Codagenix (United States)

Human parainfluenza virus type 3 (HPIV3) is a major pediatric respiratory pathogen lacking available vaccines or antiviral drugs. We generated live-attenuated HPIV3 vaccine candidates by codon-pair deoptimization (CPD). HPIV3 open reading frames (ORFs) encoding the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and polymerase (L) were modified singly or in combination to generate 12 viruses designated Min-N, Min-P, Min-M, Min-FHN, Min-L, Min-NP, Min-NPM, Min-NPL, Min-PM, Min-PFHN, Min-MFHN, and Min-PMFHN. CPD of N or L severely reduced growth in vitro and was not further evaluated. CPD of P or M was associated with increased and decreased interferon (IFN) response in vitro, respectively, but had little effect on virus replication. In Vero cells, CPD of F and HN delayed virus replication, but final titers were comparable to wild-type (wt) HPIV3. In human lung epithelial A549 cells, CPD F and HN induced a stronger IFN response, viral titers were reduced 100-fold, and the expression of F and HN proteins was significantly reduced without affecting N or P or the relative packaging of proteins into virions. Following intranasal infection in hamsters, replication in the nasal turbinates and lungs tended to be the most reduced for viruses bearing CPD F and HN, with maximum reductions of approximately 10-fold. Despite decreased in vivo replication (and lower expression of CPD F and HN in vitro), all viruses induced titers of serum HPIV3-neutralizing antibodies similar to wt and provided complete protection against HPIV3 challenge. In summary, CPD of HPIV3 yielded promising vaccine candidates suitable for further development.

Abstract Background CoviLiv is a novel intranasal live-attenuated COVID-19 vaccine candidate, derived from SARS-CoV-2/Wuhan that was synthetically engineered utilizing Codagenix’ codon pair deoptimization platform. CDX-CoV-001 was a randomized, double-blind, placebo-controlled dose-escalation study in healthy adults, that established its safety and tolerability (NCT04619628). Here, we characterize the immune response to CoviLiv in participants in the high-dose cohort of 5x106 pfu (n=6/group). Methods Humoral immune responses were characterized in sera (days 1, 29 and 57) with a spike-specific IgG ELISA and both microneutralization (MNT) and pseudovirus neutralization (PVN) assays. T cell functionality was analyzed in PBMCs (days 1 and 36) using interferon-γ (IFNγ) ELISpot and intracellular cytokine staining (ICS) after restimulation with peptide pools for SNMO or spike, as well as T cell receptor (TCR) sequencing combined with in silico mapping to TCRs with known antigen specificity. Results After 2 doses of CoviLiv, all participants exceeded a 2-fold increase in spike-specific IgG with a geometric mean fold rise of 19.5 (95% CI 3.4-113.8) on day 57. Neutralizing antibodies at this timepoint were induced 2.6-fold (CI 1.0-7.0) and 4.9-fold (CI 1.4-16.6) using MNT and PVN. On day 36 post-vaccination, IFNγ response by ELISpot after restimulation with the SNMO peptide pool increased 4.5-fold (CI 2.8-7.4) in the 2-dose cohort and 2.5-fold (CI 1.4-4.2) in the 1-dose cohort. No increase in IFNγ response was observed after placebo vaccination or in any group after restimulation with spike-only peptides. ICS confirmed significant regimen-dependent SNMO-specific increases in IFNγ, IL-2 and TNFα response especially in the CD4+ T cell subset, including induction of polyfunctional CD4+ T cells. TCR repertoire mapping revealed increases in both depth and breadth of CD4+ T cell clones specific for spike, nucleocapsid phosphoprotein and membrane glycoprotein. CD8+ T cell responses were less pronounced with CoviLiv-induced responses directed against nucleocapsid and ORF1ab. Conclusion Two doses of CoviLiv induced a poly-antigenic immune response with specificity to targets beyond spike, including highly conserved viral proteins, highlighting the value of this differentiated vaccine candidate. Disclosures Johanna K. Kaufmann, PhD, Codagenix Inc.: Salaried employee|Codagenix Inc.: Ownership Interest Keri Wyllie, MPA, Codagenix Inc.: Salaried employee|Codagenix Inc.: Ownership Interest Yiwen Zhao, PhD, Codagenix Inc.: Salaried employee Lasmy Tea, MS, MPH, Codagenix Inc.: Salaried employee|Codagenix Inc.: Ownership Interest Sybil Tasker, MD, MPH, FIDSA, Codagenix Inc.: Advisor/Consultant|Codagenix Inc.: Salaried employee|Codagenix Inc.: Ownership Interest|Inventprise: Salaried employee|Inventprise: Ownership Interest Leena R. Yeolekar, PhD, Serum Institute of India Pvt. Ltd.: Salaried employee Rajeev Dhere, PhD, Serum Institute of India Pvt. Ltd.: Salaried employee Steffen Mueller, PhD, Codagenix Inc.: Board Member|Codagenix Inc.: Patent owner|Codagenix Inc.: Salaried employee|Codagenix Inc.: Ownership Interest

