Source: CORNELL UNIVERSITY submitted to NRP
PARTNERSHIP: AGRICULTURAL BIOSECURITY PARTNERSHIP: RAPID RESPONSE TOOLS FOR HIGHLY PATHOGENIC AVIAN INFLUENZA DISEASE PREPAREDNESS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032588
Grant No.
2024-67015-42753
Cumulative Award Amt.
$800,000.00
Proposal No.
2023-08028
Multistate No.
(N/A)
Project Start Date
Aug 1, 2024
Project End Date
Jul 31, 2028
Grant Year
2024
Program Code
[A1181]- Tactical Sciences for Agricultural Biosecurity
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
(N/A)
Non Technical Summary
Avian influenza (AI) is among the most economically important diseases affecting poultry and poses a significant threat to the sustainability of the poultry industry worldwide. Over the past two decades, outbreaks of AI, especially involving highly pathogenic avian influenza (HPAI), have caused severe economic losses in many countries, including the United States (U.S.). Notably, the spread of HPAI in the U.S. in 2014-2015 affected 50 million domestic birds and resulted in an estimated loss of $3.3 billion. Currently, the U.S. has been facing another devastating outbreak of HPAI that began in 2022 and as of August 3, 2023 has affected 58.7 million birds in 47 states, surpassing the previous record of 2014-2015 outbreakand standing out as the most significant animal disease event in the history of the U.S.Vaccination is the most cost-effective and humane method for the control and prevention of viral diseases in livestock and poultry. However, viruses like avian influenza virus (AIV), which are constantly mutating, require vaccine platform(s) that can be rapidly developed and deployed in response to emerging strains. Thus, flexible vaccine platforms that enable rapid updates on AIV antigens to match circulating viruses in endemic areas or newly emerging viruses in non-endemic countries are needed. We will tackle this problems by developing novel vaccine delivery platforms for HPAI based on a new condorpox virus vector and on a self-amplifying RNA technology platform. Additionally, we will also develop serological assays that will enable differentiation of vaccinated from infected animals. The outcomes of the proposed project will have a significant impact on animal health and on the sustainability of the poultry industry worldwide.
Animal Health Component
70%
Research Effort Categories
Basic
10%
Applied
70%
Developmental
20%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3114099110180%
3114099109020%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4099 - Microorganisms, general/other;

Field Of Science
1090 - Immunology; 1101 - Virology;
Goals / Objectives
Our long-term goal is to develop rapid response vaccine delivery platforms and companion DIVA diagnostics for effective control of HPAI in endemic and non-endemiccountries. The specific aims of our project are:Aim 1: To develop novel rapid response vaccine delivery platforms for effective protection against HPAI in poultry. We aim to develop two innovative plug-and-play vaccine platforms, combining the versatility of CRISPR/Cas9 with a novel CDPV vector or the power of RNA technology with a novel self-amplifying Senecavirus A-RNA platform. These platforms will be engineered to incorporate the haemagglutinin gene (HA) of contemporary H5N1 viruses, and their capacity to express HA will be evaluated in vitro (Aim 1a and 1b) while their immunogenicity will be assessed in chickens in vivo (Aim 1c). The protective efficacy of the CRISPox- and SASVex platforms will be compared through immunization/challenge experiments (Aim 1d). The vaccine candidate demonstrating the highest level of protection in these experimental challenge trials will be further evaluated in a field trial conducted in poultry farms with high prevalence of avian influenza outbreaks (Aim 1e); andAim 2: To develop and validate a companion DIVA diagnostic for HPAI. To complement the vaccine platforms developed in Aim 1, we will develop DIVA compatible serological assays (indirect and blocking ELISAs) that will enable differentiation of antibody responses elicited by the vaccines or by natural infection with AIV.
Project Methods
The overall goal of the proposed projectis to developrapid response vaccine delivery platforms and companion DIVA diagnostic assays to improve our ability to combat HPAI.The aims of the proposed project and approaches used to achieve these aims are outlines below:Aim 1: To develop novel rapid response vaccine delivery platforms for effective prevention of HPAI in poultry.In this aim, we propose to develop two novelvaccine delivery platforms consisting of a novela novel poxvirus vector (CDPV) combined with CRISPR/Cas9 editing technology; anda self-amplifying viral RNA platform.Aim 2: To develop and validate a companion DIVA diagnostic for HPAI.In this objective we will focus on the development and validation of reliable HPAI H5-specific diagnostic assays that are DIVA compatible and complementary to the vaccine platforms developed in Aim 1. Our efforts will focus on the development and validation of ELISA-based serological assays.

