Progress 09/01/08 to 08/31/13
Outputs
Target Audience: Scientific community andstudents Changes/Problems: 1. We had proposed to use a well characterized hybridoma cell line to generate an epitope-masking single chain antibody (scFv). However, our attempts to obtain the cell line were not successful due to license and intellectual property issues. We decided to generate our own BVDV neutralizing mAbs and produced several hybridoma clones that produced mAbs reacting to BVDV. A phage display library was generated, panned to identify BVDV neutralizing scFv for generating motifs for masking BVDV MLV vaccine. We also concurrently generated BVDV BVDV neutralizing epitope-specific VH-VL motifs from B cells isolated from a steer hyper-immunized against diverse BVDV strains. 2. Initial protein expression by adenovirus constructs encoding novel mosaic BVDV protective antigens were not stable and the BVDV genes had to be modified to generate stable virus. Further modifications were necessary to improve protein expression. 3. TAMU Institutional Biosafety Committee (IBC) requirements and limited BSL2-Ag space resulted in modifications to our schedule of experiments. We carried out IBC mandated studies to determine the length of period a recombinant adenovirus persists in cattle following intradermal inoculation. This had never been done before and based on the data generated, IBC directed that animals inoculated with recombinant adenovirus should be held at BSL2-Ag containment for one week and in an approved BSL1 facility thereafter. This mandated requirement for elevated biocontainment placed unbudgeted cost burden on the project and necessitated sourcing for extra funds to support in vivo studies. 4. We could only use newly weaned calves that have not been vaccinated with a multi-valent vaccine containing BVDV vaccine and thus we had to raise one-day old calves to meet our needs. The above unexpected outcomes coupled with the severe drought in 2011 exerted a huge cost burden on this project. Additional funds were provided by TAMU but this was not enough. What opportunities for training and professional development has the project provided? 1. One postdoctoral fellow, a minority, received training 2. Three PhD students received hands-on training in the lab 3. One visiting international scholar received short-term training 3. Six Master level students, a majorit of them women from minority background, received hands-on training in the lab 4. Six undergraduate student, amojority of them women, received hands-on training in the lab How have the results been disseminated to communities of interest? 1. Publications 2. Presentations at meetings 3. Patent will allow technology developed to deployed for generation of improved vaccines What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
1. Developed a DC-targeting motif, designated CC98, generated a bi-specific conjugate and tested its ability to protect live BVD virus against antibody-mediated destruction and target the masked virus to bovine dendritic cell CD205 antigen receptor. A control motif, designated 3G4, was also generated. 2. E2-specific bovine VH-VL motifs were cloned from B cells isolated from a steer hyper-immunized with diverse BVDV isolates. 3. Generated and tested adenovirus-based BVDV vaccine. Due to poor performance, the vaccine was redesigned using modified genes encoding novel mosaic antigens, designated NEEE, NS2-31, and NS2-33, that capture the antigen repertoire present in the BVDV1&2 isolates. The genes were used to generate recombinant adenoviruses, designated AdNEEE, AdNS2-31, and AdNS2-33, that were then evaluated for protein expressionand authenticity validated using BVDV-specific mAbs, polyclonal sera, and T cells from a BVDV immunized cow. Efficacy was tested in calves and shown to confer better protection compared a coomercial MLV vaccine following challenge. 4. Generated recombinant constructs expressing DC-targeting motif fused to NEEE, NS2-31, and NS2-33 for development of a protein-based BVDV subunit vaccine.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Njongmeta, M. L., J. Bray, C. J. Davies, W. C. Davis, C. J. Howard, J. C. Hope, G. H. Palmer, W. C. Brown, and W. Mwangi. 2012. CD205 antigen targeting combined with dendritic cell recruitment factors and antigen-linked CD40L activation primes and expands significant antigen-specific antibody and CD4+ T cell responses following DNA vaccination of outbred animals. Vaccine. 30(9):1624-35. PMID: 22240344
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Wang, F., D. C. Ekiert, I. Ahmad, W. Li, Y. Zhang, A. Torkamani, T. Raudsepp, W. Mwangi, M. F. Criscitiello, I. A. Wilson, P. G. Schultz, and V. V. Smider. 2013. Reshaping Antibody Diversity. Cell 153: 1379-1393. PMID: 23746848
- Type:
Journal Articles
Status:
Submitted
Year Published:
2013
Citation:
Fang, G., J. Bray, L. M. Njongmeta, S. Lokhandwala, S. D. Waghela, and W. Mwangi. 2013. Priming broadly protective BVDV-specific immunity using a multi-component mosaic antigen. Vaccine journal-to be submitted
