using ELISA. Among the constructs, rNDV/BRSV-F+NDV F-TMCT and rNDV/BRSV-pre-F+NDV F-TMCT will be selected for animal studies in the next year.
Publications
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Progress 07/01/19 to 06/30/20
Outputs
Target Audience: Veterinarians Bovine practitioners Cattle farmers Scientific community Researchers working on animal diseases Researches working on bovine diseases Changes/Problems:The COVID-19 crisis has affected our research plan negatively, particularly the animal study. Therefore, we have desided to adjust our objectives according to new unpredicted situation. Our objectives in this research will be: 1. Construction, recovery, and characterization of recombinant NDVs containing the F gene of BRSV. F protein exhibits pre-fusion and post-fusion structures. We will first make mutations to stabilize the pre-fusion form of the F protein of BRSV strain A51908 by different modifications. The wild type and stabilized-pre-fusion F proteins will be expressed and evaluated using NDV vector. The antibody specific for recognizing pre-fusion form of human RSV F protein will be used to verify the pre-fusion form of BRSV F protein. 2. Evaluation of immunogenicity and protective efficacy of recombinant NDVs expressing BRSV F protein in calves. The protective efficacies of BRSV pre-fusion and fusion protein expressed by NDV vector will be evaluated by intranasally immunizing two to four-week-old, BRSV-seronegative calves. Calves will be challenged four weeks after vaccination by a virulent BRSV strain. Animals will be euthanized seven days after challenge to determine virus replication in lungs and to evaluate pulmonary pathology. What opportunities for training and professional development has the project provided?1. This study will support and train graduate student and post-doctoral scientists 2. This study will initiate collaboration with researchers at other universities How have the results been disseminated to communities of interest?The results were presented in Confrence f Research Workers in Animal Diseases (CRWAD). What do you plan to do during the next reporting period to accomplish the goals?Evaluation of immunogenicity and protective efficacy of recombinant NDVs expressing BRSV F protein in calves. Thirty BRSV- seronegative calves (2-4 weeks old) will be obtained from a local animal supplier. After arrival of animals, during the week of acclimatization, all the calves will be treated with procaine benzyl penicillin. The calves will be randomized into four groups (six calves in each group) so that the mean body weight is similar in each group. This study will be performed in a blind manner. All calves in Group I will be vaccinated with a commercial MLV BRSV vaccine according to the manufacturer's instructions. Each animal in groups II, III and IV will be inoculated either with 3 ml of allantoic fluid containing 106 PFU of rNDV by IN route. Each animal in Group II will be immunized with rNDV expressing pre-fusion F protein. Group III will be inoculated with control rNDV expressing F protein. Group IV will act as mock vaccinated control and will be inoculated with PBS. 3-4 weeks after the immunization, each calf will be challenged with a virulent BRSV (104 PFU/ml) by aerosolization with a nebulizer. This BRSV strain and the challenge procedure has been shown to produce moderate to severe disease in calves. Clinical signs will be evaluated and scored daily using the system previously developed by Gershwin et al. The observed clinical signs will include temperature, respiratory rate, anorexia, conjunctivitis, ocular discharge, nasal discharge, dyspnea, mouth breathing, cough and auscultation. Usually, infected calves develop a fever on day 4-5 after infection with cough beginning as early as day 3. Abnormal lung sounds are auscultated from day 4 through day 10. In severe cases, open mouth breathing and dyspnea on day 5-6. Virus is usually shed in nasal secretion from day 3 through day 7. Heparinized and unmodified blood samples will be collected from all calves. Peripheral blood mononuclear cells (PBMC) and serum will be extracted from heparinized and unmodified blood, respectively. PBMC will be used in lymphocyte proliferation assay. After challenge, nasal secretions will be collected daily using sterilized cotton swabs (NS) and tampons (NT). On day 7 post challenge the calves will be euthanized humanely, the upper and lower respiratory tracts will be removed and examined for gross lesions. Lungs will be photographed. The percentage area of lesional lung will be determined by computer software. Histopathology of each lung lobe at a pneumonic area will be conducted in a blind manner. BAL samples will be collected post-mortem from lung of all challenged calves. BRSV RNA coding for N protein in nasal secretions and in BAL cells will be quantified by RT-PCR to determine virus load. Isolation of BRSV present in nasal secretion and in BAL cells will be attempted in bovine turbinate cells. BRSV-specific IgG1 and IgA and NDV-specific IgG and IgA will be determined by commercial ELISA. Neutralizing antibody titer to BRSV and NDV will be determined by plaque reduction assay. Cellular responses will be determined by BRSV-specific lymphocyte proliferation assay and flow cytometric analysis of BRSV-specific IFN producing lymphocytes. Bovine IL-4 and interferon gamma (IFN-γ) will be detected in supernatant in re-stimulated lymphocytes.
Impacts
What was accomplished under these goals?
