Source: NORTH DAKOTA STATE UNIV submitted to NRP
INTEGRATING VACCINE EFFICACY AND SAFETY BY DIRECTED SUICIDAL REPLICATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1025793
Grant No.
2021-67015-34419
Cumulative Award Amt.
$500,000.00
Proposal No.
2020-06395
Multistate No.
(N/A)
Project Start Date
Jul 1, 2021
Project End Date
Jun 30, 2026
Grant Year
2021
Program Code
[A1221]- Animal Health and Production and Animal Products: Animal Health and Disease
Recipient Organization
NORTH DAKOTA STATE UNIV
1310 BOLLEY DR
FARGO,ND 58105-5750
Performing Department
Microbiological Sciences
Non Technical Summary
Porcine reproductive and respiratory disease syndrome virus (PRRSV) and porcine circovirus type 3 (PCV3) are economically important swine viruses which cause reproductive failure and respiratory disease. While vaccines against PCV3 are yet to be developed, it is known that attenuated vaccines against PRRSV are more effective than inactivated vaccines. However, attenuated PRRSV vaccines and live vaccines in general are associated with the risk of reversion to virulence and recombination with field strains. Hence, the primary goal of this project is to develop a rapid-attenuation method targeting enhanced vaccine safety while retaining or improving vaccine efficacy. The proposed method involves harnessing the natural property of rapid mutation in viruses to induce suicidal replication of the vaccine construct in the host. The primary objectives of the project are therefore to develop directed suicidal vaccines against PRRSV and PCV3, and test the efficacy and safety of the test vaccines in a dual PCV3 and PRRSV coinfection model. In addition, the data generated is expected to contribute to the current understanding pathogenesis in PCV3 and PRRSV coinfections. The expected project outputs include the development of improved, safe and effective vaccines against PCV3 and PRRSV.
Animal Health Component
60%
Research Effort Categories
Basic
20%
Applied
60%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3113510110150%
3113510104030%
3113510109020%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3510 - Swine, live animal;

Field Of Science
1040 - Molecular biology; 1090 - Immunology; 1101 - Virology;
Goals / Objectives
Porcine productive and respiratory disease syndrome virus (PRRSV) continues to remain the leading cause of economic losses to the pork industry for over 30 years, and PRRS is characterized by severe respiratory distress and reproductive failure in breeding animals Despite significant investment, efficacy and safety of current PRRSV vaccines remain questionable. While modified live vaccines (MLVs) against PRRSV are generally the most effective, they are associated with significant safety concerns such as recombination with field strains and reversion to virulence. The primary goal of this project is to develop a novel method to improve the safety of live attenuated vaccines. The targeted approach will harness the naturally high mutation rates of viruses such as PRRSV to direct the suicidal replication of the modified live vaccine and result in effective immunity and rapid clearance from the host to integrate vaccine safety and efficacy. Porcine circovirus type 3 (PCV3) is a recently discovered virus which also causes reproductive failure, respiratory illness and the porcine dermatitis and nephropathy syndrome. PCV3 and PRRSV are frequently co-detected in reproductive failure, underscoring the urgent need to better understand and prevent PCV3, especially in the context of coinfections. Vaccines against PCV3 are not yet available. Although porcine circoviruses are DNA viruses, their mutation rates are similar to that of RNA viruses. Given the similarities in the clinical manifestations of PCV3 and PRRSV, the other goals of the project are to develop a PCV3 and PRRSV dual infection model and apply the directed suicidal vaccine approach to develop modified live vaccines against both PCV3 and PRRSV, and assess protection against both viral infections using the dual infection model.The objectives of the project are to:Aim 1 - To develop and test a DS-PRRSV MLV To test the hypothesis that a DS-PRRSV-MLV will elicit superior efficacy and safety compared to a commercial PRRSV MLV, the DS-PRRSV-MLV candidate developed by us will be tested in a piglet vaccination and heterologous PRRSV challenge model.Aim 2 - To develop and test a DS-PCV3-MLV vaccine against singular PCV3 challenge and dual PCV3 and PRRSV challenge. Objective 2.1 To optimize singular and dual PCV3 and PCV3 and PRRSV swine infection models respectively. To test the hypothesis that dual infection of weanling piglets with recombinant PCV3 and PRRSV will result in exacerbation of clinical signs, swine will be infected with either PCV3 alone or with PCV3 and PRRSV, and clinical and immunological parameters evaluated.?Objective 2.2. To develop and test the safety and efficacy of a DS-PCV3-MLV against single and dual PCV3 and PRRSV challenge. To test the hypothesis that the DS-PCV3-MLV can protect against PCV3 and also mitigate the effects of PRRSV in coinfections, the DS-PCV3-MLV will be tested by vaccination of piglets and challenge with PCV3 alone or PCV3+PRRSV.
Project Methods
1. Development of the directed suicidal PRRSV and PCV3 vaccines will be achieved using recombinant DNA and cloning techniques in the backbone of infectious clones of both viruses. The strategy will be assessed by success in rescuing the modified live vaccine constructs and reactivity to PRRSV and PCV3 specific antibodies. The targeted accumulations of deleterious mutations will be assessed by placing the constructs under immune pressure and subjecting them to deep sequencing.2. Development of the PRRSV and PCV3 coinfection model will be achieved by dual infection of piglets with both viruses, while singly infected pigs will serve as controls. The expected exacerbation of infection in coinfected pigs will be assessed by the observation of clinical signs, qPCR to determine if viral replication is increased in coinfection and by the gross and microscopic pathological examination of infected pigs.3. Assessment of vaccine safety and efficacy will be achieved by vaccinating piglets and challenging them singly or dually with PCV3 and PRRSV. Assessments of the desired outcomes of improved safety and efficacy will be achieved by measuring viral replication by qPCR and prevention of disease manifestation as clinical signs and by pathological examination after euthanasia.