- Type:
Journal Articles
Status:
Submitted
Year Published:
2013
Citation:
Njongmeta, M. L., J. Bray, S. D. Waghela, and W. Mwangi. 2013. Evaluation of the immunogenicity and protective efficacy of an adenovirus-vectored BVDV mosaic antigen in cattle. Vaccine journal-to be submitted
- Type:
Journal Articles
Status:
Submitted
Year Published:
2013
Citation:
Stafforini, G., J. Bray, L. M. Njongmeta, C. Ashley, Z. Browning, S. D. Waghela, D. M. P. Filgueira, and W. Mwangi. 2013. Replication-incompetent Recombinant Adenovirus-5 inoculated intradermally is short-lived but primes robust immune response in calves: Biosafety implication. Vaccine journal-to be submitted
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Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: This study has two components: 1) Protect modified live virus (MLV) and contemporary BVDV vaccines against destruction by immune mechanisms directed by pre-existing maternal antibodies and target the masked vaccine to dendritic cells (DCs) for induction of T cell responses in neonates. Single chain antibody (scFv) specific to both BVDV-1 and BVDV-2 have been generated and shown to have neutralizing activity against both viruses. These scFv motifs will shield MLV vaccine against antibody-mediated destruction. We have developed a DC-targeting motif, generated a bi-specific conjugate and tested its ability to protect live BVD virus against antibody-mediated destruction and target the masked virus to bovine dendritic cell CD205 antigen receptor. 2) Three chimeric genes encoding conserved BVDV 1 & 2 antigenic determinants were designed from Npro, E2, and NS2-3, and used to generate three synthetic genes. The genes were cloned into pCMV expression vector which has a CD5-based secretory signal and a FLAG tag. Positive clones were identified by sequencing and three constructs with authentic sequences were identified and designated pBVDV1, pBVDV2, and pBVDV3. Protein expression was verified by immunocytometric analysis of 293A cells transfected with the DNA constructs using anti-FLAG mAb and BVDV-specific mAbs and polyclonal antibodies. The genes were also subcloned into adenovirus vector and three positive clones of each construct were sequenced and protein expression evaluated as above. One positive clone of each construct, designated pAdBVDV1, pAdBVDV2, and pAdBVDV3, was used to generate recombinant adenoviruses, and protein expression by the recombinant viruses was validated as above. The AdBVDV3 was evaluated in calves in a dose escalation study to determine the titer required to induce optimal immune responses following a single dose immunization. Due to sub-optimal protein expression levels and the fact that adenovirus encoding the chimeric BVDV antigens were unstable, the genes were re-designed and shortened to improve adenovirus assembly and protein expression. The modified genes, designated NEEE, NS2-31, and NS2-33, capture the antigen repertoire present in the BVDV1&2 isolates. The genes were used to generate recombinant adenoviruses, designated AdNEEE, AdNS2-31, and AdNS2-33, that were then evaluated for protein expression as above and authenticity validated using BVDV-specific mAbs, polyclonal sera, and T cells from a BVDV immunized cow. In addition, the dendritic cell antigen targeting motif, designated CC98, was added at the N-terminal to generate chimeric genes designated CC98NEEE, CC98NS2-31, and CC98NS2-33, respectively, which were then cloned into the pCMV vector. Protein expression by the resultant constructs was validated as above and the chimeric genes from clones expressing the proteins were subcloned into a baculovirus vector for large scale protein generation. Patent filed: Overriding pre-existing vaccine-specific neutralizing antibodies to prime and or boost adaptive immunity. Patent application: TAMU 17023. PARTICIPANTS: Participants in this research project were my co-investigator - Dr. Surya Waghela; and a close collaborator Dr. Luc Berghman. In addition, post-doctoral associates Drs. Daad Abi-Ghanem, Leo Njongmeta, and Vanitha Vinodkumar. Graduate students: Shehnaz Lokhandwala and George Fang. Jocelyn Bray has been providing valuable technical assistance. TARGET AUDIENCES: Target audiences include the dairy and the beef industry for a successful strategy to vaccinate at an early age for complete protection. The success of this strategy would indicate its application for other diseases, such as bovine respiratory disease, and would be of interest to commercial firms that supply or make vaccines for animal health. The basic principle for this strategy in vaccine design will provide immunologists; particularly those involved in ruminant immunology a platform to increase efficacy of live vaccines and live-vectored vaccines to override pre-existing antibody barrier. We will provide our results to our peers and colleagues through presentations at