BackgroundMultiple oncolytic viruses have been shown to induce beneficial changes in the tumor microenvironment (TME) by increasing immune cell infiltration and activating stimulatory immune responses, which ultimately support induction of anti-tumor immunity and efficacy. We have previously demonstrated that CodaLytic™, a codon-modified influenza virus, can activate the cancer immunity cycle at various steps in EMT6, a murine triple-negative breast cancer model with moderate responsiveness to immunotherapies, leading to 67% complete tumor regressions. Here, we are confirming the mechanisms of action of the virus alone and in combination with immune checkpoint inhibition in several murine models and human tumor explants with different baseline immune contexture.MethodsEfficacy after intratumoral injection of 108 PFU CodaLytic 3x/week for up to 12 doses as a monotherapy and/or in combination with systemic anti-PD-1 checkpoint blockade was determined in EMT6 breast cancer, CT26 colon cancer and B16F10 melanoma mouse models. Pharmacodynamic changes in the TME after treatment were characterized using flow cytometry. Anti-tumor immune memory was assessed by interferon-γ ELISpot in splenocytes of long-term survivors. Human breast cancer tumoroids maintaining the orignal patient TME were incubated ex vivo with 108 PFU CodaLytic, 10 μg/ml pembrolizumab or both. Tumor cell killing (TCK) was detected by high-content imaging at 72h and the TME was characterized by flow cytometry, cytokine release and RNA sequencing at 24h and 48h.ResultsCodaLytic monotherapy led to significant tumor growth inhibition (TGI) across tumor models, including 76% in EMT6 with moderate, immunologically active infiltration [1] and 69% TGI in poorly infiltrated B16F10 melanoma resistant to PD-1 blockade. In this model, efficacy further improved to 86% with addition of anti-PD-1. In all models, CodaLytic treatment increased infiltration of CD45+ leukocytes, CD8+ T cells and cross-presenting dendritic cells. Ex vivo recall responses to tumor cell lysate (EMT6) or AH1 peptide (CT26) were observed in long-term survivors, confirming generation of an anti-tumor T cell response. Efficacy of the CodaLytic/anti-PD-1 combination was confirmed in well-infiltrated human breast cancer tumoroids (51% TCK vs 24% with pembrolizumab alone, 6% with CodaLytic alone, and 2% in poorly-infiltrated tumoroids), a system in which priming and immune cell recruitment from a systemic reservoir are absent.ConclusionsCodaLytic treatment induced favorable changes in multiple murine tumor models independently of their intrinsic immune contexture and sensitized B16F10 melanomas to PD-1 blockade. Ongoing work investigates correlates of TCK in human tumoroid cultures beyond baseline immune infiltration to support future development of CodaLytic for immuno-virotherapy.AcknowledgementsWe would like to thank Soner Altiok, MD, PhD and the team and Nilogen Oncosystems for their expert support of this work.ReferenceYu JW, Bhattacharya S, Yanamandra N, et al. Tumor-immune profiling of murine syngeneic tumor models as a framework to guide mechanistic studies and predict therapy response in distinct tumor microenvironments. PLoS ONE. 2018;13(11):e0206223.Ethics ApprovalThe animal work in this study was approved after MisPro Biotech Services IACUC review, protocols 2019-01-17-COD-1 and 2022-COD-02.The human tissue component of this study was approved by Vanderbuilt University’s Ethics review board; approval no. 031078.

