Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523
Performing Department
Clinical Science
Non Technical Summary
Avian influenza virus (AIV) represents a significant threat to poultry health and production economics worldwide. Current vaccines for AIV have many shortcomings, leaving an unmet need for effective methods for controlling AIV outbreaks. An ideal alternate or complementary tool would be an antiviral technology generated in a scalable process, amenable to mass aerosolization, and efficacious against all AIV strains and subtypes. In fulfillment of this need, our team is developing a novel patent-pending antiviral (referred to as "SiVEC-AIV") for treatment and prevention of AIV in poultry. This technology combines RNA interference with a delivery platform that utilizes non-pathogenic bacteria to generate and deliver silencing RNAs to respiratory tissues, thereby inhibiting viral replication at the site of infection to prevent viral spread. Early proof-of-concept studies demonstrated safe and effective use against avian influenza in commercial chickens (applied intranasally as a prophylactic). Recently completed aerosolization trials indicated that this antiviral can be administered using an industry-standard fan sprayer to provide a significant reduction in viral shedding titers in commercial chickens (applied as sprayed aerosol as a prophylactic). However, we lack data demonstrating efficacy as a treatment for existing AIV infection in poultry. This is an important aspect, as the CSU-owned pending patent claims antiviral use as both a prophylactic and a treatment. Moreover, USDA and several global animal health companies have highlighted the value in showing our technology can be aerosolized to treat an existing AIV infection in poultry, as this could provide significant utility as part of an outbreak response strategy. This is a critical next-step in our continued research and development efforts. This work increases the likelihood of securing an earlier licensing agreement with a high potential to generate a stable revenue stream for CVMBS and position CSU as a global leader in avian flu outbreak response and preparedness.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
0%
Goals / Objectives
Avian influenza virus (AIV) represents a significant threat to poultry health and production economics worldwide. Current vaccines for AIV have many shortcomings which limit their use, leaving an unmet need for effective methods for controlling AIV outbreaks. An ideal alternate or complementary tool would be an antiviral technology generated in a scalable process, amenable to mass aerosolization, and efficacious against all AIV strains and subtypes. In fulfillment of this need, our team is developing a novel patent-pending antiviral (referred to as "SiVEC-AIV") for treatment and prevention of AIV in poultry. This technology combines RNA interference with a delivery platform that utilizes non-pathogenic bacteria to generate and deliver silencing RNAs to respiratory tissues, thereby inhibiting viral replication at the site of infection to prevent viral spread.Early proof-of-concept studies using the SiVEC-AIV antiviral demonstrated safe and effective use against avian influenza in commercial chickens (applied intranasally as a prophylactic). Recently completed aerosolization trials indicated that this antiviral can be administered using an industry-standard fan sprayer to provide a significant reduction in viral shedding titers in commercial chickens (applied as sprayed aerosol as a prophylactic). Showing efficacy as a prophylactic (to prevent AIV) and efficacy when applied as an aerosol (for mass administration) are both key development milestones. However, we lack data demonstrating efficacy as a treatment for existing AIV infection in poultry. This is an important aspect as the pending patent claims antiviral use as both a prophylactic and a treatment. Additionally, conversations with USDA and several global animal health companies (Zoetis, Merck, Boehringer Ingelheim) have highlighted the value in showing our technology can be aerosolized to treat an existing AIV infection in poultry as this could provide significant utility as part of an outbreak response strategy.We hypothesize that the SiVEC-AIV antiviral can be administered as an aerosol to treat an ongoing infection, and thereby reduce viral shedding titers by 1 log or more and significantly reduce the duration of viral shedding in treated compared to non-treated chickens. This hypothesis will be addressed with the following two specific aims:Aim 1: Demonstrate a reduction in viral shedding titers in commercial chickens when the SiVEC-AIV antiviral is administered after AIV inoculation.Commercial chickens will be treated with SiVEC-AIV antiviral at 24 and 72 hours post AIV inoculation. We will use an industry-standard fan sprayer to administer the antiviral and evaluate the ability to reduce viral shedding in treated chickens at each time (24 and 72 hours) post inoculation.Aim 2: Demonstrate a reduction in the duration of viral shedding in commercial chickens when the SiVEC-AIV antiviral is administered after AIV inoculation.In addition to monitoring viral shedding titers in Aim 1, we will determine if the antiviral treatment reduces the duration of infection by monitoring the proportion of chickens shedding virus at each sampling timepoint during the study.
