Progress 07/01/22 to 02/29/24

Outputs
Target Audience:Our target audience includes producers and major animal health pharmaceutical companies. We are in discussions with several animal health pharmaceutical companies regarding the development of animal R2 vaccines including our BoHV-1 R2 vaccine. Changes/Problems:A no cost extension was requesteddue to difficulties establishing a stable clone of our BoHV-1 isolate (Aim 1). The challenge of establishing this clone was inconsistent with our prior experience producing clones of other alphaherpesviruses and was unexpected. We were ultimately successful and Aims 2 & 3 progressed as intended following this initial delay and have been completed. What opportunities for training and professional development has the project provided?A full-time Thyreos lab technician was responsible for producing the BoHV-1 R2 vaccine and has expanded his skill set. How have the results been disseminated to communities of interest?When the animal studies have been completed and all data have been analyzed we intend to publish the results in a peer-reviewed journal and post the results on the Thyreos Inc. website. What do you plan to do during the next reporting period to accomplish the goals?After final analysis study results will be prepared for dissemination.

Impacts
What was accomplished under these goals? The three objectives were completed: A native isolate of BoHV-1 was acquired by Thyreos along with all rights to its use. A full-length infectious clone of the BoHV-1 isolate was produced as a self-excising BAC. Codon substitutions were introduced into the R2 coding region of UL37 and the Us9 gene was deleted. All mutations were sequence validated. Live-attenuated vaccine virus was recovered from the clone and grown to high titers. A safety study was conducted using six colostrum-deprived (CD) castrated male Holstein calves that were two-three weeks old when received.Animals were randomly divided into two groups of three. After acclimation to a 14:10 light:dark cycle in a BSL2 room the facility, the approximately four week old calves were inoculated with native BoHV-1 or with BoHV-1 R2 vaccine, both modified to express enhanced green fluorescent protein (EGFP). Animals received one ml of virus applied to the conjunctiva of each eye and one ml/nostril at 6.0 x 10e8 pfu/ml. Ninety-six hours after the inoculation, the trigeminal ganglia (TG) were dissected by a pathologist at the Nebraska Veterinary Diagnostic Center (NVDC). One TG from each calf was placed in cold 4% paraformaldehyde. Serial 40 µm sections through the entire TG were examined for EGFP labeled neurons. BoHV-1 gB PCR analysis was performed on the other TGs. Each left TG from the three native BoHV-1 inoculated calves contained several hundred infected sensory neurons (660, 419, 313); each right TG was gB PCR positive with levels indicating a moderate number of genome copies. In contrast, the three calves inoculated with the live-attenuated R2 vaccine had gB PCR values of the right TGs that were 0 or undetectable. The results supported that the BoHV-1 R2 vaccine is safe and does not enter the nervous system after peripheral inoculation, consistent with previous studies that include over two hundred TGs from experimental animals including calves. Although additional calves were not available to repeat this study, the lack of TG invasion following R2 vaccine inoculation was confirmed in the challenge study. A challenge study was conducted using twelve colostrum-deprived (CD) castrated male Holstein calves that were two-three weeks old when received. Animals were randomly divided into two groups of six. After acclimation to a 14:10 light:dark cycle in a BSL2 room at the facility, the approximately four week old calves were either mock-vaccinated with phosphate buffered saline (PBS) (1 ml/nostril) or vaccinated with BoHV-1 R2 (1 ml/nostril; 2x10e8 pfu/ml). Three weeks later vaccinated calves received an intramuscular BoHV-1 R2 boost (2 ml of 2x10e8 pfu/ml) and mock-vaccinated calves received a mock boost (2 ml PBS). Three weeks later all calves were challenged intranasally with BoHV-1 strain Cooper obtained from the USDA (1 ml/nostril; 5x10e6 pfu/ml). Two weeks after the challenge all calves were euthanized and TGs were collected for gB PCR analysis by facility staff. Nasal swabs and blood were collected after all inoculations and body temperature was recorded daily after BoHV-1 challenge. The two primary goals of this challenge study were: 1) to reconfirm that Thyreos Inc BoHV-1 R2 vaccine did not enter the nervous system of its natural host; and 2) to demonstrate that vaccination with Thyreos Inc BoHV-1 R2 prevented native BoHV-1 from infecting the nervous system following a subsequent challenge. Both goals were successful. Seven of twelve TGs from the mock-vaccinated calves had detectable BoHV-1 genomes after challenge with BoHV-1 Cooper whereas none of the 12 TGs from BoHV-1 R2 vaccinated has detectable BoHV-1. Thus, Thyreos Inc BoHV-1 R2 vaccine did not infect the nervous system and, more importantly, BoHV-1 R2 vaccination prevented native BoHV-1 from infecting the nervous system (p < 0.005; Fishers exact test, two-tailed). The body temperature of mock-vaccinated calves was elevated above baseline at 3, 4 and 5 days post-challenge; BoHV-1 R2 vaccinated calves had a slight increase in body temperature above baseline only on day 4 and this increase was less than that observed in mock-vaccinated animals (103.2 ± 0.30 oF, mock n=6 vs 102.4 ± 0.08 oF, R2 n=6; p = 0.0221). Neutralizing antibody titers rose in the BoHV-1 R2 vaccinated animals from undetectable prior to vaccination to an average of 5.3 x 10e1 after the second vaccination (boost). Nasal swabs indicated that BoHV-1 R2 vaccinated calves shed decreasing levels of virus for nine days after intranasal vaccination. We conclude that Thyreos Inc BoHV-1 R2 vaccine is safe, does not infect the host nervous system, and is an efficacious vaccine that prevents establishment of latent infections by blocking invasion of the nervous system.

