Safety and immunogenicity of a recombinant DNA COVID-19 vaccine containing the coding regions of the spike and nucleocapsid proteins: Preliminary results from an open-label, phase 1 trial in healthy adults aged 19-55 years

BackgroundWe investigated the safety and immunogenicity of two recombinant COVID-19 DNA vaccine candidates in first-in-human trials. GX-19 contains plasmid DNA encoding SARS-CoV-2 spike protein, and GX-19N contains plasmid DNA encoding SARS-CoV-2 receptor binding domain (RBD) foldon and nucleocapsid protein (NP) as well as plasmid DNA encoding SARS-CoV-2 spike protein. MethodsTwo open-label phase 1 trials of GX-19 and GX-19N safety and immunogenicity were performed in healthy adults aged 19-55 years. GX-19 trial participants received two vaccine injections (1{middle dot}5 mg or 3{middle dot}0 mg, 1:1 ratio) four weeks apart. GX-19N trial participants received two 3{middle dot}0 mg vaccine injections four weeks apart. FindingsBetween June 17 and July 30 and December 28 and 31, 2020, 40 and 21 participants were enrolled in the GX-19 and GX-19N trials, respectively. Thirty-two participants (52{middle dot}5%) reported 80 treatment-emergent adverse events (AE) after vaccination. All solicited AEs were mild except one case of moderate fatigue reported in the 1{middle dot}5 mg GX-19 group. Binding antibody responses increased after vaccination in all groups. The geometric mean titers (GMTs) of spike-binding antibodies on day 57 were 85{middle dot}74, 144{middle dot}20, and 201{middle dot}59 in the 1{middle dot}5 mg, 3{middle dot}0 mg GX-19 groups and the 3{middle dot}0 mg GX-19N group, respectively. In GX-19N group, neutralizing antibody response (50% neutralizing titer using FRNT) significantly increased after vaccination, but GMT of neutralizing antibody on day 57 (37.26) was lower than those from human convalescent serum (288.78). GX-19N induced stronger T cell responses than GX-19. The magnitude of GX-19N-induced T cell responses was comparable to those observed in the convalescent PBMCs. GX-19N induced both SARS-CoV-2 spike- and NP-specific T cell responses, and the amino acid sequences of 15-mer peptides containing NP-specific T cell epitopes identified in GX-19N-vaccinated participants were identical with those of diverse SARS-CoV-2 variants InterpretationGX-19N is safe, tolerated and induces humoral and broad SARS-CoV-2-specific T cell response which may enable cross-reactivity to emerging SARS-CoV-2 variants. FundingThis research was supported by Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (HQ20C0016, Republic of Korea). Research in contextO_ST_ABSEvidence before this studyC_ST_ABSTo overcome the COVID-19 outbreak, the development of safe and effective vaccines is crucial. Despite the successful clinical efficacy of the approved vaccines, concerns exist regarding emerging new SARS-CoV-2 variants that have mutated receptor binding domains in the spike protein. We searched PubMed for research articles published up to May 1, 2021, using various combinations of the terms "COVID-19" or "SARS-CoV-2", "vaccine", and "clinical trial". No language or data restrictions were applied. We also searched the ClinicalTrials.gov registry and World Health Organization (WHO) draft landscape of COVID-19 candidate vaccines for ongoing trials of COVID-19 vaccines up to May 1, 2021. Ten DNA-based vaccines, including the vaccine candidate reported here, are in ongoing clinical trials. Among these, safety and immunogenicity results were reported from only one phase 1 trial of a DNA vaccine against SARS-CoV-2 (INO-4800). INO-4800 demonstrated favorable safety and tolerability and was immunogenic, eliciting humoral and/or cellular immune responses in all vaccinated subjects. There is only one ongoing clinical trial of a vaccine against SARS-CoV-2 variants (mRNA-1273.351). Added value of this studyThis is the first-in-human phase 1 trial in healthy adults of a recombinant DNA vaccine for COVID-19 (GX-19N) containing the coding regions of both the spike and nucleocapsid proteins. This trial showed that GX-19N is safe, tolerated, and able to induce both humoral and cellular responses. A two-dose vaccination of 3{middle dot}0 mg GX-19N (on days 1 and 29) induced significant humoral and cellular responses. The neutralizing geometric mean titers in individuals vaccinated with GX-19N were lower than those of human convalescent sera. However, the GX-19N group showed increased T cell responses, which was similar to those analyzed using convalescent PBMCs. Furthermore, GX-19N induced not only SARS-CoV-2 spike-specific T cell responses but also broad nucleocapsid-specific T cell responses, which were also specific to SARS-CoV-2 variants. Implications of all the available evidenceIt is important to note that GX-19N contains a plasmid encoding both the spike and nucleocapsid proteins, and that it showed broad SARS-CoV-2-specific T cell responses, which may allow cross-reactivity with emerging SARS-CoV-2 variants. Based on these safety and immunogenicity findings, GX-19N was selected for phase 2 immunogenicity trials.

