Recipient Organization
CODAGENIX INC.
7 GAY DR
GREAT NECK,NY 11024
Performing Department
Vaccine Development
Non Technical Summary
This Phase II SBIR seeks to further commercialize a live-attenuated Foot-and-Mouth-Disease Virus (FMDV) vaccine candidate that we succesffully constructed in Phase I via a collaboration with the USDA-Plum Island Animal Disease Center (PIADC; CRADA 58-3K95-4-1688-M). In proof of concept studies we demonstrated that Codagenix's vaccine platform was capable of yielding a highly attenuated and immunogenic FMDV vaccine candidate using a laboratory strain. Now, meeting all of our Phase I deliverables, we have successfully applied our platform to serotype-A24, yielding a field and commerically relevant FMDV candidate vaccine. In Phase II we will focus on the swine testing of our A-24 vaccine strain, as well as use our A24 backbone to construct live vaccine candidates for serotypes O and Asia-1, two prevelant serotypes. With A-24, O, and Asia-1 combined we will havea commerically viable vaccine product that covers >74% of all circulating FMDV. Our technology termed Synthetic Attenuated Virus Engineering (SAVE), "de-optimzies" viral genomes thereby reducing viral translation efficency via hundred of stable silent mutations. This SAVE-deoptimization retains 100% identity to the wildtype protein sequence - yielding a vaccine strain that is highly attenuated and a PERFECT antigenic match to your target. By being disadvantaged for translation within host cells, SAVE-deoptimized vaccine strains are avirulent while preserving highly immunogenic properties since they are antigenically identical to the parental wild type strain. FMDV is endemic in many countries around the globe and has proven to be devastating to livestock, with small outbreaks of FMDV causing nearly $1.5 billion in economic losses. A fast-acting and effective FDMV vaccine is needed to help halt the spread of an outbreak and protect cloven-hoofed livestock such as cattle, sheep, and swine. This is best accomplished with a live attenuated vaccine, such as the one proposed here. To differentiate between between vaccinated from naturally infected animals, our deoptimized A24 vaccine strain contains proprietary DIVA markers developed by our collaborators at PIADC. Since FMDV is a Biosafety-level-3+ pathogen and a select agent, all virus work will be conducted in direct collaboration with the PIADC at their Plum Island facilities.
Animal Health Component
50%
Research Effort Categories
Basic
(N/A)
Applied
50%
Developmental
50%
Goals / Objectives
This Phase II SBIR seeks to further commercialize a live-attenuated Foot-and-Mouth-Disease Virus (FMDV) vaccine candidate that we successfully constructed in Phase I via a collaboration with the USDA-Plum Island Animal Disease Center (PIADC; CRADA 58-3K95-4-1688-M). In proof of concept studies we demonstrated that Codagenix's vaccine platform was capable of yielding a highly attenuated and immunogenic FMDV vaccine candidate using a laboratory strain. Now, meeting all of our Phase I deliverables, we have successfully applied our platform to serotype-A24, yielding a field and commerically relevant FMDV candidate vaccine. In Phase II our goals are tofocus on the swine testing of our A-24 vaccine strain by testing the attenuation, immunogenicity and efficacy of our vaccine candidate in swine. Additionally, we willuse our A24 backbone to construct live vaccine candidates for serotypes O and Asia-1, two prevalent serotypes that occur globally. With A-24, O, and Asia-1 combined we will yield a commercial product that covers >74% of all circulating FMDV, thus producing a trivalent vaccine in Phase II that is commercially viable.
Project Methods
Swine efficacy testing ofA24-Deopt (USDA-PIDAC). Swine attenuation experiments. 20 Yorkshire gilts (five weeks old and weighing approximately 18-23 kg each) will be divided into 5 groups of 4 animals each housed in individual rooms. Three animals of each of 4 groups will be IDHB inoculated in the right hind foot with doses of either 103, 104, 105 or 106 pfu/animal of A24-Deopt mutant virus. One additional naïve animal will be cohoused in each room for contact transmission evaluation. An additional group of 4 animals will be inoculated with 105 of parental WT A24 FMDV as control for evaluation of full virulence. Following each FMDV inoculation, clinical scores will be evaluated by determining the number of toes presenting FMDV lesions and the presence of lesions in the snout and/or mouth. Blood, serum, and nasal swab samples will be collected daily. The % of lymphocytes in the white cell population from whole blood collected in EDTA will be measured using a Hemavet cell counter (Drew Scientific-Erba Diagnostics, FL). Virus will be measured in nasal secretions and blood using plaque titration in BHK cells andrRT-PCR. Sufficient attenuation will be characterized by a lack of lesions, no decrease in lymphocytes in the white cell population from whole blood collected, and reduced or absence of virus in blood and nasal secretions. At 21 dpi, animals that did not get sick will be challenged with A24-WT virus by IDHB inoculation in the left hind foot to assess possible efficacy and potency.Swine Efficacy. In a second experiment, 12 swine will be divided into 3 groups of 4 animals each. Two groups will be SC inoculated with A24 deopt at doses at least 100x lower than the dose causing disease in the experiments described in task 1.1 i. e. 102 and 103 or One group will be maintained as control for challenge with WT virus. Clinical and serological parameters will be evaluated as in task 1.1 also including measurement of humoral