relevant meetings and in peer-reviewed journals. PROJECT MODIFICATIONS: We had proposed to use a well characterized hybridoma cell line to generate an epitope-masking single chain antibody (scFv). However, our attempts to obtain the cell line were not successful due to license and intellectual property issues. We decided to generate our own BVDV neutralizing mAbs and we have produced several hybridoma clones that produce mAbs reacting to BVDV. A phage display library was generated and panned to identify BVDV neutralizing scFv as mentioned above for generating motifs for masking BVDV MLV vaccine. Initial expression adenovirus constructs were not stable and the BVDV genes had to be modified/trimmed to generate stable virus. Further modifications were necessary to improve protein expression. TAMU Institutional Biosafety Committee (IBC) requirements and limited BSL2 space have resulted in modifications to our schedule of experiments. We have carried out IBC mandated studies to determine the length of period a recombinant adenovirus persists in cattle following intradermal inoculation. Based on the data generated, IBC directed that animals inoculated with recombinant adenovirus should be held at BSL2 containment for one week and in an approved BSL1 facility thereafter. This mandated requirement for elevated biocontainment has placed unbudgeted cost burden on the project and necessitated sourcing for extra funds to support in vivo studies. We can only use newly weaned calves that have not been vaccinated with a multi-valent vaccine containing BVDV vaccine and thus we have to raise one-day old calves to meet our needs. The above unexpected outcomes coupled with drought have exerted a huge cost burden on this project. Additional funds have been received from TAMU but this is not enough.
Impacts
BVDV-specific scFvs were identified for the development of a motif capable of protecting MLV vaccine against antibody-mediated destruction. A bovine DC-targeting motif was made and shown to target antigens to CD205 antigen receptor in vitro and in situ (Njongmeta, L., et. Al., 2012 Vaccine). We have generated a BVDV-bovine DC bi-specific conjugate and shown that it protects the BVDV from destruction by antibody-dependent immune mechanisms and target the virus to the CD205 antigen receptor. Three codon-optimized synthetic chimeric genes encoding multiple B and T cell epitopes from defined BVDV-1 & 2 Npro, E2, and NS2-3 antigenic determinants were generated and used to develop eukaryotic expression constructs designated pBVDV1, pBVDV2, and pBVDV3. These constructs were shown to express the encoded mosaic antigens as determined by immunocytometric analysis using anti-FLAG mAb and more importantly, the expressed antigens are recognized by mAbs and polyclonal antibodies against BVDV-1 & 2. The adenovirus constructs pAdBVDV1, pAdBVDV2, and pAdBVDV3 are also expressing the encoded antigens and the expressed antigens are recognized by BVDV1&2-specific mAbs, polyclonal antibodies, and BVDV-specific T cells. Recombinant adenoviruses generated using the pAdBVDV1 and pAdBVDV2 constructs were unstable and failed to assemble and produce viable viral particles. The recombinant adenovirus generated using the pAdBVDV3 construct was stable and assembled viable virus expressing the encoded antigen but the protein yield was low. The expressed BVDV3 mosaic antigen is strongly recognized by polyclonal antibodies against BVDV-1 & 2 and by mAbs against BVDV-2, but recognition by BVDV-1-specific mAb is moderate. These outcomes suggest that these mosaic antigens will likely induce broad B cell responses against BVDV-1 & 2. The AdBVDV3 virus was titrated in calves in a dose-escalation study to determine the dose required for optimal induction of immune responses in neonatal calves in the presence of BVDV-specific maternal antibodies. The outcome showed that the vaccine needs optimization to improve efficacy. The modified genes, NEEE, NS2-31, and NS2-33, were used to generate recombinant adenoviruses, AdNEEE, AdNS2-31, and AdNS2-33, that were then shown to have improved protein expression and also shown to contain authentic BVDV B and T cell targets as validated using BVDV-specific mAbs, polyclonal sera, and T cells from a BVDV hyper-immunized animal. The recombinant adenoviruses were used to immune calves and this study is on-going. To further improve the subunit vaccine, the NEEE, NS2-31, and NS2-33 genes were further modified to contain dendritic cell targeting motif, CC98, to improve priming. Constructs encoding these genes were shown to express the BVDV antigens and the expressed protein was shown to bind to the dendritic cell antigen receptor CD205 that is recognized by the CC98 motif. In addition, the expressed antigens were shown to contain authentic BVDV B and T cell determinants. Generation of recombinant protein using the baculovirus system is underway and the generated proteins will be used to conduct vaccine efficacy studies in calves.