Progress 07/01/23 to 06/30/24

Outputs
Target Audience:The target audience includes animal health researchers, swine producers, vaccine manufacturers and funding agencies Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One research scientist is involved in the project. She are being trained in the laboratory techniques and methodology involved in the project. Shewill be provided with opportunities to present data and participate in scientific meetings as the research progresses. How have the results been disseminated to communities of interest?The data from the described studies was presented at CRWAD and related data regarding PCV2 was disseminated via publications. What do you plan to do during the next reporting period to accomplish the goals?The analysis of samples and pathological evaluation for the animal studies will be completed. The data will be published in peer-reviewed journals

Impacts
What was accomplished under these goals? Aim 1: The DS-PRRSV-MLV was tested in a swine vaccination and challenge model. The vaccine induced robust antibody responses. Samples generated from the study are currently being analyzed for viral loads and pathological lesions. Aim2: Objective 2.1: Pigs were infected with PCv3 virus rescued from the infectious clone, either alone or in combination with PRRSV. In the singular infection model, sero-conversion to PCV3 occurred and correlated with the detection of viral DNA in serum and viral antigen in PBMCs by flow cytometry. PCV3 virus was re-isolated from the serum of infected pigs and was viable on serial passages. Pathological lesions and detection of PCV3 in tissues is currently under process. The data indicates that the developed reverse genetics system is effective in generating viable PCV3 which is infective to pigs. The coinfection of PCV3 with PRRSV did not appear to enhance PRRSV induced pathological lesions in the experimental model of infection. Objective 2.2: Pigs were vaccinated with the DS-PCV3-MLV and challenged with PCV3 derived from the infectious clone. The vaccine was cleared from infected pigs within a week of vaccination, indicating the directed suicidal vaccine strategy was very effective in improving vaccine safety. Vaccinated pigs mounted significant binding and virus neutralizing antibody responses to PCV3. Vaccination with the DS-PCV3-MLV induced sterilizing immunity in the vaccinated pigs while the unvaccinated controls experienced significant PCV3 viremia. Pathological evaluation of lesions due to the challenge virus is currently under process.

Publications

  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Ssemadaali, M.; Islam, M.; Fang, W.; Aboezz, W.; Webb, B.; Ramamoorthy, S. Trans-replicase Helper Activity of Porcine Circoviruses Promotes The Synergistic Replication of Torque Teno Virus. Front Microbiol. 2024 Jan 23;15:1326696.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: A minimally replicative PRRSV vaccine targeting improved safety and efficacy. Fang W and Ramamoorthy. S. Annual Meeting of the Council of Research Workers in Animal Diseases.2024. Chicago, IL.