Construction, recovery, and characterization of recombinant NDVs containing the F gene of BRSV. We have made four cDNAs, including BRSV pre-F, BRSV F, ectodomain of BRSV pre-F or F gene that were fused with NDV F protein transmembrane and cytoplasmic tail. These cDNAs all contained the NDV gene-start and gene-end sequences. The F protein transcriptional units have been inserted at the unique Pme I site between P and M genes of the full-length cDNA of a chimeric NDV strain Beaudette C (BC) backbone with the ectodomains of F and HN proteins in NDV LaSota strain, which has been constructed using a pBR322 plasmid. Infectious NDV recombinants have been generated using our standard reverse genetics technique. The rNDVs expressing BRSV-pre-F, BRSV F, BRSV pre-F ectodomain fused with NDV F protein transmembrane and cytoplasmic tail and BRSV F ectodomain fused with NDV F protein transmembrane and cytoplasmic tail have been designated as rNDV/BRSV-preF, rNDV/BRSV-F, rNDV/BRSV-preF+NDV F-TMCT and rNDV/BRSV-F+NDV F-TMCT, respectively. The presence of transcription cassettes containing BRSV genes in rNDVs genomes was confirmed by RT-PCR.Briefly, a primer pair set: a) NDV P gene 2841 forward primer and b) NDV M gene 3322 reverse primer was used to amplify the transcription cassettes containing BRSV genes from cDNA synthesized from extracted rNDVs genome. Thirty-five cycles of PCR at 94°C for 30s of denaturation, 56°C for 30s of annealing, and 68°C for 150s of elongation using the TAKARA LA Tag polymerase was used to amplify the fragments. The correct sequence of BRSV genes were confirmed by sequence analysis using a set of primers for fully sequencing the F and Pre-F genes of BRSV. We also successfully generated three other recombinant viruses as described above. The recovered viruses have been characterized in vitro for their ability to replicate and their expression levels of BRSV F protein and BRSV pre- F protein. rNDVs expressing BRSV proteins were passaged in eggs. The allantoic fluids containing high titers of each recombinant NDV virus were aliquoted in vials and stored at -70°C. The titer of each virus was determined by plaque forming unit (PFU) assay on DF1 cells in presence of Dulbecco's minimal essential medium (DMEM) containing 0.8% methylcellulose and 10% fresh allantoic fluid over layer. All rNDVs replicated in eggs efficiently with titers of 28 HAU/50µl. All rNDVs developed almost similar plaques sizes in monolayers of DF1 cells, suggesting insertion of BRSV F protein does not affect the replication capacity of recombinant NDV. The expression of BRSV F and pre-F proteins was detected in DF1 cells by Western blot analysis using F-specific antibody. Motavizumab monoclonal antibody (mAb) recognizes human RSV F and pre-F proteins and 8G12 mAb interacts with BRSV F and pre-F proteins. In denaturing condition, the motavizumab mAb detected an approximately 64 kDa, an approximately 50 kDa and a an approximately 20 kDa bands which respectively represents a uncleaved F0, and cleaved F1 and F2 subunits of F protein, expressed by rNDV/BRSV-F and rNDV/BRSV-F+NDV F-TMCT. However, a slightly higher level of F protein expressed by rNDV/BRSV-F+NDV F-TMCT was detected than that of rNDV/BRSV-F. The motavizumab mAb also detected an approximately 64 kDa, an approximately 50 kDa which respectively represents a uncleaved F0, and cleaved F1 subunit of pre-F protein, expressed by rNDV/BRSV-pre-F and rNDV/BRSV-pre-F+NDV F-TMCT. There was no approximately 20 kDa band detected for F2 subunit. Similarly, a higher level of F protein expressed by rNDV/BRSV-pre-F+NDV F-TMCT was detected than that of rNDV/BRSV-pre-F. In non-denaturing conditions, an approximately 180 kDa band was detected for F protein expressed by rNDV/BRSV-F and rNDV/BRSV-F+NDV F-TMCT, but not for pre-F protein expressed by rNDV/BRSV-pre-F and rNDV/BRSV-preF+NDV-FTMCT using motavizumab or 8G12 mAb. An approximately 70 kDa band was also detected for HN protein of rNDVs using a mAb against HN protein of NDV. The expressions of BRSV F and pre-F proteins in embryonated chicken eggs were assessed by Western blot analysis using motavizumab mAb. In denaturing condition, the motavizumab mAb detected an approximately 50 kDa band for cleaved F1 of F protein expressed by rNDV/BRSV-F, rNDV/BRSV-F+NDV F-TMCT, rNDV/BRSV-pre-F and rNDV/BRSV-pre-F+NDV F-TMCT. However, a higher level of F protein expressed by rNDV/BRSV-F+NDV F-TMCT was detected than that of rNDV/BRSV-F. Similarly, the level of F protein expressed by rNDV/BRSV-pre-F+NDV F-TMCT was higher than that of rNDV/BRSV-pre-F. An approximately 70 kDa band was also detected for HN protein of rNDVs using a mAb against HN protein of NDV. Therefore, the Western blot analysis showed that rNDVs expressed F and pre-F proteins of BRSV. However, rNDV/BRSV-F+NDV F-TMCT and rNDV/BRSV-F+NDV pre-F-TMCT expressed higher levels of F and pre-F proteins, respectively. The pattern and the level of expression detected for F and pre-F protein showed some