key: cord-322345-rq5gh710 authors: Zheng, Fang; Zhou, Yanwen; Zhou, Zhiguo; Ye, Fei; Huang, Baoying; Huang, Yaxiong; Ma, Jing; Zuo, Qi; Tan, Xin; Xie, Jun; Niu, Peihua; Wang, Wenlong; Xu, Yun; Peng, Feng; Zhou, Ning; Cai, Chunlin; Tang, Wei; Xiao, Xinqiang; Li, Yi; Zhou, Zhiguang; Jiang, Yongfang; Xie, Yuanlin; Tan, Wenjie; Gong, Guozhong title: A Novel Protein Drug, Novaferon, as the Potential Antiviral Drug for COVID-19 date: 2020-04-29 journal: nan DOI: 10.1101/2020.04.24.20077735 sha: doc_id: 322345 cord_uid: rq5gh710 Abstract Background Novaferon, a novel protein drug approved for the treatment of chronic hepatitis B in China, exhibits potent antiviral activities. We aimed to determine the anti-SARS-CoV-2 effects of Novaferon in vitro, and conducted a randomized, open-label, parallel group study to explore the antiviral effects of Novaferon for COVID-19. Methods In laboratory, the inhibition of Novaferon on viral replication in cells infected with SARS-CoV-2, and on SARS-CoV-2 entry into healthy cells was determined. Antiviral effects of Novaferon were evaluated in COVID-19 patients with treatment of Novaferon, Novaferon plus Lopinavir/Ritonavir, or Lopinavir/Ritonavir. The primary endpoint was the SARS-CoV-2 clearance rates on day 6 of treatment, and the secondary endpoint was the time to the SARS-CoV-2 clearance in COVID-19 patients Results Novaferon inhibited the viral replication in infected cells (EC50=1.02 ng/ml), and protected healthy cells from SARS-CoV-2 infection (EC50=0.1 ng/ml). Results from the 89 enrolled COVID-19 patients showed that both Novaferon and Novaferon plus Lopinavir/Ritonavir groups had significantly higher SARS-CoV-2 clearance rates on day 6 than the Lopinavir/Ritonavir group (50.0% vs.24.1%, p = 0.0400, and 60.0% vs.24.1%, p = 0.0053). Median time to SARS-CoV-2 clearance were 6 days, 6 days, and 9 days for three groups respectively, suggesting a 3-dayreduction of time to SARS-CoV-2 clearance in both Novaferon and Novaferon plus Lopinavir/Ritonavir groups compared with Lopinavir/Ritonavir group. Conclusions Novaferon exhibited anti-SARS-CoV-2 effects in vitro and in COVID-19 patients. These data justified the further evaluation of Novaferon. Key words: COVID-19, SARS-CoV-2, Novaferon, Antiviral drug, Lopinavir/Ritonavir Lopinavir/Ritonavir groups had significantly higher SARS-CoV-2 clearance rates on day 6 than the Lopinavir/Ritonavir group (50.0% vs.24.1%, p = 0.0400, and 60.0% vs.24.1%, p = 0.0053). Median time to SARS-CoV-2 clearance were 6 days, 6 days, and 9 days for three groups respectively, suggesting a 3-dayreduction of time to SARS-CoV-2 clearance in both Novaferon and Novaferon plus Lopinavir/Ritonavir groups compared with Lopinavir/Ritonavir group. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 29, 2020. . https://doi.org/10.1101/2020.04.24.20077735 doi: medRxiv preprint COVID-19 by the infection of a novel coronavirus, SARS-CoV-2, has become a global pandemic and caused more than1,000,000 confirmed cases and over 60,000 deaths [1] [2] [3] [4] . Effective antiviral drugs for COVID-19 might lead to earlier clearance of SARS-CoV-2 that in turn helps to slow down or stop the progress of disease course for patients. The elimination of virus in patients at early stage would also contribute to the reduction of transmission of this deadly virus. With immediate availability and established safety profiles, approved antiviral drugs were targeted with the desire to find effective anti-SARS-CoV-2 drugs in the shortest time possible [5] . However, none of the most promising antiviral drugs for COVID-19 has been proved effective yet. Most published findings for the antiviral treatment of COVID-19 were based on the individual case reports or cellular antiviral results [6, 7] . Despite the lack of convincing evidence, Lopinavir/Ritonavir was quickly selected and recommended as an antiviral drug for COVID-19 in China since January. So far, only limited observations of Lopinavir/Ritonavir for coronavirus in SARS patients were reported [8] . Results from a recently completed trial of Lopinavir/Ritonavir in patients with severe COVID-19 generated disappointed outcomes and showed no significant antiviral effects [9] . Without effective antiviral drugs for COVID-19, health care workers have no alternative choices but mainly rely on the supportive and symptomatic treatments to manage COVID-19 patients. The rapid spread and daily increase of large death numbers in over 100countries perhaps represent one of the biggest medical and humanity challenges, it is even more critical than two months ago to find antiviral drugs with clinical evidence for COVID-19 patients [10, 11] . Novaferon, a novel antiviral protein drug which has been approved for the treatment . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 29, 2020. [12] . Novaferon has similar properties of human interferon, but its antiviral activities were greatly improved and are at least 10 times more potent than human interferon alpha-2b [13] . The antiviral efficacy of Novaferon was demonstrated by clinical studies conducted in China. In April 2018, Novaferon was approved in China by former CFDA (Chinese Food and Drug Administration) for the treatment of chronic hepatitis B. Novaferon protein's nonproprietary name was temporarily defined as "recombinant cytokine gene-derived protein injection" by Chinese Pharmacopeia Committee, and the recommended international nonproprietary name (rINN) by WHO is not available yet. For convenience purposes, Novaferon was used as the drug name in our study. In the present study, we specifically focused on observing the antiviral effects of Novaferon. We first determined whether Novaferon was able to inhibit SARS-CoV-2 at cellular level, and then conducted a randomized, open-label, parallel group trial to explore the antiviral effects of Novaferon in patients with COVID-19 by observing the SARS-CoV-2 clearance rates at different times during the treatment period. The primary endpoint was the SARS-CoV-2 clearance rates on day 6, and the secondary endpoint was the median time to reach SARS-CoV-2 clearance in patients with COVID-19. As a popular and standard antiviral drug for COVID-19 in China, Lopinavir/Ritonavir was included in this study to serve as a control for comparison. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 29, 2020. All the in vitro experiments were conducted in a biosafety level-3 (BSL3/P3) laboratory at Chinese Center for Disease Control and Prevention (Chinese CDC). We is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 29, 2020. We further observed whether the previous treatment of Vero E6 cells with Novaferon protected the cells from viral entry through exposure of the pre-treated cells to SARS-CoV-2 later. Detailed operation procedures were identical to the above description, except that the step orders were changed to allow the observation of the preventive effects of Novaferon. Briefly, blank Vero E6 cells were incubated with series concentrations of Novaferon for 2 hours, and the supernatants containing Novaferon were then removed. The pre-treated Vero E6 cells were exposed to SARS-CoV-2 by incubation with C-Tan-nCov Wuhan strain 01 (100PFU) for 2 hours, and the supernatants containing SARS-CoV-2 were then removed. Fresh medium was added, and the cells were incubated for 48 hours. 100 μ L of supernatants were taken from each well, and the total viral RNA in the supernatants was measured using the same methods described above. The Ct number obtained from Vero E6 cells without pre-treatment of Novaferon was considered as 100%, and the decreased Ct numbers obtained from the pre-treated Vero E6 cells with various concentrations of Novaferon were used to calculate the inhibition percentages. The preventive effects of Novaferon were then determined by observing the viral RNA reduction inthe cells pre-treated with Novaferon. The EC 50 of Novaferon for the observed preventive effects was decided accordingly. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 29, 2020. The study was originally designed as a multi-center study across hospitals in Changsha city and in other cities of Hunan Province, China. However, per government order, all patients from hospitals in Changsha city had to be relocated to the First Hospital of Changsha, a designated treatment center for all COVID-19 patients in Changsha city, and hospitals in other cities of Hunan Province were not able to participate due to various . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 29, 2020. Antiviral effects were assessed on day 3, day 6, and day 9 after starting drug administration. For the above antiviral drugs involved in the study, Novaferon (temporary non-proprietary name: recombinant cytokine gene-derived protein injection) was . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 29, 2020. Grading Scale for Determining the Severity. The peak levels of SARS virus were around 10 days after onset and then the viral level began to decrease without effective antiviral treatment in SARS patients [14] . Considering the homology of gene sequences of SARS-CoV-2 and SARS was over 90% [2] , we assumed that the intervention of antiviral drugs in COVID-19 patients would likely enhance or shorten the time of viral clearance. In this regarding, the primary endpoint for this study was decided as the SARS-CoV-2 clearance rates in COVID-19 patients assessed on day 6 of antiviral treatment. The secondary endpoint was the time till the SARS-CoV-2 clearance. