Progress 08/01/16 to 01/31/17
Outputs
Target Audience:Currently, Codagenix is in communication with a large agribusiness company to potentially produce and market our SIV vaccine. Additionally, our Series-A investors at TopSpin Partners have expressed interest regardingthe results of our Phase I pre-clinical research and are interested in discussing continued investment in this project after completion of our Phase II project. Therefore, our main target audience for the Phase I research were our investors and potential partners in marketing a SIV vaccine. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
SAVE 'De-optimization' and de novo synthesis of vaccine strains based on currently circulating H1N2 and H3N2 strains of SIV and rescue by reverse genetics. Codagenix obtained viral genomic sequences of H1N1, H1N2, and H3N2 viruses isolated in the last year from the USDA and other databases. The specific strain identity for each subtype is confidential. We then applied our SAVE-deoptimization approach, i.e. making segments of the virus dense in under-represented mammalian codon-pairs, to the H1N1-, H1N2-, and H3N2-SIV genomes. The other 6 genomic segments of the viruses were maintained as wild-type. We then synthesized these deoptimized genes and molecularly cloned these synonymously re-coded genomic segments into DNA vectors. We then transfected these DNA vectors containing all eight genomic segments of the SIV-strains into susceptible cells and recovered replicating SIV-H1N1, -H1N2, and -H3N2 vaccine strain in our BSL-2 lab via reverse genetics (Table 1). Success with Human-CodaVax had demonstrated that targeting the two specific genomic segments is the optimal approach to derive live-attenuated vaccine candidates and we therefore succeeded in recovering similar live-attenuated SIV vaccine strains in the H1N1, H1N2, and H3N2 background. All vaccine strains grow to about 5 x 106 TCID50/ml in MDCK cells, a titer suitable for downstream manufacturing (Table 1, Column 3). Phase I, Aim 1 Deliverable Met: Efficiently replicating SAVE de-optimized Swine Influenza Virus (SIV) vaccine strain candidates based on H1N1, H1N2, and H3N2 SIV strains ready for use in safety and efficacy testing in swine Phase II In vivo characterization of SAVE- 'de-optimized' H1N1 vaccine candidate for safety and efficacy in pigs. In Phase I, Aim 2 we tested our previously deoptimized H1N1 HUMAN vaccine strain (CodaVax) in swine. CodaVax has previously been shown to be highly attenuated in vivo in mice and ferrets with similar replicative fitness to WT in vitro. Additionally, low doses (5 PFU in mice, 5 x 103 TCID50 in ferrets) have been observed to be protective against challenge with wild-type H1N1. In Phase I, Aim 2 we performed assays similar to our pilot studies in ferrets but in pigs. Our human Influenza vaccine candidate CodaVax, which is currently entering Phase I human clinical trials, was tested for attenuation, immunogenicity, and efficacy in swine. Specifically, groupings of 19-20 swine were vaccinated intranare, a single time, with either: CodaVax High (105 PFU), CodaVax Low (104), or Mock. At Day 7 post vaccination, 10 pigs were euthanized from the CodaVax high group and lung lesions were measured to assess safety (Figure 1C). A lung lesion score of <1% was observed, indicating a high margin of safety for CodaVax at dose of 105. At Day 28 post-vaccination, pigs were bled and then intra-nare challenged with 108 PFU of a 1988-H1N1-SIV (i.e. a non-homologous H1N1 challenge virus). First, we measured the immune response engendered by CodaVax using the Day 28 blood from vaccinated pigs. We found that CodaVax was highly immunogenic in vaccinated pigs as determined by significantly elevated levels of hemagglutination inhibiting (HAI) antibodies, with 19/20 pigs seroconverting (i.e. a HAI >= 40) to the CodaVax-homologous CA/09/2009 virus (Figure 1A, Column 2, green). Interestingly, when we also measured the serum from CodaVax vaccinated-swine against another human H1N1 virus we found that the serum was also cross-reactive against the new human seasonal H1N1 strain MI/45/15 as shown by similar levels of HAI antibodies (Figure 1A, Column 3, blue). The seroconversion of swine by CodaVax against another Influenza virus indicates CodaVax was able to produce a broad immune response - a trait we can anticipate in our SIV-specific vaccine strains. We did not test the HAI titer of serum from CodaVax vaccinated-swine against the challenge virus 1988-H1N1-SIV because we did not have it in our lab (it was only at the challenge site and it could not be shipped to us). Efficacy of CodaVax to protect against wild-type 1988-H1N1-SIV virus challenge was clinically determined via RT-PCR of searching for challenge virus in nare swabs of challenged pigs (Figure 1B). It was found that CodaVax was able to prevent nasal replication of the 1998-H1N1-SIV challenge virus in 19 out of 20 pigs in the CodaVax low group when using this highly sensitive RT-PCR compared to detection of challenge virus in 100% of mock vaccinated animals which were PCR positive by Day 31 (Figure 1B). We also assessed efficacy by looking counting Lung Lesion Scores of lung tissue for lesions associated with the wild-type 1988-H1N1-SIV challenge virus at 5 days post-challenge (Figure 1C). Clinical evaluation of lung tissue from vaccinated animals showed a 50.0-65.2% reduction in the lesion score as well as compared to the mock vaccinated group (Figure 1C). At 7 days post-vaccination with the low dose of CodaVax, the mean lung lesion score was 6.79% compared to 19.51% in mock vaccinated pigs (Figure 1C), indicating CodaVax was efficacious against challenge. Given that CodaVax is based on the human H1N1 Influenza CA/09/2009, we deemed it better to design a Swine specific SIV-H1N1 as opposed to including CodaVax as the H1N1 component in the future trivalent formulation. The success of Phase I Aim 2 demonstrates that a SAVE-deoptimized live, attenuated Influenza vaccine is safe and efficacious against no matching challenge viruses. Phase I Completed: Demonstration of efficacy in Swine and the in vitro construction of a Tri-valent SIV-specific vaccine ready for in vivo testing in Phase II.
Publications
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