immune response at different times post inoculation. At 21 days post inoculation animals that did not develop disease will be challenged with 105 pfu of FMDV A24 WT. Challenged animals will be clinically assess for presentation of FM disease symptoms, number of toes presenting FMDV lesions, and the presence of lesions in the snout and/or mouth. Additionally, blood, serum, and nasal swab samples will be collected to monitor replication of challenge virus using plaque titration and rRT-PCR or for determining serum neutralizing antibodies titers.De novo synthesis of 'de-optimized' FMDV Serotypes O1 and Asia-1 (Codagenix, Inc.), insertion into the marker A24-Deopt backbone by genetic engineering, and rescue of viable virus progeny (USDA-PIDAC).O1 and Asia-1 capsid synthesis. Using relevant field sequence data, the capsid sequence from serotypes O1 and Asia-1 will besynthesized with flanking restriction enzyme sites to facilitate cloning into the A24-Deopt cDNA backbone.Cloning of the O1 and Asia-1 serotypes capids into the A24 backbone. These wild type capsids from the O1 and Asia-1 serotype viruses will be molecularly cloned into the A24-Deopt full-length-cDNA backbone constructed during Phase I. This SAVE deoptimized sequence encodes the same amino acids as the wild type sequence; however, it now uses less favorable codons and/or under-represented codon-pairs. These O1 and Asia-1 capsid fragments will be used to substitute the one corresponding to A24 via conveniently engineered unique restriction sites flanking the capsid. Recovery of replicating O1-A24-Deopt and Asia1-A2-4Deotp vaccine strains via reverse genetics. Linearized cDNAs will each be used as templates for in vitro RNA synthesis with T7 polymerase using the MegaScript kit (Life Technologies). In a BSL-3 laboratory at PIADC, the in vitro transcribed full-length RNAs for O1-A24-Deopt and Asia1-A24-Deopt will then be electroporated into BHK-21 cells and replicating virus will be derived after repeated passage(Rieder et al 2005). Recovered viral RNA for each strain will be sequenced for verification. Next, we will concentrate replicating vaccine candidates by polyethylene glycol precipitation and purify each candidate by density gradient centrifugation in a 10-50% sucrose gradient (Bachrach, 1964). The concentrate and purified stock will be plaque titrated in BHK21 monolayers. These novel chimeric vaccine strains O1-A24-Deopt and Asia1-A24-Deopt will contain the DIVA marker (Differentiating Infected from Vaccinated Animals that was previously developed at PIADC) as part of the A24 backbone developed in Phase I.In vitro characterization of SAVE- 'de-optimized' FMDV strains O1-A24-Deopt and Asia1-A24-Deopt candidates. The deoptimized FMDV strains O1-A24-Deopt and Asia1-A24-Deopt will be examined in culture by single-step growth curve and plaque assay to assess replication and spread of resultant virus. Cultured cell monolayers will be infected with O1-A24-Deopt and Asia1-A24-Deopt viruses at a multiplicity of infection of 10. After 1h adsorption at 37ºC, unabsorbed virus will be removed by washing the cells with a solution containing 150mM NaCl in 20mM morpholineethanesulfonic acid (MES) pH=6.0, before adding MEM and proceeding with incubation at 37ºC in 5% CO2. Cell culture supernatants and scraped cells will be collected after 1, 3, 6 and 24 hours post infection,frozen at -80C, and titrated on BHK-21 cells.From the 6 and 24 hour supernatant and cell samples, viral proteins and RNA will be extracted using standard techniques. Western blot analysis will be performed to evaluate kinetics of viral protein synthesisIn vivo testing of attenuation and immunogenicity in swine of multivalent 'de-optimized' FMDV vaccine candidate.Evaluation of attenuation and immunogenicity in swine. 20 Yorkshire gilts (five weeks old and weighing approximately 18-23 kg each) will be divided into 5 groups of 4 animals each. In 4 groups, 3 animals will be IDHB inoculated with the different vaccine candidates and one animal will be maintained naïve for evaluation of contact transmission. The different groups will treated under the following schedule: 1) 3 pigs will be inoculated with 102 or 103pfu O1-A24Deopt based on the results of Tasks 1.1 and 1.2 2) 3 pigs will be inoculated with 105pfu O1-A24Deopt based on the results of Task 1.1 and 1.2 3) 3 pigs will be inoculated with 102 or 103pfu Asia1-A24Deopt 4) 3 pigs will be inoculated with 105pfu Asia1-A24Deopt and one animal will be maintained naïve; 5) 4 pigs will be inoculated with a trivalent combination of 102 o 103 pfu each A24Deopt, O1-A24Deopt and Asia1-A24Deopt. Clinical scores will be evaluated by determining the number of toes presenting FMDV lesions and the presence of lesions in the snout and/or mouth. Blood, serum, and nasal swab samples will be collected daily for 7 dpi. The % of lymphocytes in the white cell population from whole blood collected in EDTA will be measured using a Hemavet cell counter (Drew Scientific-Erba Diagnostics, FL). Virus will be measured in nasal secretions and blood using plaque titration in BHK cells and rRT-PCR. Neutralizing antibody titers will be determined in swine serum samples by end-point titration at 0, 5, 7, 14, and 21 dpi to test for immunogenicity to all of the serotypes. Predictive protection of the trivalent formulation will be characterized in group 5) by the detection of high titer of neutralizing antibodies against all three serotypes (A24, O1 and Asia1).Data analyses. Data handling, analysis and graphic representations will be performed using Prism 5.0 (GraphPad Software, San Diego, CA) or Microsoft Excel (Microsoft, Redmond, WA).