Publications

Progress 07/01/22 to 01/30/24

Outputs
Target Audience:Our target audience includes producers and major animal health pharmaceutical companies. We are in discussions with several animal health pharmaceutical companies regarding the development of animal R2 vaccines including our BoHV-1 R2 vaccine. Changes/Problems:A no cost extension was requesteddue to difficulties establishing a stable clone of our BoHV-1 isolate (Aim 1). The challenge of establishing this clone was inconsistent with our prior experience producing clones of other alphaherpesviruses and was unexpected. We were ultimately successful and Aims 2 & 3 progressed as intended following this initial delay and have been completed. What opportunities for training and professional development has the project provided?A full-time Thyreos lab technician was responsible for producing the BoHV-1 R2 vaccine and has expanded his skill set. How have the results been disseminated to communities of interest?When the animal studies have been completed and all data have been analyzed we intend to publish the results in a peer-reviewed journal and post the results on the Thyreos Inc. website. What do you plan to do during the next reporting period to accomplish the goals?After final analysis study results will be prepared for dissemination.

Impacts
What was accomplished under these goals? The three objectives were completed: A native isolate of BoHV-1 was acquired by Thyreos along with all rights to its use. A full-length infectious clone of the BoHV-1 isolate was produced as a self-excising BAC. Codon substitutions were introduced into the R2 coding region of UL37 and the Us9 gene was deleted. All mutations were sequence validated. Live-attenuated vaccine virus was recovered from the clone and grown to high titers. A safety study was conducted using six colostrum-deprived (CD) castrated male Holstein calves that were two-three weeks old when received from J.R. Livestock Inc., Lake Mills, IA. Animals were randomly divided into two groups of three. After acclimation to a 14:10 light:dark cycle in a BSL2 room in the Life Science Annex (LSA) at the University of Nebraska-Lincoln (UNL), the approximately four week old calves were inoculated with native BoHV-1 or with BoHV-1 R2 vaccine, both modified to express enhanced green fluorescent protein (EGFP). Animals received one ml of virus applied to the conjunctiva of each eye and one ml/nostril at 6.0 x 10e8 pfu/ml. Ninety-six hours after the inoculation, calves were euthanized by an overdose of pentobarbital and the trigeminal ganglia (TG) were dissected by a pathologist at the Nebraska Veterinary Diagnostic Center (NVDC). One TG from each calf was placed in cold 4% paraformaldehyde. Serial 40 µm sections through the entire TG were examined for EGFP labeled neurons. The NVDC performed BoHV-1 gB PCR analysis on the other TGs. Each left TG from the three native BoHV-1 inoculated calves contained several hundred infected sensory neurons (660, 419, 313); each right TG was gB PCR positive with levels indicating a moderate number of genome copies. In contrast, the three calves inoculated with the live-attenuated R2 vaccine had gB PCR values of the right TGs that were 0 or undetectable. However, a miscommunication resulted in one of the R2 vaccinated animals becoming exposed to residual native BoHV-1 (native inoculations were mistakenly performed before the R2 vaccine