Oestrogen inhibits salt-dependent hypertension by suppressing GABAergic excitation in magnocellular AVP neurons.

AIMS: Abundant evidence indicates that oestrogen (E2) plays a protective role against hypertension. Yet, the mechanism underlying the antihypertensive effect of E2 is poorly understood. In this study, we sought to determine the mechanism through which E2 inhibits salt-dependent hypertension. METHODS AND RESULTS: To this end, we performed a series of in vivo and in vitro experiments employing a rat model of hypertension that is produced by deoxycorticosterone acetate (DOCA)-salt treatment after uninephrectomy. We found that E2 prevented DOCA-salt treatment from inducing hypertension, raising plasma arginine-vasopressin (AVP) level, enhancing the depressor effect of the V1a receptor antagonist (Phenylac1,D-Tyr(Et)2,Lys6,Arg8,des-Gly9)-vasopressin, and converting GABAergic inhibition to excitation in hypothalamic magnocellular AVP neurons. Moreover, we obtained results indicating that the E2 modulation of the activity and/or expression of NKCC1 (Cl- importer) and KCC2 (Cl- extruder) underpins the effect of E2 on the transition of GABAergic transmission in AVP neurons. Lastly, we discovered that, in DOCA-salt-treated hypertensive ovariectomized rats, CLP290 (prodrug of the KCC2 activator CLP257, intraperitoneal injections) lowered blood pressure, and plasma AVP level and hyperpolarized GABA equilibrium potential to prevent GABAergic excitation from emerging in the AVP neurons of these animals. CONCLUSION: Based on these results, we conclude that E2 inhibits salt-dependent hypertension by suppressing GABAergic excitation to decrease the hormonal output of AVP neurons.

Excessive maternal salt intake gives rise to vasopressin-dependent salt sensitivity of blood pressure in male offspring.

Salt sensitivity of blood pressure (SSBP) is a trait carrying strong prognostic implications for various cardiovascular diseases. To test the hypothesis that excessive maternal salt intake causes SSBP in offspring through a mechanism dependent upon arginine-vasopressin (AVP), we performed a series of experiments using offspring of the rat dams salt-loaded during pregnancy and lactation with 1.5% saline drink ("experimental offspring") and those with normal perinatal salt exposure ("control offspring"). Salt challenge, given at 7-8 weeks of age with either 2% saline drink (3 days) or 8% NaCl-containing chow (4 weeks), had little or no effect on systolic blood pressure (SBP) in female offspring, whereas the salt challenge significantly raised SBP in male offspring, with the magnitude of increase being greater in experimental, than control, rats. Furthermore, the salt challenge not only raised plasma AVP level more and caused greater depressor responses to V1a and V2 AVP receptor antagonists to occur in experimental, than control, males, but it also made GABA excitatory in a significant proportion of magnocellular AVP neurons of experimental males by depolarizing GABA equilibrium potential. The effect of the maternal salt loading on the salt challenge-elicited SBP response in male offspring was precluded by maternal conivaptan treatment (non-selective AVP receptor antagonist) during the salt-loading period, whereas it was mimicked by neonatal AVP treatment. These results suggest that the excessive maternal salt intake brings about SSBP in male offspring, both the programming and the expression of which depend on increased AVP secretion that may partly result from excitatory GABAergic action.

Excitatory GABAergic Action and Increased Vasopressin Synthesis in Hypothalamic Magnocellular Neurosecretory Cells Underlie the High Plasma Level of Vasopressin in Diabetic Rats.

Diabetes mellitus (DM) is associated with increased plasma levels of arginine-vasopressin (AVP), which may aggravate hyperglycemia and nephropathy. However, the mechanisms by which DM may cause the increased AVP levels are not known. Electrophysiological recordings in supraoptic nucleus (SON) slices from streptozotocin (STZ)-induced DM rats and vehicle-treated control rats revealed that γ-aminobutyric acid (GABA) functions generally as an excitatory neurotransmitter in the AVP neurons of STZ rats, whereas it usually evokes inhibitory responses in the cells of control animals. Furthermore, Western blotting analyses of Cl- transporters in the SON tissues indicated that Na+-K+-2Cl- cotransporter isotype 1 (a Cl- importer) was upregulated and K+-Cl- cotransporter isotype 2 (KCC2; a Cl- extruder) was downregulated in STZ rats. Treatment with CLP290 (a KCC2 activator) significantly lowered blood AVP and glucose levels in STZ rats. Last, investigation that used rats expressing an AVP-enhanced green fluorescent protein fusion gene showed that AVP synthesis in AVP neurons was much more intense in STZ rats than in control rats. We conclude that altered Cl- homeostasis that makes GABA excitatory and enhanced AVP synthesis are important changes in AVP neurons that would increase AVP secretion in DM. Our data suggest that Cl- transporters in AVP neurons are potential targets of antidiabetes treatments.