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

Outputs
Target Audience:The target audience includes animal health researchers, swine producers, vaccine manufacturers and funding agencies Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One research scientist is involved in the project. They are being trained in the laboratory techniques and methodology involved in the project. They will be provided with opportunities to present data and participate in scientific meetings as the research progresses. How have the results been disseminated to communities of interest?A poster was presented in CRWAD on the development and testing of the PCV3 infectious clone. What do you plan to do during the next reporting period to accomplish the goals?Once the required virus stocks are prepared, the plan is to test the DS-PRRSV vaccine. We anticipate that we will be able to schedule the study in the fall of 2023?

Impacts
What was accomplished under these goals? Currently, other samples from both objective 2.1 and 2.2 are being processed to assess PCV3 lesion scores, seroconversion, and viral loads.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Rescue and characterization of porcine circovirus type 3 (PCV3) from a recombinant infectious clone. Sheela Ramamoorthy. 102nd Annual Meeting of the Council of Research Workers in Animal Diseases.2023. Chicago, IL


Progress 07/01/21 to 06/30/22

Outputs
Target Audience:The target audience will consist of pork producers, scientists, vaccine manufacturers and granting agencies Changes/Problems:The first animal study was scheduled to start in the 2nd week of September 2022. However, a recent adverse weather event at South Dakota (derecho) resulted in damage to the animal testing facilities, which now require the air handlers to be replaced. It is likely that the facility will be closed in the fall and the therefore, the study may be conducted in early spring 2023 after the repairs are completed. What opportunities for training and professional development has the project provided?One research scientist and graduate student are involved in the project. They are being trained in the laboratory techniques and methodology involved in the project. They will be provided with opportunities to present data and participate in scientific meetings as the research progresses. How have the results been disseminated to communities of interest?As the project is in the first year, a poster was presented at the NA PRRS/NC 229 symposium in CRWAD and the PI provided an invited talk at the same conference What do you plan to do during the next reporting period to accomplish the goals?Once the required virus stocks are prepared, the plan is to complete the animal studies for the PCV3 and PRRSV model and to test the DS-PRRSV vaccine. We anticipate the 1st study to be conducted in spring 2023, and complete study readouts by summer 2023.

Impacts
What was accomplished under these goals? Aim1: The DS-PPRSV-MLV constructs were cloned and rescued successfully using the PRRSV VR2385 as a backbone. The DS-PRRSV-N gene constructs could be cultured to higher titers than the DS-PRRSV M&N gene constructs. Currently, in vitro vaccine stability tests, growth curvesand sequencing to ensure the integrity of the constructs are being conducted Aim 2: The DS-PCV3-MLV construct was prepared and rescued successfully. To prepare the challenge virus, an infectious clone of PCV3 was constructed and recombinant virus successfully rescued by transfection. Both viruses maintained high titers for up to the 7 passages tested in cell culture. Currently, efforts are underway to sequence the passaged virus to ensure stability and to dimerize the infectious clones to facilitate the preparation of virus cultures for the animal study Pregnant sows from the NDSU sow barn were bled and tested by PCV and ELISA to ensure a negative status for PCV3 and PRRSV. Planning of thestudy to optimize the PCV3 and PRRSV coinfection model and testing of the DS vaccines in piglets is currently underway

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Rakibuzzaman, A.; Pineyro, P.; Pillatzki, A.; Ramamoorthy, S. Harnessing the genetic plasticity of PCV2 to target suicidal replication. Viruses 2021. 13, 1676. https://doi.org/10.3390/v13091676
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Targeting suicidal replication to enhance the safety of attenuated viral vaccines- 2021. Sheela Ramamoorthy  Invited speaker assignment. North American PRRS symposium, Chicago, IL
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Targeting suicidal replication to enhance the safety of attenuated viral vaccines . AGM Rakibuzzaman, Pablo Pineyro, Angela Pillatzki, Sheela Ramamoorthy .2021. Poster for the NIFA-PD presentation in the North American PRRS symposium, Chicago, IL
  • Type: Journal Articles Status: Published Year Published: 2021 Citation: Rakibuzzaman, AGM.; Ramamoorthy, S. Comparative immunopathogenesis and biology of recently discovered porcine circoviruses. Transboundary and emerging diseases 2021, 10.1111/tbed.14244,
  • Type: Book Chapters Status: Awaiting Publication Year Published: 2021 Citation: Pineyro, P.; Ramamoorthy, S. Circoviridae. In Veterinary Microbiology, Fourth ed.; McVey, S., Kennedy, M., M.M. Chengappa, M.M., Wilkes, R., Eds. Wiley Blackwell: 2021; In Press.