Progress 08/01/24 to 07/31/25

Outputs
Target Audience:During the reporting period results of the project were presened at the 2025 USDA NIFA Ag Biossecurity PD meeting. Additionally, we have submitted an abstract to the Conference for Research Workers in Animal Diseases describing the development of a DIVA multiplex luminex assay for differentiating infected from vaccinated birds. This abstract will be presented in the 2026 CRWAD meeting in January of 2026. Therefore, the results of the project reached the scientific community, veterinary community, biological industry and government agencies during the reporting period. Changes/Problems:As explained in the accomplishment section we encounter an unexpected challenge with the SVA-based saRNA platform as the virus was not able to infect avian cells. This led us to change the platform to SINV. We have now confirmed that SINV can indeed infect avian cells and efficiently delivers heterologous genes in these cells. Due to the introduction of a new and better technology in our lab (Luminex) we decided to switch the platform of the DIVA assay in Aim 2 from an ELISA to a Luminex assay. This resulted in increased sensitivity and testing efficacy, as the luminex assay anables multiplexing and testing of the sample against multiple antigens in a single well. While the ELISA assay requires multiple wells, in which each antigen is tested individually. Both changes - resulted from troubleshooting - and provide better alternatives to the platforms originally proposed in our project. What opportunities for training and professional development has the project provided?During the reporting period the proposed project enable training of 1 PhD student which was engaged in HPAI protein expression and purification and 3 postdoctoral students which were involved in protein expression purification, Luminex DIVA assay development and optimization and recombinant vaccine development and purification. How have the results been disseminated to communities of interest?We have submitted an abstract to be presented as a poster at the CRWAD 2026 meeting. Additionally, we presented the progress of our project in the Ag Biossecurity PD meeting organized by NIFA. What do you plan to do during the next reporting period to accomplish the goals?We plan to complete the selection of the recombinant CDPV-HA vaccine candidates as well as the development of the CRISPR cell lines for rapid selection of the virus. We will also perform immunizatin studies with this vector in avian species. Additionally, we will complete the development of the saSINV-HA platform and include it in the immunization studies. Once immunization studies have been conducted we will complete the validation of the DIVA Luminex assay in avian species.

Impacts
What was accomplished under these goals? During the reporting period we made progress towards achieving both aims of our project. Below we provide details of the accomplishments of our team: Aim 1:To develop novel rapid response vaccine delivery platforms for effective protection against HPAI in poultry.We are making major progress on the development of of the poxvirus and self amplifying RNA vaccine plaforms for HPAI. We have designed and selected a hemmagglutinin sequenced based on the currently circulating HPAI H5N1 viruses of the clade 2.3.4.b. This construct was synthesized and is now being introduced into the genome of the poxvirus condorpoxvirus vector (CDPV). We are working on establishing cell lines expressing the Cas9 and guide RNAs targeting the TK gene of CDPV, which will then be used for high efficient selection of the CDPV-HA recombinant virus. Selection of the virus using traditional plaque assay purification is ongoing and the recombinant CPDV-HA virus is close to be purified. This vector will be soon used for immunization studies in chickens and quail. We will establish quail as a model for immunization and efficacy trials for HPAI. the use of this species will allow rapid screen of multiple vaccine constructs prior to conducting more costly trials in chickens. We have also attempted to generate the self-amplifying replicon system based on senecavirus A (SVA), however, upon testing the delivery and infectivity of SVA in cells of chicken origin we learned that SVA does not efficiently infect avian cells, which precludes its use in this species. To circumvent this unexpected proble, we adopted another RNA replicon system based on the alphavirus sindbis virus (SINV). We have confirmed that SINV replicon can infet avian cells and efficiently deliver genes of interest in avian cells. Additionally, we have developed stable cell lines expressing the SINV capsid proteins, which will allos us to recover the saSINV replicon from these cells that can then be used directly for immunization of avian species against HPAI. Our team is now working to insert the HPAI HA insert into the SINV genome. This is expected to be completed soon and the saSINV-HA will be tested side by side with the CDPV-HA vector in avian species soon. Aim 2: To develop and validate a companion DIVA diagnostic for HPAI.To complement the vaccine platforms developed in Aim 1, we have develop DIVA compatible serological assays based on a multiplex Luminex assay. From the time we submitted the proposal to NIFA until the present moment, we have acquired a Luminex instrument in our lab and have established procedures for serological assay development using this technology. The main advantages of the Luminex platform is that it is highly sensitive and it can test for the presence of antibodies against multiple antigens. In our case, we have developed a multiplex Luminex assay that can detect antibodies against HA, NA, NP and a second assay that also includes M1. We have tested this assay in serum samples from birds known to be infected with influenza viruses and the assay worked really well. While we do not have samples from vaccinated birds, we tested the assay with samples from vaccinated cattle and cats, and the assay was able to differentiate antibodies elicited by infection from those elicited by immunization. Additionally, we have also developed monoclonal antibody reagents against HPAI H5 HA and NA proteins. These antibodies can be used to develop ELISA-based DIVA assays, in the unlikely case that the luminex assay will not work properly.

Publications