Publications
- Leo M. Njongmeta, Jocelyn Bray, Christopher J. Davies, William C. Davis, Christopher J. Howard, Jayne C. Hope, Guy H. Palmer, Wendy C. Brown, and Waithaka Mwangi. 2012. CD205 antigen targeting combined with dendritic cell recruitment factors and antigen-linked CD40L activation primes and expands significant antigen-specific antibody and CD4+ T cell responses following DNA vaccination of outbred animals. Vaccine. 30(9):1624-35. PMID: 22240344.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: This study has two major components: 1) Protect current and contemporary BVDV vaccines against destruction by immune mechanisms directed by pre-existing maternal antibodies and target the masked vaccine to dendritic cells (DCs) for induction of potent T cell responses. A monoclonal antibody that is specific to both BVDV-1 and BVDV-2 has been generated and shown to have neutralizing activity against both viruses. We are generating a single chain antibody (scFv) from the hybridoma secreting the neutralizing mAb for protecting modified live virus (MLV) BVDV vaccine against antibody-mediated destruction. We have developed a DC-targeting motif, generated a bi-specific conjugate and tested its ability to protect live BVD virus against antibody-mediated destruction and target the masked virus to bovine dendritic cell CD205 antigen receptor. In addition, we have developed an agonistic anti-bovine CD40 mAbs for the development of a CD40-based polyvalent adjuvant. Three chimeric genes encoding defined and predicted B and T cell epitopes of most available BVDV 1 & 2 isolates were designed from the major antigenic proteins namely: Npro, E2, and NS2-3, and used to generate three synthetic genes of approximately 4.5 kb each. The genes were cloned into pCMV eukaryotic expression vector which has a CD5-based secretory signal and a FLAG tag. Positive clones were identified by DNA sequencing and three constructs with authentic sequences were identified and designated pBVDVI, pBVDVII, and pBVDVIII. Protein expression was verified by immunocytometric analysis of 293A cells transfected with the DNA constructs using anti-FLAG mAb and BVDV-specific mAbs and polyclonal antibodies. These genes were also subcloned into adenovirus generation vector and three positive clones of each construct were sequenced and protein expression evaluated as above. One positive clone of each construct, designated pAdBVDVI, pAdBVDVII, and pAdBVDVIII, was used to transfect 293A cells to generate recombinant adenovirus, and protein expression by the recombinant virus was validated as above. The AdBVDVIII is being evaluated in calves in a dose escalation study to determine the titer that can induce optimal immune responses following a single dose immunization. Due to poor protein expression levels and the fact that adenovirus encoding the chimeric BVDV antigens were unstable, the genes were re-designed and shortened to improve adenovirus assembly and protein expression. In addition, the dendritic cell antigen targeting motif was added at the N-terminal. The modified constructs were evaluated for protein expression as above. Patent filed: Overriding pre-existing vaccine-specific neutralizing antibodies to prime and or boost adaptive immunity. Patent application Number: TAMU 17023. PARTICIPANTS: Participants in this research project were my co-investigator - Dr. Surya Waghela; a close collaborator Dr. Luc Berghman. In addition, post-doctoral associates Drs. Daad Abi-Ghanem, Leo Njongmeta, and Vanitha Vinodkumar. Finally, Ms. Jocelyn Bray has been providing valuable technical assistance. TARGET AUDIENCES: Target audiences include the dairy and the beef industry for a successful strategy to vaccinate at an early age for complete protection. The success of this strategy would indicate its application for other diseases, such as bovine respiratory disease, and would be of interest to commercial firms that supply or make vaccines for animal health. The basic principle for this strategy in vaccine design will provide immunologists; particularly those involved in ruminant immunology a platform to