S100A8/A9 as a biomarker for synovial inflammation and joint damage in patients with rheumatoid arthritis

S100A8 and S100A9 are major leukocyte proteins, known as damage-associated molecular patterns, found at high concentrations in the synovial fluid of patients with rheumatoid arthritis (RA). A heterodimeric complex of S100A8/A9 is secreted by activated leukocytes and binds to Toll-like receptor 4, which mediates downstream signaling and promotes inflammation and autoimmunity. Serum and synovial fluid levels of S100A8/A9 are markedly higher in patients with RA than in patients with osteoarthritis or miscellaneous inflammatory arthritis. Serum levels of S100A8/A9 are significantly correlated with clinical and laboratory markers of inflammation, such as C-reactive protein, erythrocyte sedimentation rate, rheumatoid factor, and the Disease Activity Score for 28 joints. Significant correlations have also been found between S100A8/A9 and radiographic and clinical assessments of joint damage, such as hand radiographs and the Rheumatoid Arthritis Articular Damage score. In addition, among known inflammatory markers, S100A8/A9 has the strongest correlation with total sum scores of ultrasonography assessment. Furthermore, baseline levels of S100A8/A9 are independently associated with progression of joint destruction in longitudinal studies and are responsive to change during conventional and biologic treatments. These findings suggest S100A8/A9 to be a valuable diagnostic and prognostic biomarker for RA.

MRP8 promotes Th17 differentiation via upregulation of IL-6 production by fibroblast-like synoviocytes in rheumatoid arthritis

Myeloid-related protein (MRP)8/MRP14 is an endogenous Toll-like receptor 4 (TLR4) ligand and is abundant in synovial fluid (SF) of rheumatoid arthritis (RA) patients. Belonging to damage-associated molecular patterns, it amplifies proinflammatory mediators and facilitates a wide range of inflammatory and autoimmune diseases. Interleukin (IL)-17-producing T-helper (Th)17 cells have a crucial role in RA pathogenesis, and IL-6 is the key factor promoting Th17 differentiation. We investigated whether the level of MRP8/MRP14 is positively associated with IL-6 and IL-17 levels in RA SF and found that MRP8/MRP14 level had a significant correlation with IL-6 and IL-17 levels in RA SF. We also observed that MRP8-induced IL-17 production by peripheral blood mononuclear cells but MRP14 did not. Upon stimulation with MRP8, IL-6 production was enhanced by RA fibroblast-like synoviocytes (FLS) and was further elevated by coculturing RA FLS with activated CD4+ T cells. Moreover, we demonstrated that MRP8-activated IL-6 production by RA FLS promoted differentiation of Th17 cells using the coculture system consisting of CD4+ T cells and RA FLS. In addition, IL-6 blockade attenuated Th17 polarization of CD4+ T cells in the cocultures. Inhibitor studies revealed that MRP8 increased IL-6 production in RA FLS via TLR4/phosphoinositide 3-kinase/nuclear factor-kappaB and mitogen-activated protein kinase signaling pathways. Our results show that MRP8 has a crucial role in stimulating IL-6 expression by RA FLS, and subsequently promotes Th17 differentiation in RA, suggesting that neutralizing MRP8 level in RA synovium may be an effective therapeutic strategy in RA treatment.

Reciprocal Regulation of TH17 and Regulatory T Cells by Methotrexate and Its Therapeutic Effects in Collagen-induced Arthritis (CIA)

OBJECTIVE: Methotrexate is the first-line drug in treatment of rheumatoid arthritis (RA) exhibiting higher efficacy and better tolerability than most other DMARDs. To have a better understanding of the anti-arthritic mechanism of methotrexate, we investigated the effect of methotrexate on suppressing the autoimmune inflammatory and destructive arthritis in collagen-induced arthritis (CIA) mice. METHODS: The effects of methotrexate on joint inflammation were assessed by clinical scoring and histologic analysis. Levels of cytokines and autoreactive antibodies were analyzed by immunohistochemistry and ELISA. The population of TH17 and Foxp3+ regulatory T (Treg) cells and phosphorylation of their critical transcription activators, STAT3 and STAT5, were examined by fluorescence microscopy and flow cytometry, respectively. RESULTS: Treatment with methotrexate significantly alleviated joint inflammation and cartilage destruction in CIA. Serum levels of total immunoglobulins G, G1, G2a specific to type II collagen were also reduced considerably in methotrexate-treated mice. The drug inhibited the expression of proinflammatory cytokines such as IL-1beta, TNF-alpha, IL-6 and IL-17 in arthritic joints ex vivo as well as by splenocytes in vitro. Moreover, methotrexate treatment resulted in reciprocal modulation of TH17 cells and Foxp3+ regulatory T (Treg) cells in spleen tissues, in which TH17 cells were decreased and Treg cells in number were increased. Subsequent analysis of CD4+T cells showed that phosphorylation of STAT3 was decreased whereas phosphorylation of STAT5 was increased in methotrexate-treated mice. CONCLUSION: Methotrexate treatment effectively suppressed autoimmune arthritis and restored homeostasis of the immune system by reciprocal regulation of TH17 and Treg cells in a mouse model of collagen-induced arthritis.