inoculations instead of after, and residual inoculum was present in the nebulizer when proceeding to the first calf of the R2 vaccine group). Although the gB PCR did not detect virus in the right TG of this animal, 85 cells were labeled in the left TG. The remaining two animals of this group had no EGFP labeled neurons. Factoring in this technical error, the results supported that the BoHV-1 R2 vaccine is safe and does not enter the nervous system after peripheral inoculation, consistent with previous studies that include over two hundred TGs from experimental animals including calves. Although additional calves were not available to repeat this study, the lack of TG invasion following R2 vaccine inoculation was confirmed in the challenge study. A challenge study was conducted using twelve colostrum-deprived (CD) castrated male Holstein calves that were two-three weeks old when received from J.R. Livestock Inc., Lake Mills, IA. Animals were randomly divided into two groups of six. After acclimation to a 14:10 light:dark cycle in a BSL2 room in the LSA at UNL, the approximately four week old calves were either mock-vaccinated with phosphate buffered saline (PBS) (1 ml/nostril) or vaccinated with BoHV-1 R2 (1 ml/nostril; 2x10e8 pfu/ml). Three weeks later vaccinated calves received an intramuscular BoHV-1 R2 boost (2 ml of 2x10e8 pfu/ml) and mock-vaccinated calves received a mock boost (2 ml PBS). Three weeks later all calves were challenged intranasally with BoHV-1 strain Cooper obtained from the USDA (1 ml/nostril; 5x10e6 pfu/ml). Two weeks after the challenge all calves were euthanized and TGs were collected for gB PCR analysis by the NVDC. Nasal swabs and blood were collected after all inoculations and body temperature was recorded daily after BoHV-1 challenge. The two primary goals of this challenge study were: 1) to reconfirm that Thyreos Inc BoHV-1 R2 vaccine did not enter the nervous system of its natural host; and 2) to demonstrate that vaccination with Thyreos Inc BoHV-1 R2 prevented native BoHV-1 from infecting the nervous system following a subsequent challenge. Both goals were successful. Seven of twelve TGs from the mock-vaccinated calves had detectable BoHV-1 genomes after challenge with BoHV-1 Cooper whereas none of the 12 TGs from BoHV-1 R2 vaccinated has detectable BoHV-1. Thus, Thyreos Inc BoHV-1 R2 vaccine did not infect the nervous system and, more importantly, BoHV-1 R2 vaccination prevented native BoHV-1 from infecting the nervous system (p < 0.005; Fishers exact test, two-tailed). The body temperature of mock-vaccinated calves was elevated above baseline at 3, 4 and 5 days post-challenge; BoHV-1 R2 vaccinated calves had a slight increase in body temperature above baseline only on day 4 and this increase was less than that observed in mock-vaccinated animals (103.2 ± 0.30 oF, mock n=6 vs 102.4 ± 0.08 oF, R2 n=6; p = 0.0221). Neutralizing antibody titers rose in the BoHV-1 R2 vaccinated animals from undetectable prior to vaccination to an average of 5.3 x 10e1 after the second vaccination (boost). Nasal swabs indicated that BoHV-1 R2 vaccinated calves shed decreasing levels of virus for nine days after intranasal vaccination. We conclude that Thyreos Inc BoHV-1 R2 vaccine is safe, does not infect the host nervous system, and is an efficacious vaccine that prevents establishment of latent infections by blocking invasion of the nervous system.