Foot-and-mouth disease (FMD) is one of the most economically important viral diseases that can affect livestock. In the last 70 years, use of an inactivated whole antigen vaccine has contributed to the eradication of disease from many developed nations. However, recent outbreaks in Europe and Eastern Asia demonstrated that infection can spread as wildfire causing economic and social devastation. Therefore, it is essential to develop new control strategies that could confer early protection and rapidly stop disease spread. Live attenuated vaccines (LAV) are one of the best choices to obtain a strong early and long-lasting protection against viral diseases. In proof of concept studies, we previously demonstrated that “synonymous codon deoptimization” could be applied to the P1 capsid coding region of the viral genome to derive attenuated FMDV serotype A12 strains. Here, we demonstrate that a similar approach can be extended to the highly conserved non-structural P2 and P3 coding regions, providing a backbone for multiple serotype FMDV LAV development. Engineered codon deoptimized P2, P3 or P2, and P3 combined regions were included into the A24Cruzeiro infectious clone optimized for vaccine production, resulting in viable progeny that exhibited different degrees of attenuation in cell culture, in mice, and in the natural host (swine). Derived strains were thoroughly characterized in vitro and in vivo. Our work demonstrates that overall, the entire FMDV genome tolerates codon deoptimization, highlighting the potential of using this technology to derive novel improved LAV candidates.

Currently, influenza vaccine manufacturers need to produce 1–5 x 107 PFU of each vaccine strain to fill one dose of the current live-attenuated-influenza-vaccine (LAIV). To make a single dose of inactivated vaccine (15 ug of each hemagglutinin), the equivalent of 1010 PFU of each vaccine strains need to be grown. This high dose requirement is a major drawback for manufacturing as well as rapidly sourcing sufficient doses during a pandemic. Using our computer-aided vaccine platform Synthetic Attenuated Virus Engineering (SAVE), we created a vaccine candidate against pandemic H1N1 A/CA/07/2009 (CodaVax-H1N1) with robust efficacy in mice and ferrets, and is protective at a much lower dose than the current LAIV. CodaVax-H1N1 is currently in Phase I/II clinical trials. The hemagglutinin (HA) and neuraminidase (NA) gene segments of A/California/07/2009 (H1N1) (CA07) were “de-optimized” and a LAIV was generated ex silico using DNA synthesis. In DBA/2 mice, vaccination at a very low dose (100 or approximately 1 PFU) with CodaVax-H1N1 prevented disease after lethal challenge with wild-type H1N1. In BALB/c mice, as little as 103 PFU was protective against lethal challenge with mouse-adapted H1N1. In ferrets, CodaVax-H1N1 was more potent compared to currently licensed LAIV and still effective at a low dose of 103 PFU at preventing replication of challenge virus.

