ABSTRACT
Background: SARS CoV 2 infection alters the immunological profiles of natural killer (NK) cells. However, whether NK anti-viral functions (direct cytotoxicity and/or antibody-dependent cell cytotoxicity (ADCC)) are impaired during severe COVID-19 and what host factors modulate these functions remain unclear. Method(s): Using functional assays, we examined the ability of NK cells from SARS-CoV-2 negative controls (n=12), mild COVID-19 patients (n=26), and hospitalized COVID-19 patients (n=41) to elicit direct cytotoxicity and ADCC [NK degranulation by flow] against cells expressing SARS-CoV-2 antigens. SARS-CoV- 2 N antigen plasma load was measured using an ultra-sensitive Simoa assay. We also phenotypically characterized the baseline expression of NK activating (CD16 and NKG2C), maturation (CD57), and inhibitory (NKG2A and the glyco-immune negative checkpoint Siglec-9) by flow cytometry. Finally, an anti-Siglec-9 blocking antibody was used to examine the impact of Siglec-9 expression on anti-SARS-CoV-2-specific ADCC [degranulation and target cell lysis]. Result(s): NK cells from hospitalized COVID-19 patients degranulate less against SARS-CoV-2-antigen-expressing cells (in direct cytolytic and ADCC assays) than did cells from mild COVID-19 patients or negative controls (Fig. 1A). The lower NK degranulation was associated with higher plasma levels of SARS-CoV-2 N-antigen (P<=0.02). Phenotypic and functional analyses showed that NK cells expressing Siglec-9 elicited higher ADCC than Siglec-9- NK cells (P<0.05;Fig. 1B). Consistently, Siglec-9+ NK cells expressed an activated and mature phenotype with higher expression of CD16, CD57, and NKG2C, and lower expression of NKG2A, than Siglec-9- NK cells (P<=0.03). These data are consistent with the concept that the NK cell subpopulation expressing Siglec-9 is highly activated and cytotoxic. However, the Siglec-9 molecule itself is an inhibitory receptor that restrains NK cytotoxicity during cancer and other infections. Indeed, blocking Siglec-9 significantly enhanced the ADCC-mediated NK degranulation and lysis of SARS-CoV-2-antigen-positive target cells (P<=0.05;Fig. 1C). Conclusion(s): These data support a model (Fig. 1D) in which the Siglec-9+ CD56dim NK subpopulation is cytotoxic even while being restrained by the inhibitory effects of Siglec-9. However, alleviating the Siglec-9-mediated restriction on NK cytotoxicity can further improve NK immune surveillance and presents an opportunity to develop novel immunotherapeutic tools against SARS-CoV-2 infected cells. (Figure Presented).
ABSTRACT
Background: Novel coronaviruses (CoV) caused 3 global outbreaks over the past 2 decades: SARS-CoV (2002), MERS-CoV (2012), and 2019-nCoV in Wuhan, China. Each caused pneumonia with mortality of 10%, 35% and 2%, respectively (2019-nCoV estimated). GLS-5300 DNA vaccine targeting MERS-CoV Spike (S) was first to enter clinical trial, was safe and immunogenic (Lancet ID;2019). In Phase I, a 3 dose series at Day0, 4 and 12 weeks of GLS-5300 at either 0.67, 2 or 6mg was given IM followed by electroporation (EP, IM+EP) with CELLECTRA-5P device. GLS-5300 induced antibodies (Abs) in 94%, Tcell response in 76%, and neutralizing Abs in 50% of participants. No dose response was observed. GLS-5300 response was similar to those recovered from natural MERS-CoV infection. The absence of dose response and prior experience showing benefits of ID+EP vs IM+EP (JID;2019) led us to design this trial of lower ID dosing with an arm for a 2-dose regimen. We report results from MERS-002, the ongoing Phase I/IIa study of GLS-5300. Methods: MERS-002 is an open label, dose ranging, phase I/IIa study of GLS-5300. Participants were enrolled at 2 Korean sites into 3 groups receiving GLS-5300 ID+EP with the CELLECTRA-3P device: Group 1 received three 0.3mg doses at Day0 and weeks 4 and 12;Group 2 received three 0.6mg doses at Day0 and weeks 4 and 12;Group 3 received two 0.6mg doses at Day0 and week 8. Safety and tolerability of GLS-5300 was evaluated at each visit. Samples were collected at baseline, before each dose, and at both 2 and 4 weeks post dose 2 and post dose 3. Study data through 4 weeks after the primary series for a subset of immunoassays were included here. Findings: GLS-5300 given ID+EP was well-tolerated with no vaccine-associated SAEs. Preliminary results were available for: full length S (flS) ELISA, EMC2012-Vero neutralization (MERS-neut) and MERS-CoV S IFNg ELISPOT. GLS-5300 at 0.6mg induced MERS-CoV-specific Abs by flS ELISA and MERS-neut in 74% and 48%, respectively, after 1 dose. After the 2 or 3 dose vaccine series at 0.6mg per dose, flS ELISA response was seen in 100% and 92% of participants, respectively. MERS-neut response was 92% in both 2 and 3 dose 0.6mg groups. Antibody responses and rates were higher during and after primary series in 0.6mg group regardless of regimen than 0.3mg per dose. GLS-5300 induced Tcell responses via MERS-CoV IFNg ELISPOT in 60% and 84% receiving 0.6mg after the 2 or 3 dose series, respectively. Compared to 0.67mg of GLS-5300 given IM+EP in the first trial, 0.6mg of GLS-5300 given ID+EP in MERS-002, binding Abs appeared sooner and neutralizing Abs were observed in a higher fraction of participants (92% vs 50%) while Tcell reactivity was similar between vaccination schema. Conclusions: GLS-5300 was well tolerated with no vaccine-associated SAEs. Like prior studies, DNA vaccines given by ID+EP had fewer injection-related AEs relative to IM+EP. In MERS-002, 0.6mg of GLS-5300 in a 2-dose regimen spanning 8 weeks had similar reactivity and rate to the longer 3-dose regimen. GLS-5300 was safe and immunogenic when given IM+EP and, similarly, when given ID+EP in both 2- and 3-dose regimens in this ongoing MERS-002 Phase I/IIa trial. A Phase II clinical evaluation of the use of GLS-5300 to prevent MERS-CoV infection in endemic regions is planned.