Publications

Progress 07/01/22 to 06/30/23

Outputs
Target Audience:Our primary target audience includes producers (consumers) and animal health pharmaceutical compaines that manufactureand market vaccines. Ourcompany continues to have discussions with producers and animal health pharmaceutical companies regarding the development of animal R2 vaccines including our BoHV-1 R2 vaccine. Changes/Problems:In 2022, a one year, no cost extension was requested due to difficulties establishing a stable clone of our BoHV-1 isolate (Aim 1). The challenge of establishing this clone was inconsistent with our prior experience producing clones of other alphaherpesviruses and was unexpected. We were ultimately successful and Aim2 & Aim 3 progressed as intended following this initial delay. Since the request for the no cost extension in 2022, wehave no new changes or problems to report. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Results have not yet been diseminated to communities of interest. When the animal studies have been completed and all data have been analyzed we intend to either publish the results in a peer-reviewed journal or post the results on the Thyreos Inc. website. What do you plan to do during the next reporting period to accomplish the goals?As described above, we will complete neuroinvasion study.

Impacts
What was accomplished under these goals? The over-arching goal of this Phase I SBIR project is to construct a non-neuroinvasive BoHV-1 R2 vaccine. The objectives are: 1: Establish a self-excising bacterial artificial chromosome (BAC) infectious clone from a BoHV-1 isolate. This new clone be used to produce a vaccine free of intellectual property constraints. This objective has been completed. 2: Introduce R2 mutations into the BAC and delete the US9 gene to make a marker vaccine derivative. Our established recombination methods will be used to modify the BoHV-1 BAC clone first to mutate the UL37 gene in the R2 effector coding region, and second to delete the US9 gene. Propagation of the recovered viruses will be assessed in cell culture. This objective has been completed. 3: Validate the safety and efficacy of the BoHV-1 live-attenuated vaccine in calves. A Go/No-Go decision to advance the BoHV-1 vaccine will be based on confirmation that the R2 mutations ablate the neuroinvasive property of the virus and its virulence, while also providing unprecedented protection against native BoHV-1 challenge. This objective is in progress and nearly complete. The project is nearly completed and detailed outcomereporting for all three objectives will be submitted in the final report.A challenge study was conducted and very recently completed. Weincreased the group size from 4 calves/group to 6 calves/group. Calves were vaccinated with BoVH-1 R2 ?Us9 or mock-vaccinated with PBS intranasally followed three weeks later by an imtramuscular BoVH-1 R2 ?Us9 boost or mock boost. Three weeks later all calves were challenged intranasally with BoHV-1 strain Cooper and euthanized two weeks later. We are waiting for final lab results from swabs and serum. Initial results are encouraging; none of the BoVH-1 R2 ?Us9 vaccinated calves had detectable BoHV-1 in their trigeminal ganglia (TG) (0 of 12 TGs) confirming that the BoVH-1 R2 ?Us9 vaccine did not invade the nervous system as expected. In addition, the BoVH-1 R2 ?Us9 vaccine prevented wild-type challenge virus from infecting the nervous system. In the six mock-vaccinated calves, 4 of 12 TGs had detectable BoHV-1 indicating that the challenge BoHV-1 infected their nervous system. The results are significantly different (0 of 12 R2 TGs infected vs 4 of 12 mock TGs infected; p < 0.05, Fisher's exact test). The neuroinvasion study using more stringent conditions is scheduled and will be completed by the end of the year. A final progress report will be submitted in early 2024.

Publications