increase efficacy of live vaccines and live-vectored vaccines to override pre-existing antibody barrier. We will provide our results to our peers and colleagues through presentations at relevant meetings and in peer-reviewed journals. PROJECT MODIFICATIONS: We had proposed to use a well characterized hybridoma cell line to generate an epitope-masking single chain antibody (scFv). However, our attempts to obtain the cell line were not successful due to license and intellectual property issues. We decided to generate our own BVDV neutralizing mAbs and we have produced several hybridoma clones that produce mAbs reacting to BVDV. A BVDV neutralizing mAb has been identified and once it has been well characterized, we will prepare scFv genes for generating motifs for masking BVDV MLV vaccine. TAMU Institutional Biosafety Committee (IBC) requirements and limited BSL2 space have resulted in modifications to our schedule of experiments. We have carried out IBC mandated studies to determine the length of period a recombinant adenovirus persists in cattle following intradermal inoculation. Based on the data generated, IBC directed that animals inoculated with recombinant adenovirus should be held at BSL2 containment for one week and in an approved BSL1 facility thereafter. This mandated requirement for elevated biocontainment has placed unbudgeted cost burden on the project and necessitated sourcing for extra funds to support in vivo studies. We can only use newly weaned calves that have not been vaccinated with a multi-valent vaccine containing BVDV vaccine and thus we have to raise one-day old calves to meet our needs.
Impacts
A panel of monoclonal antibodies against BVDV-1 and 2 were generated and one clone that is neutralizing BVDV-1 and 2 was identified. cDNA has been generated for the development of a single chain antibody (scFv) capable of protecting modified live virus (MLV) BVDV vaccine against antibody-mediated destruction. We have developed a bovine DC-targeting motif and shown that it targets antigens to DC CD205 antigen receptor in vitro and in situ. We have generated a BVDV-bovine DC bi-specific conjugate and shown that it protects the BVD virus from destruction by antibody-dependent immune mechanisms and targets the BVD virus to the bovine CD205 antigen receptor. An agonistic anti-bovine CD40 mAb has been developed and shown to be a potent stimulant of bovine antigen presenting cells. This mAb will be used for generating a polyvalent adjuvant. A non-agonistic anti-bovine CD40 mAb has also been generated to serve as a negative control. Three codon-optimized synthetic chimeric genes encoding multiple B and T cell epitopes from defined BVDV-1 & 2 Npro, E2, and NS2-3 antigenic determinants have been generated and used to develop eukaryotic expression constructs designated pBVDVI, pBVDVII, and pBVDVIII. These constructs are expressing the encoded mosaic antigens as determined by immunocytometric analysis using anti-FLAG mAb and more importantly, the expressed antigens are recognized by mAbs and polyclonal antibodies against BVDV-1 & 2. The adenovirus constructs pAdBVDVI, pAdBVDVII, and pAdBVDVIII are also expressing the encoded antigens and the expressed antigens are recognized by mAbs and polyclonal antibodies against BVDV-1 & 2. Recombinant adenoviruses generated using the pAdBVDVI and pAdBVDVII constructs were unstable and failed to assemble and produce viable viral particles. The recombinant adenovirus generated using the pAdBVDVIII construct was stable and assembled viable virus expressing the encoded antigen but the protein yield was low. The expressed BVDVIII mosaic antigen is strongly recognized by polyclonal antibodies against BVDV-1 & 2 and by mAbs against BVDV-2, but recognition by BVDV-1-specific mAb is moderate. These outcomes suggest that these mosaic antigens will likely induce broad B cell responses against BVDV-1 & 2. The AdBVDVIII virus is being titrated in calves in a dose-escalation study to determine the dose required for optimal induction of immune responses in neonatal calves in the presence of BVDV-specific maternal antibodies.