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 29, 2020. Statistical analysis was performed on an intent-to-treat basis, and all patients randomized and treated at least once with the study medications were included for the primary analysis. For patient demographics information and baseline disease characteristics, qualitative variables were compared among treatment groups with the use of Chi-square test, and quantitative variables were compared with the use of an ANOVA model. Only the overall differences among the three treatment groups were tested (based on null hypothesis, "all three groups were the same", against an alternative hypothesis, "at least one group was different") and therefore, no pairwise comparison was performed for baseline characteristics. For the primary endpoint, SARS-CoV-2 clearance rate, estimates of the rates were calculated based on a binomial distribution. Difference between treatment groups was tested using the Chi-square test. To control the overall significance level for the study, the three pairwise comparisons for the primary endpoint were performed at the two-sided alpha = 0.05 using a closed testing procedure according to the following order: Novaferon plus Lopinavir/Ritonavir vs. Lopinavir/Ritonavir alone; Novaferon alone vs. Lopinavir/Ritonavir alone; Novaferon plus Lopinavir/Ritonavir vs. Novaferon alone. For the secondary endpoint, time to SARS-CoV-2 clearance, median time for each group was estimated with the use of the Kaplan-Meier method and treatment differences were tested using log-rank test. All tests were two-sided, with a p value of less than 0.05 considered to indicate statistical significance. Analysis was conducted using SAS V9.2. For missing SARS-CoV-2 clearance status, Last Observation Carried Forward (LOCF) analysis was presented as the primary analysis. For purpose of sensitivity analyses, complete case analysis and worst case imputation methods were also performed. For the worst case imputation, missing SARS-CoV-2 status was replaced with 'positive'. The planned sample size of 90 patients (30 patients per group) was not determined . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 29, 2020. based on statistical consideration. Adverse events were reported and graded using WHO Toxicity Grading Scale for determining the severity. Incidence of adverse events was summarized descriptively without a formal statistical test. As shown in fig.1 These data indicated that Novaferon effectively inhibited the viral replication within SARS-CoV-2-infected cells. In addition, healthy Vero E6 cells that were previously treated with Novaferon obtained the ability of resisting the viral entry, as indicated by the reduction of viral RNA after exposure of the treated cells to SARS-CoV-2 later. Novaferon exhibited this preventive effect efficiently with the EC 50 (0.1 ng/ml), lower than the EC 50 for inhibiting SARS-CoV-2 replication in the infected cells. These data suggested that Novaferon inhibited viral replication in SARS-CoV-2infected cells and enabled healthy cells to resist the viral entry. A total of 92 patients with moderate or severe illness were assessed for the eligibility criteria and 3 patients were excluded. 89 patients were randomized into the study from 1 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 29, 2020. severe ill. There were no apparent differences among the groups with respect to any of the patient demographics and the baseline characteristics. Table 2 summarized the complete RT-PCR test results of all 89 patients on day 3, day 6, and day 9 after starting drug administration. The negative results of SARS-CoV-2 nucleic acid detection in the tested samples served as the indicator of in vivo SARS-CoV-2 clearance in patients. The SARS-CoV-2 clearance rates on day 3, day 6, and day 9 in three treatment groups were presented and compared ( is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 29, 2020. Novaferon plus Lopinavir/Ritonavir group, and 51.7% (15/29) in Lopinavir/Ritonavir group. There were no statistically significant differences between the groups. The median time to SARS-CoV-2 clearance were 6 days, 6 days, and 9 days for Analyses based on both complete case analysis and worst case imputationforSARS-CoV-2 clearance rates showed little differences with the LOCF analysis, and the statistical conclusions for all the treatment comparisons remained the same. No severe adverse events (SAE) associated with the tested antiviral drugs were observed. Reported grade1, and grade 2 adverse events (AE) were described below. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 29, 2020. during the observation period. No matter whether exhibiting good, poor or none anti-COVID-19 effects, Lopinavir/Ritonavir in this study served as the control and allowed us to assess the The antiviral effects of Novaferon for COVID-19 patients were in consistence with the laboratory findings. Inhibition of the viral replication by Novaferon at cellular level was very efficient as indicated by the low EC 50 (1.02 ng/ml). More interestingly, healthy cells that were pre-treated by Novaferon obtained the ability, in the absence of Novaferon, to resist the viral entry into cells when the treated cells were exposed to SARS-CoV-2 later (EC 50 0.1 ng/ml). It might be worth to explore the potential use of Novaferon as a preventive agent for high risk population, especially for health care workers who have to routinely contact COVID-19 patients. The viral loads in COVID-19 patients were reported to reach peak levels around 5 6 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 29, 2020. in COVID-19 patients with Novaferon treatment were unlikely related to the natural disease course. Rather, the observed enhancement of SARS-CoV-2 clearance apparently reflected the antiviral effects of Novaferon in observed COVID-19 patients. Our study has several limitations. First, all observations were done at one hospital in one city. Second, the sample size was relatively small and was not based on statistical consideration as limited by the availability of COVID-19 patients in Changsha city. Third, the unexpected difficulties associated with the COVID-19 outbreak compromised the quality of this study. For example, it's highly possible that adverse events were underreported due to the lack of medical staff and the risk situation. However, these limitations shouldn't change the overall conclusion for thisrandomized trial because the antiviral assessments were strictly performed according to a vigorous standard. Novaferon exhibited anti-SARS-CoV-2 effects at cellular level and in patients with . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted April 29, 2020. . https://doi.org/10.1101/2020.04.24.20077735 doi: medRxiv preprint 1 8 WHO. Coronavirus disease 2019 (COVID-19) Situation Report-76. Accessed A Novel Coronavirus from Patients with Pneumonia in China Genomic characterization and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding A new coronavirus associated with human respiratory disease in China A pneumonia outbreak associated with a new coronavirus of probable bat origin International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity Therapeutic options for the 2019 novel coronavirus First Case of 2019 Novel Coronavirus in the United States Role of lopinavir/ritonavir in the treatment of SARS: initial virological and clinical findings A Trial of Lopinavir-Ritonavir in Adults Hospitalized with Severe Covid-19 Potential Interventions for Novel Coronavirus in China: A Systematic Review Recombinant human interferon-like proteins (Patent No. US 7,625,555 B2) Novaferon, a novel recombinant protein produced by DNA-shuffling of IFN-alpha, shows antitumor effect in vitro and in vivo Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study We thank all the medical and management staff, who came from hospitals across Changsha City and worked at the First Hospital of Changsha, for their courage and dedication to COVID-19 patient care and overall operations of the hospital during the difficulty time. We especially thank Dr. Charlie Chen of SRD ClinMax Corporation for conducting the statistical analysis. Author Contributors: Drs F Zheng, Y Zhou, Z Zhou and F Ye contributed equally to this article and share first authorship. Drs Y Jiang, YXie, W Tan, GGong should be considered as co-correspondence authors, have full access to all the data in the study and take responsibility for the integrity and accuracy of the data.Competing interests: none. Ethical approval: The study protocol was approved by the ethics committee of First Hospital of Changsha.. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted April 29, 2020. . https://doi.org/10.1101/2020.04.24.20077735 doi: medRxiv preprint 1 9The copyright holder for this preprint this version posted April 29, 2020. . https://doi.org/10.1101/2020.04.24.20077735 doi: medRxiv preprint 2 0. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted April 29, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 29, 2020. *LPV/ r: Lopinavir/Ritonavir. †p value: Based on null hypothesis, "all three groups were the same", against an alternative hypothesis, "at least one group was difference"; 'sex' and 'disease stage' were tested with the use of a Chi-square test, and 'age' with an ANOVA model;. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 29, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 29, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 29, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)The copyright holder for this preprint this version posted April 29, 2020. . https://doi.org/10.1101/2020.04.24.20077735 doi: medRxiv preprint