Project Methods
Aim 1: Demonstrate a reduction in viral shedding titers in commercial chickens when the AIV-antiviral is administered after AIV inoculation.Animals: One-day-old layer chicks (n=48) will be sourced from a commercial hatchery. All chicks will receive standard vaccinations including Rispen and HVT/IBD, administered by the grower. Chicks will be transferred to the Painter Center animal research facility at Colorado State University and gang-housed in a temperature and humidity-controlled room. Chick grower feed and water will be provided ad libitum, and aspen shavings and scratch offered for enrichment. The birds will be randomly split into three identical rooms at three weeks, representing three treatment groups (n=16 each). At four weeks of age, birds will be individually identified with plastic color-coded and numbered leg bands and a baseline oropharyngeal (OP) swab will be collected for real-time PCR (described below).Infection: At four weeks of age, all chickens will be inoculated with 1x106 EID50/0.1 mL of A/chicken/Texas/473-2/2010 (H6N2) LPAIV stock, administered as 50 μL per nostril.Antiviral Administration: On the day of administration, the SiVEC-AIV antiviral will be prepared as previously described (9) to a concentration of 108 CFU/mL in a 110mL volume with the addition of a sterile green indicator dye. At 24 hours post H6N2 inoculation, one group of n=16 chickens will be treated with 110mL of SiVEC-AIV at 108 CFU/mL, delivered with a Desvac fan sprayer (Ceva; Saint-Barthélemy-d'Anjou, France). At 72 hours post H6N2 inoculation, one group of n=16 chickens will be treated with the same concentration and volume of SiVEC-AIV using the Desvac fan sprayer. The final group of n=16 chickens will remain untreated and will represent the positive control group for making viral shedding comparisons.Sample Collection: An OP swab will be collected from all chickens at 2, 3, 5, 7, 9 and 11 days post-inoculation (DPI). A sterile polyester-tipped swab will be inserted into the oral cavity and rubbed over the posterior opening of the pharynx and roof of the oropharynx, taking care to swab the choanal slit. The swab will be placed into a sterile tube with 1 mL of brain-heart-infusion (BHI) medium and vigorously agitated to release collected mucus and cells. The sample tubes will be stored chilled prior to and immediately following collection and then frozen at -80°C once returned to the lab. Following sample collection on 11 DPI, all chickens will be euthanized by CO2 inhalation.Viral Shedding Titers: All OP swab samples collected over the infection period will be processed by quantitative real-time PCR (RT-qPCR) to quantify the H6N2 viral titers. According to the manufacturer's recommendations, total RNA will be extracted using the RNeasy 96 QIAcube HT Kit (Qiagen) and processed using the QIAcube HT automated instrument (Qiagen). RT-qPCR will be performed with primers and probe specific for conserved sequences in avian influenza matrix (M) gene in accordance with the NAHLN (National Animal Health Laboratory Network) AIV Matrix RT-qPCR protocol and as previously described (13). The standard curve for virus quantification will be generated in triplicate using a series of 10-fold dilutions from 1x101 to 1x1010 of the H6N2 stock virus from which EID50 equivalent per mL (EID50 eq/mL) sample medium for each RNA sample will be calculated. The limit of detection has been determined to be 101 EID50/ml (1 log10 EID50/ml) per reaction.Sample Size and Statistical Analysis: Descriptive statistics, including median viral titers with 95% CI and fold-reductions in shedding titers will be calculated. Differences in the median viral titers between treated and untreated chickens will be analyzed using the Wilcoxon rank-sum test (p<0.05). All statistical analyses will be performed using the statistical computing software STATA 10 IC (StataCorp 2009, Stata Statistical Software: Release 10). A sample size of n=16 per group was calculated to achieve at least 90% power with a 95% level of significance. The most important quantitative measurement is viral titer (EID50/mL), a value that represents viral shedding. Therefore, the power calculation was based on expected EID50/mL measurements and a minimum viral titer reduction of 1-log EID50/mL.Aim 2: Demonstrate a reduction in the duration of viral shedding in commercial chickens when the AIV-antiviral is administered after AIV inoculation.The same treatment groups and methods outlined above in Aim 1 above will be utilized to determine if the antiviral treatment reduces the duration of viral shedding in commercial chickens.Statistical Analysis: Differences in the proportion of chickens shedding virus at each DPI will be analyzed using Fisher's exact test (p<0.05).