Publications
- Leo M. N., Bray,J., Davies,C.J., Davis,W.C., Howard, C.J., Hope, J.C., Palmer, G.H., Brown, W.C.,and Mwangi, w. 2011. CD205 Antigen Targeting at a Dendritic Cell-Enriched Immunization Site Significantly Enhances Priming and Expansion of Antigen-Specific Antibody and CD4+ T cell Responses Following DNA Vaccination of Outbred Animals. Vaccine (Accepted).
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: This study has two major components: 1) Protect current and contemporary BVDV vaccines against destruction by immune mechanisms directed by pre-existing maternal antibodies and target the masked vaccine to dendritic cells (DCs) for induction of potent T cell responses. We have developed a panel of monoclonal antibodies (mAbs) against BVDV-1 [BVDV-2-specific mAbs are also being developed] to identify neutralizing mAbs from which we will generate a single chain antibody (scFv) capable of protecting modified live virus (MLV) BVDV vaccine against antibody-mediated destruction. We have developed a DC-targeting motif and generated a bi-specific chimera for targeting BVD virus to bovine dendritic cell CD205 antigen receptor. In addition, we have developed anti-bovine CD40 mAbs for the development of a CD40-based polyvalent adjuvant. Three chimeric genes encoding defined and predicted B and T cell epitopes of most available BVDV 1 & 2 isolates were designed from the major antigenic proteins namely: Npro, E2, and NS2-3 and used to generate three synthetic genes. The genes were cloned into pCMV eukaryotic expression vector which has a CD5-based secretory signal and a FLAG tag. Positive clones were identified by DNA sequencing and three constructs with authentic sequences were identified and designated pBVDVI, pBVDVII, and pBVDVIII. Protein expression was verified by immunocytometric analysis of 293A cells transfected with the DNA constructs using anti-FLAG mAb and BVDV-specific mAbs and polyclonal antibodies. These genes were also subcloned into adenovirus generation vector and three positive clones of each construct were sequenced and protein expression evaluated as above. One positive clone of each construct, designated pAdBVDVI, pAdBVDVII, and pAdBVDVIII, was used to transfect 293A cells to generate recombinant adenovirus, and protein expression by the recombinant virus was tested as above. In addition, a construct encoding BVDV E2 antigen was generated for expression of recombinant antigen in prokaryotic cells for use in assays for analysis of immune response. PARTICIPANTS: Participants in this research project were my co-investigator - Dr. Surya Waghela; a close collaborator Dr. Luc Berghman. In addition, post-doctoral associates Drs. Daad Abi-Ghanem, Leo Njongmeta, and Vanitha Vinodkumar, as well as a graduate student L. Akoolo whose PhD research is a part of this project. Finally, Ms. Jocelyn Bray has been providing valuable technical assistance. Three student workers received training in techniques for performing routine tasks. TARGET AUDIENCES: Target audiences include the dairy and the beef industry for a successful strategy to vaccinate at an early age for complete protection. The success of this strategy would indicate its application for other diseases and would be of interest to commercial firms that supply or make vaccines for animal health. The basic principle for this strategy in vaccine design will provide immunologists; particularly those involved in ruminant immunology a platform to increase efficacy of live vaccines and live-vectored vaccines to override pre-existing antibody barrier. We will provide our results to our peers and colleagues through presentations at relevant meetings and in peer-reviewed journals. PROJECT MODIFICATIONS: We had proposed to use a well characterized hybridoma cell line to generate an epitope-masking single chain antibody (scFv). However, our attempts to obtain the cell line were not successful due to license and intellectual property issues. We decided to generate our own BVDV neutralizing mAbs and we have produced several hybridoma clones that produce mAbs reacting to BVDV. We are selecting clones that produce BVDV neutralizing mAbs and once they are well characterized, we will prepare scFv genes for generating motifs for masking BVDV MLV vaccine. TAMU Institutional Biosafety Committee (IBC) requirements and limited BSL2 space have resulted in modifications to our schedule of experiments. We are at present carrying out studies to find out the length of period a recombinant adenovirus will persist in a bovine host and IBC will use this data to determine when it is safe to move the adenovirus-exposed calves to a lower BLS containment. We can only use newly weaned calves that have not been vaccinated with a multi-valent vaccine containing BVDV vaccine and thus we have to raise one-day old calves to meet our needs.
