Identification of a Papain-Like Protease Inhibitor with Potential for Repurposing in Combination with an Mpro Protease Inhibitor for Treatment of SARS-CoV-2

SARS-CoV-2 requires two cysteine proteases for viral polypeptide processing to allow maturation and replication: the 3C-like protease also known as the Main protease (Mpro) and the papain-like protease (PLpro). In addition to its critical role in viral replication, PLpro removes post-translational modifications like ubiquitin and interferon-stimulated gene product 15 (ISG15) from host proteins through its deubiquitinase domain, leading to host immunosuppression and increased ability of the virus to evade the host antiviral immune response. Through screening of a custom clinical compound library, we identified eltrombopag (DDL-701), a thrombopoietin receptor agonist, as having PLpro inhibitory activity that is sustained in the presence of the Mpro inhibitor nirmatrelvir. DDL-701 also suppressed both the deubiquitinase and ISG15 cleavage activities of PLpro. In addition, DDL-701 partially restored interferon-{beta} induction - an element of the host immune response - in an in vitro model system. Further, modeling and docking studies suggest DDL-701 interacts with the active site region of the PLpro enzyme and pilot pharmacokinetic studies indicate it is brain permeable. DDL-701 is already approved for treatment of thrombocytopenia and has previously been shown to achieve human plasma levels after oral dosing that is above the IC50 needed for it to exert its PLpro inhibitory activity in vivo. In addition, it has also been reported to have antiviral efficacy against SARS-CoV-2. DDL-701 thus represents a drug that can immediately be repurposed and undergo clinical evaluation as a PLpro inhibitor that may be most effectively used in a protease inhibitor cocktail with an Mpro inhibitor such as nirmatrelvir (Paxlovid) for the treatment of COVID-19.

Single Prime hAd5 Spike (S) + Nucleocapsid (N) Dual Antigen Vaccination of Healthy Volunteers Induces a Ten-Fold Increase in Mean S- and N- T-Cell Responses Equivalent to T-Cell Responses from Patients Previously Infected with SARS-CoV-2

In response to the need for a safe, efficacious vaccine that elicits vigorous T cell as well as humoral protection against SARS-CoV-2 infection, we have developed a dual-antigen COVID-19 vaccine comprising both the viral spike (S) protein modified to increase cell-surface expression (S-Fusion) and nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to enhance MHC class I and II presentation and T-cell responses. The antigens are delivered using a human adenovirus serotype 5 (hAd5) platform with E1, E2b, and E3 regions deleted that has been shown previously in cancer vaccine studies to be safe and effective in the presence of pre-existing hAd5 immunity. The findings reported here are focused on human T-cell responses due to the likelihood that such responses will sustain efficacy against emerging variants, a hypothesis supported by our in silico prediction of T-cell epitope HLA binding for both the first-wave SARS-CoV-2 A strain and the B.1.351 strain K417N, E484K, and N501Y spike and T201I N variants. We demonstrate the hAd5 S-Fusion + N-ETSD vaccine antigens expressed by previously SARS-CoV-2-infected patient dendritic cells elicit Th1 dominant activation of autologous patient T cells, indicating the vaccine antigens have the potential to elicit immune responses in previously infected patients. For participants in our open-label Phase 1b study of the vaccine (NCT04591717; https://clinicaltrials.gov/ct2/show/NCT04591717), the magnitude of Th-1 dominant S- and N-specific T-cell responses after a single prime subcutaneous injection were comparable to T-cell responses from previously infected patients. Furthermore, vaccinated participant T-cell responses to S were similar for A strain S and a series of spike variant peptides, including S variants in the B.1.1.7 and B.1.351 strains. The findings that this dual-antigen vaccine elicits SARS-CoV-2-relevant T-cell responses and that such cell-mediated protection is likely to be sustained against emerging variants supports the testing of this vaccine as a universal booster that would enhance and broaden existing immune protection conferred by currently approved S-based vaccines.

Complete Protection of Nasal and Lung Airways Against SARS-CoV-2 Challenge by Antibody Plus Th1 Dominant N- and S-Specific T-Cell Responses to Subcutaneous Prime and Thermally-Stable Oral Boost Bivalent hAd5 Vaccination in an NHP Study

We have developed a dual-antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike (S-Fusion) protein and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) with the potential to increase MHC class I/II responses. The adenovirus serotype 5 platform used, hAd5 [E1-, E2b-, E3-], previously demonstrated to be effective in the presence of Ad immunity, can be delivered in an oral formulation that overcomes cold-chain limitations. The hAd5 S-Fusion + N-ETSD vaccine was evaluated in rhesus macaques showing that a subcutaneous prime followed by oral boosts elicited both humoral and Th1 dominant T-cell responses to both S and N that protected the upper and lower respiratory tracts from high titer (1 x 106 TCID50) SARS-CoV-2 challenge. Notably, viral replication was inhibited within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge. ONE SENTENCE SUMMARYhAd5 spike + nucleocapsid SC prime/oral boost vaccine elicits humoral and T-cell responses and protects rhesus macaques from SARS-CoV-2 challenge.