The Flavivirus genus of the Flaviviridae family encompasses numerous enveloped plus-strand RNA viruses. Dengue virus (DENV), a flavivirus, is the leading cause of serious arthropod-borne disease globally. The genomes of DENV, like the genomes of yellow fever virus (YFV), West Nile fever virus (WNV), or Zika virus (ZIKV), control their translation by a 5'-terminal capping group. Three other genera of Flaviviridae are remarkable because their viruses use internal ribosomal entry sites (IRESs) to control translation, and they are not arthropod transmitted. In 2006, E. Harris' group published work suggesting that DENV RNA does not stringently need a cap for translation. They proposed that instead DENV translation is controlled by an interplay between 5' and 3' termini. Here we present evidence that the DENV or ZIKV 5' untranslated regions (5'-UTRs) alone have IRES competence. This conclusion is based, first, on the observation that uncapped monocistronic mRNAs 5' terminated with the DENV or ZIKV 5'-UTRs can efficiently direct translation of a reporter gene in BHK and C6/36 cells and second, that either 5'-UTR placed between two reporter genes can efficiently induce expression of the downstream gene in BHK cells but not in C6/36 cells. These experiments followed observations that uncapped DENV/ZIKV genomic transcripts, 5' terminated with pppAN… or GpppAN…, can initiate infections of mammalian (BHK) or mosquito (C6/36) cells. IRES competence of the 5'-UTRs of DENV/ZIKV raises many open questions regarding the biology and control, as well as the evolution, of insect-borne flaviviruses.IMPORTANCE Members of the genus Flavivirus of Flaviviridae are important human pathogens of great concern because they cause serious diseases, sometimes death, in human populations living in tropical, subtropical (dengue virus [DENV], Zika virus [ZIKV], and yellow fever virus), or moderate climates (West Nile virus). Flaviviruses are known to control their translation by a cap-dependent mechanism. We have observed, however, that the uncapped genomes of DENV or ZIKV can initiate infection of mammalian and insect cells. We provide evidence that the short 5' untranslated region (5'-UTR) of DENV or ZIKV genomes can fulfill the function of an internal ribosomal entry site (IRES). This strategy frees these organisms from the cap-dependent mechanism of gene expression at an as yet unknown stage of proliferation. The data raise new questions about the biology and evolution of flaviviruses, possibly leading to new controls of flavivirus disease.

The latest Zika virus (ZIKV) pandemic caused great international concern from explosively proliferating throughout the Americas. Currently, there is no vaccine to prevent Zika virus infection and available tests rely on antibodies or RNA. Unfortunately, antibody-based detection systems can result in false positive results and RNA-based detection systems are costly, time-consuming, and impractical for testing in remote regions. In this study, a potential point-of-care (POC) diagnostic system was developed using a chip-based potentiometric sensor to detect Zika virus using a 3D molecular imprinting technique. This chip-based potentiometric sensor system was able to detect 10-1 PFU mL-1 ZIKV in a buffered solution under 20 minutes without any sample manipulation. This sensor was tested against Dengue virus at clinical viral loads and showed no sign of cross-reactivity. When tested against human saliva samples containing clinical viral loads, this sensor was able to detect 10 PFU mL-1 ZIKV among the pool of bio-macromolecules. The high sensitivity and high selectivity demonstrated here proved that this lab-on-a-chip diagnostic has the potential to become a POC detection system for rapid and accurate screening of flaviviruses.

Recoding viral genomes by numerous synonymous but suboptimal substitutions provides live attenuated vaccine candidates. These vaccine candidates should have a low risk of deattenuation because of the many changes involved. However, their genetic stability under selective pressure is largely unknown. We evaluated phenotypic reversion of deoptimized human respiratory syncytial virus (RSV) vaccine candidates in the context of strong selective pressure. Codon pair deoptimized (CPD) versions of RSV were attenuated and temperature-sensitive. During serial passage at progressively increasing temperature, a CPD RSV containing 2,692 synonymous mutations in 9 of 11 ORFs did not lose temperature sensitivity, remained genetically stable, and was restricted at temperatures of 34 °C/35 °C and above. However, a CPD RSV containing 1,378 synonymous mutations solely in the polymerase L ORF quickly lost substantial attenuation. Comprehensive sequence analysis of virus populations identified many different potentially deattenuating mutations in the L ORF as well as, surprisingly, many appearing in other ORFs. Phenotypic analysis revealed that either of two competing mutations in the virus transcription antitermination factor M2-1, outside of the CPD area, substantially reversed defective transcription of the CPD L gene and substantially restored virus fitness in vitro and in case of one of these two mutations, also in vivo. Paradoxically, the introduction into Min L of one mutation each in the M2-1, N, P, and L proteins resulted in a virus with increased attenuation in vivo but increased immunogenicity. Thus, in addition to providing insights on the adaptability of genome-scale deoptimized RNA viruses, stability studies can yield improved synthetic RNA virus vaccine candidates.