Impacts
A panel of monoclonal antibodies against BVDV-1 has been generated and is being screened to identify neutralizing mAbs from which we will generate a single chain antibody (scFv) capable of protecting modified live virus (MLV) BVDV vaccine against antibody-mediated destruction. We have developed a bovine DC-targeting motif and shown that it targets antigens to DC CD205 antigen receptor in vitro and in situ. We have generated a BVDV-bovine DC bi-specific chimera that targets BVD virus to the bovine CD205 antigen receptor and protects the virus from recognition/destruction by antibody-dependent immune mechanisms. An agonistic anti-bovine CD40 mAb has been developed and shown to be a potent stimulant of bovine antigen presenting cells. This mAb will be used for generating a polyvalent adjuvant. A non-agonistic anti-bovine CD40 mAb has also been generated to serve as a negative control. Three codon-optimized synthetic chimeric genes encoding multiple B and T cell epitopes from defined BVDV-1&2 Npro, E2, and NS2-3 antigenic determinants have been generated and used to develop eukaryotic expression constructs designated pBVDVI, pBVDVII, and pBVDVIII. These constructs are expressing the encoded mosaic antigens as determined by immunocytometric analysis using anti-FLAG mAb and more importantly, the expressed antigens are recognized by mAbs and polyclonal antibodies against BVDV-1 & 2. The adenovirus constructs pAdBVDVI, pAdBVDVII, and pAdBVDVIII are also expressing the encoded antigens and the expressed antigens are recognized by mAbs and polyclonal antibodies against BVDV-1 & 2. The pAdBVDVIII construct has been used to assemble a recombinant adenovirus, designated AdBVDVIII, and this virus is expressing the BVDVIII mosaic antigen. The mosaic antigen is strongly recognized by polyclonal antibodies against BVDV-1 & 2 and by mAbs against BVDV-2, but recognition by BVDV-1-specific mAb is moderate. These outcomes suggest that these mosaic antigens will likely induce broad B cell responses against BVDV-1 & 2. The AdBVDVIII virus is been titrated in calves in a dose-escalation study to determine the dose required for optimal induction of immune responses in neonatal calves in the presence of BVDV-specific maternal antibodies. The recombinant E2 antigen expressed in E. coli reacts with both reference mAb and polyclonal antibodies in Western blot.
Publications
- No publications reported this period
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Conserved DNA sequences encoding defined and predicted B and T cell epitopes of most available BVDV 1 & 2 isolates were used to design and generate chimeric constructs for inclusion in the synthesis of chimeric genes for the development of a broadly protective vaccine. The sequences were designed from the the major antigenic proteins namely: Npro, E2, and NS2-3. Because of the hypervariablity of E2 sequences, three BVDV 1 and 2 BVDV 2 chimeric constructs were designed. One of the BVDV 1 chimeric gene sequence was use to generate a synthetic gene which was then cloned into a eukaryotic expression vector with a FLAG tag and a secretory signal. Protein expression by the recombinant construct was verified by immunocytometric analysis of 293 cells transfcetd witht the construct. Protein expression was confirmed using anti-FLAG monoclonal antibody as well as polyclonal antibodies specific for BVDV. The expressed protein has been purified and currently undergoing characterization. Similarly we have produced recombinant E2 protein using prokaryotic cells for use in assays for analysis of immune response. The rE2 reacts with both reference mAb and polyclonal in Western blot. Furthermore, since we had faced difficulty in obtaining a hybridoma for an available neutralizing monoclonal antibody, we have immunized steers for developing and identifying a neutralizing recombinant antibody. We have used peripheral lymphocytes to generate the scFv genes and are in the process of developing a phage library for screening for activity. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have shown that the chimeric antigen expressed from a chimeric gene encoding conserved antigenic determinants from BVDV type 1 Npro, E2 and NS2-3 is recognized by sera from animals immunized or exposed to type 1 BVD viruses and this outcome is significant because it suggests that the chimeric protein has potential to induce protective immunity against BVDV type 1 viruses. We have similarly developed chimeras using BVDV 2 sequences and in all, three chimeric constructs with broad coverage of the existing BVDV genomes have been generated to allow us develop a broadly protective BVDV vaccine.
Publications
- No publications reported this period
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