difference: i) uncleaved F0 and cleaved F1 and F2 were detected for F protein, whereas uncleaved F0 and cleaved F1 were only detected for pre-F protein. The F2 subunit was not detected for pre-F protein, ii) F protein was detected at a oligomeric form by motavizumab and 8G12 mAbs, while an oligomer form was not found in pre-F protein and iii) F protein was detected by both motavizumab and 8G12 mAbs, but pre-F protein was detected by motavizumab mAb and was not detected or detected at very low level by 8G12 mAb. The interaction of BRSV F and pre-F proteins expressed by rNDVs with antibody specific for fusion and pre-fusion forms of HRSV F protein was also analyzed by ELISA using motavizumab anti F protein and pre-F protein of human RSV mAb or AM14 mAb anti pre-F protein of human RSV and a secondary anti-human HRP conjugated antibody. The plates were analyzed using an ELISA reader after developing with substrate peroxidase ABTS.The ELISA result showed that motavizumab mAb interacted with F and pre-F proteins expressed by rNDV/BRSV-F, rNDV/BRSV-F+NDV F-TMCT, rNDV/BRSV-pre-F and rNDV/BRSV-pre-F+NDV F-TMCT. However, compared to pre-F protein expressed by rNDV/BRSV-pre-F, F protein expressed by rNDV/BRSV-F interacted at higher level. Similarly, compared to pre-F protein provided by rNDV/BRSV-preF+NDV F-TMCT, F protein provided by rNDV/BRSV-F+NDV F-TMCT detected at a higher level. Compared to pre-F protein expressed by rNDV/BRSV-preF, pre-F protein from the rNDV/BRSV-pre-F+NDV F-TMCT interacted at higher level. Compared to F protein in rNDV/BRSV-F, F protein expressed by rNDV/BRSV-F+NDV F-TMCT was detected at higher level. AM14 mAb specific for human RSV pre-fusion antibody only detected pre-F protein provided by rNDV/BRSV-pre-F+NDV F-TMCT. In conclusion, the recombinant NDVs expressing F and pre-F proteins of BRSV were successfully generated. They replicated efficiently and provided high titers in embryonated chicken eggs. They formed similarly sized plaques in chicken DF1 cells. The expression of F or pre-F protein was detected by Western-blot and ELISA analysis. Western-blot analysis did not confirm the stabilizing pre-fusion form of the pre-F protein. However, pre-F protein was detected by a pre-fusion specific mAb using ELISA. Among the constructs, rNDV/BRSV-F+NDV F-TMCT and rNDV/BRSV-pre-F+NDV F-TMCT will be selected for animal studies in the next year as described below.
Publications
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Progress 07/01/18 to 06/30/19
Outputs
Target Audience:Our research is beneficial to students, postdocs and other research scientists working in animal health. The scientists working in vaccine development and production, the scientists working in human health, cattle producers and industries are also benefitted by our work. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?It provided training to graduate students and technicians, and other scientists. How have the results been disseminated to communities of interest?We presented the poster in CRAWD meeting in December 2018. What do you plan to do during the next reporting period to accomplish the goals?Animal experiments in cattle.
Impacts
What was accomplished under these goals?
Bovine respiratory syncytial virus (BRSV) is the major cause of pneumonia in calves. Currently available BRSV are not efficacious. The BRSV fusion protein (BRSV F) is the principal target of BRSV neutralizing antibodies in bovine sera. The F protein is present on the surface of virions in an unstable prefusion form, which upon contact with adjacent cell membrane undergoes conformational change to stable post-fusion form. Recently, it was shown that in closely related human respiratory syncytial virus (HRSV) the prefusion form of the F protein is the major neutralizing antigen. Therefore, we plan to express a stable prefusion form of BRSV F using Newcastle disease virus (NDV) as a vaccine vector. The pre-fusion form of the F protein of BRSV will be stabilized by different modifications. The wild type and stabilized-pre-fusion F proteins will be expressed and evaluated using NDV vector. The antibody specific for pre-fusion form of HRSV F protein will be used to detect the pre-fusion form of BRSV F protein. We will study the tropism, replication and immunogenicity of recombinant NDV in two to four week old NDV-seronegative calves. The neutralization ability of the vaccinated serum samples will also be determined by NDV neutralization test. The protective efficacy of BRSV F protein expressed by NDV vector will be evaluated in two to four week old, BRSV-seronegative calves. We have constructed, recovered and characterized recombinant NDVs expressing the wild type and pre-fusion forms of BRSV F protein. Our results show that the mutations identified in the F protein of HRSV can be used to stabilize the F protein of BRSV. Work is in progress to characterize the F protein of BRSV in protection.
Publications
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