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.

Performance of an Automated Fluorescence Antinuclear Antibody Image Analyzer

BACKGROUND: The gold standard for antinuclear antibody (ANA) screening is the indirect immunofluorescence (IIF) assay with human epithelial cells (HEp-2). However, a number of substantial disadvantages of manual IIF assays have highlighted the need for the automation and standardization of fluorescent ANA (FANA) testing. We evaluated the performance of EUROPattern Suite (Euroimmun AG, Germany), an automated FANA image analyzer, with regard to ANA detection and pattern recognition compared with conventional manual interpretation using the fluorescence microscopic IIF assay. METHODS: A total of 104 samples including 70 ANA-positive sera and 34 ANA-negative sera collected from September to October 2015 were included. The sensitivity, specificity, and pattern recognition function were evaluated to determine the performance of EUROPattern Suite compared with the manual IIF assay results. RESULTS: The sensitivity and specificity of EUROPattern Suite for ANA detection were 94.3% and 94.1%, respectively. The concordance rate between the two methods was 94.2%. For pattern recognition, 45.7% of the samples were assigned identical ANA patterns including simple and mixed. When major pattern matching was considered, 83.7% (41/49) and 95.2% (20/21) of the samples with simple and mixed patterns, respectively, showed concordant results between the two methods. CONCLUSIONS: EUROPattern Suite, an automated FANA image analyzer, provides a viable option for distinguishing between positive and negative results, although the ability to assign specific patterns is insufficient to replace manual microscopic interpretation. This automated system may increase efficiency in laboratories, in which a large number of samples need to be processed.

Evaluation of the Automated Blood Bank Systems Galileo NEO and QWALYS-3 for ABO-RhD Typing and Antibody Screening / 대한수혈학회지

BACKGROUND: An automation system for ABO-RhD typing and antibody screening has been developed and its use is increasing. We compared the results of ABO-RhD typing and antibody screening tests using the manual (ABO-RhD typing) or semiautomated (antibody screening) method and with the automation instruments Galileo NEO (Immucor Gamma, Norcoss, USA) and QWALYS-3 (DIAGAST, Loos Cedex, France). METHODS: A total of 332 blood samples were tested for ABO-RhD typing in comparison with routine manual tests, and 236 samples for antibody screening in comparison with DS-Screening II (Bio-Rad Laboratories, 1785 Cressier FR, Switzerland). We evaluated the performance of Galileo NEO and QWALYS-3 in terms of concordance, carryover, and sensitivity test for ABO-RhD typing and antibody screening. RESULTS: The concordance rates of ABO-RhD typing results between the manual methods and the two instruments were 99.4% for Galileo NEO and 99.1% for QWALYS-3, respectively. On antibody screening tests, a concordance rate of 97.9% was observed between the semiautomated method and Galileo NEO or QWALYS-3, because of discordance in five specimens. The carryover was not observed for ABO-RhD typing and antibody screening. The overall sensitivity of the two automation instruments appears to be parallel with that of DS-Screening II except for anti-E. CONCLUSION: The Galileo NEO and QWALYS-3 system showed good performance, it can be used with confidence for routine pre-transfusion testing in the blood bank.

p21Cip/WAF1 activation is an important factor for the ERK pathway dependent anti-proliferation of colorectal cancer cells

p21Cip/WAF1, an important regulator of cell proliferation, is induced by both p53- and extracellular signal regulated kinase (ERK) pathways. The induction of p21Cip/WAF1 occurs by prolonged activation of the ERKs caused by extracellular stimuli, such as zinc. However, not all the cells appeared to respond to ERK pathway dependent p21Cip/WAF1 induction. Here we investigated the cause of such difference using colorectal cancer cells. p21Cip/WAF1 induction and concomitant reduction of bromodeoxyuridine (BrdU) incorporation were observed by zinc treatment within HT-29 and DLD-1. However, HCT-116 cells with high endogenous p21Cip/WAF1 levels did not show any additional increment of p21Cip/WAF1 levels by zinc treatment and did maintain high BrdU incorporation level. The p21Cip/WAF1 induction by zinc depended upon prolonged activation of extracellular signal regulated kinase (ERK) was not observed in HCT-116 cells. The percentage of BrdU positive cells was 50% higher in p21Cip/WAF1 -/- HCT-116 cells compared to p21Cip/WAF1 +/+ HCT- 116 cells, and no cells induced p21Cip/WAF1 incorporated BrdU in its nucleus, yet confirming the importance of p21Cip/WAF1 induction in anti- proliferation. These results again support that p21Cip/WAF1 induction is a determinant in the regulation of colonic proliferation by the ERK pathway.

Biochemical properties of full-length hepatitis C virus RNA-dependent RNA polymerase expressed in insect cells

The hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B protein, is the key viral enzyme responsible for replication of the HCV viral RNA genome. Although several full-length and truncated forms of the HCV NS5B proteins have been expressed previously in insect cells, contamination of host terminal transferase (TNTase) has hampered analysis of the RNA synthesis initiation mechanism using natural HCV RNA templates. We have expressed the HCV NS5B protein in insect cells using a recombinant baculovirus and purified it to near homogeneity without contaminated TNTase. The highly purified recombinant HCV NS5B was capable of copying 9.6-kb full-length HCV RNA template, and mini-HCV RNA carrying both 5'- and 3'-untranslated regions (UTRs) of the HCV genome. In the absence of a primer, and other cellular and viral factors, the NS5B could elongate over HCV RNA templates, but the synthesized products were primarily in the double stranded form, indicating that no cyclic replication occurred with NS5B alone. RNA synthesis using RNA templates representing the 3'-end region of HCV minus-strand RNA and the X-RNA at the 3'-end of HCV RNA genome was also initiated de novo. No formation of dimersize self-primed RNA products resulting from extension of the 3'-end hydroxyl group was observed. Despite the internal de novo initiation from the X-RNA, the NS5B could not initiate RNA synthesis from the internal region of oligouridylic acid (U)20, suggesting that HCV RNA polymerase initiates RNA synthesis from the selected region in the 3'-UTR of HCV genome.