Th1 Dominant Nucleocapsid and Spike Antigen-Specific CD4+ and CD8+ Memory T Cell Recall Induced by hAd5 S-Fusion + N-ETSD Infection of Autologous Dendritic Cells from Patients Previously Infected with SARS-CoV-2

To address the need for a safe, efficacious vaccine against SARS-CoV-2 infection with the critical properties of enabling both blocking viral entry into cells and clearing virus from cells already infected, we have developed a bivalent, human adenovirus serotype 5 (hAd5) SARS-CoV- 2 S-Fusion + N-ETSD vaccine that is currently in clinical testing. This vaccine uses the next- generation hAd5 [E1-, E2b-, E3-] platform previously used successfully in cancer patients with pre-existing adenovirus immunity, engineered to express both SARS-CoV-2 spike (S) protein modified to improve the generation of neutralizing antibodies to block entry of the virus, and nucleocapsid (N) protein with an Enhanced T cell Stimulation Domain (ETSD) to activate CD4+ and CD8+ T cells to clear the virus and block replication by killing infected cells. The targeting of N to endosomes and lysosomes to enhance CD4+ and CD8+ T-cell responses distinguishes our vaccine. In our previously reported pre-clinical studies we showed that in mice, the hAd5 S-Fusion + N-ETSD vaccine elicits both humoral and T-cell responses that are robust and T helper cell 1 (Th1) dominant. Here we report that the hAd5 S-Fusion + N-ETSD vaccine is recognized by anti-sera and T cells from previously SARS-CoV-2 infected patients, and that the presence of N is vital for T-cell recall. The findings presented herein: (i) demonstrate specific recognition of hAd5 S-Fusion + N-ETSD infected cells by plasma antibodies from previously SARS-CoV-2 infected patients, but not antibodies from virus-naive subjects; (ii) show enhanced binding of plasma SARS-CoV-2 antibodies from previously infected patients to monocyte-derived dendritic cells (MoDCs) expressing the hAd5 S-Fusion + N-ETSD vaccine as compared to hAd5 S-Fusion alone; (iii) reveal N-ETSD localizes to vesicles associated with MHC class II antigen presentation, including endosomes, lysosomes and autophagosomes in MoDCs; (iv) demonstrate endosome/lysosome-targeted N-ETSD elicits higher interferon-{gamma} T-cell responses than cytoplasm-localized N; and (v) N-ETSD alone or in the hAd5 S-Fusion + N-ETSD construct induces both CD4+ and CD8+ T cell memory recall. This recognition of hAd5 S-Fusion + N-ETSD vaccine antigens by T cells from previously SARS-CoV-2 infected patients, together with the ability of this vaccine candidate to elicit de novo immune responses in naive mice suggests that it re-capitulates the natural immune response to SARS-CoV-2 to activate both B and T cells towards viral neutralization and recognition of infected cells, critical for prevention of COVID-19 disease. Intriguingly, our hAd5 S-Fusion + N-ETSD T-cell biased vaccine has the potential to not only provide protection for uninfected individuals, but also to be utilized as a therapeutic for already infected patients to induce rapid clearance of the virus by activating T cells to kill the virus-infected cells, thereby reducing viral replication and lateral transmission.

A Next Generation Bivalent Human Ad5 COVID-19 Vaccine Delivering Both Spike and Nucleocapsid Antigens Elicits Th1 Dominant CD4+, CD8+ T-cell and Neutralizing Antibody Responses

In response to the health crisis presented by the COVID-19 pandemic, rapid development of safe and effective vaccines that elicit durable immune responses is imperative. Recent reports have raised the concern that antibodies in COVID-19 convalescent patients may not be long lasting and thus even these individuals may require vaccination. Vaccine candidates currently in clinical testing have focused on the SARS-CoV-2 wild type spike (S) protein (S-WT) as the major antigen of choice and while pre-clinical and early clinical testing have shown that S elicits an antibody response, we believe the optimal vaccine candidate should be capable of inducing robust, durable T-cell responses as well as humoral responses. We report here on a next generation bivalent human adenovirus serotype 5 (hAd5) vaccine capable of inducing immunity in patients with pre-existing adenovirus immunity, comprising both an S sequence optimized for cell surface expression (S-Fusion) and a conserved nucleocapsid (N) antigen designed to be transported to the endosomal subcellular compartment, with the potential to generate durable immune protection. Our studies suggest that this bivalent vaccine is optimized for immunogenicity as evidenced by the following findings: (i) The optimized S-Fusion displayed improved S receptor binding domain (RBD) cell surface expression compared to S-WT where little surface expression was detected; (ii) the expressed RBD from S-Fusion retained conformational integrity and recognition by ACE2-Fc; (iii) the viral N protein modified with an enhanced T-cell stimulation domain (ETSD) localized to endosomal/lysosomal subcellular compartments for MHC I/II presentation; and (iv) these optimizations to S and N (S-Fusion and N-ETSD) generated enhanced de novo antigen-specific B cell and CD4+ and CD8+ T-cell responses in antigen-naive pre-clinical models. Both the T-cell and antibody immune responses to S and N demonstrated a T-helper 1 (Th1) bias. The antibody responses were neutralizing as demonstrated by two independent SARS-CoV-2 neutralization assays. Based on these findings, we are advancing this next generation bivalent hAd5 S-Fusion + N-ETSD vaccine as our lead clinical candidate to test for its ability to provide robust, durable cell-mediated and humoral immunity against SARS-CoV-2 infection. Further studies are ongoing to explore utilizing this vaccine construct in oral, intranasal, and sublingual formulations to induce mucosal immunity in addition to cell-mediated and humoral immunity. The ultimate goal of an ideal COVID-19 vaccine is to generate long-term T and B cell memory.