Establishment of an in‐house real‐time RT‐PCR assay for the detection of severe acute respiratory syndrome coronavirus 2 using the first World Health Organization international standard in a resource‐limited country

BackgroundThe COVID‐19 pandemic caused by SARS‐CoV‐2 remains public health burdens and many unresolved issues worldwide. Molecular assays based on real‐time RT‐PCR are critical for the detection of SARS‐CoV‐2 in clinical specimens from patients suspected of COVID‐19.ObjectiveWe aimed to establish and validate an in‐house real‐time RT‐PCR for the detection of SARS‐CoV‐2.MethodologyPrimers and probes sets in our in‐house real‐time RT‐PCR assay were designed in conserved regions of the N and E target genes. Optimized multiplex real‐time RT‐PCR assay was validated using the first WHO International Standard (NIBSC code: 20/146) and evaluated clinical performance.ResultsThe limit of detection validated using the first WHO International Standard was 159 IU/ml for both E and N target genes. The evaluation of clinical performance on 170 clinical samples showed a positive percent agreement of 100% and the negative percent agreement of 99.08% for both target genes. The Kappa value of 0.99 was an excellent agreement, the strong correlation of Ct values observed between two tests with r2 = 0.84 for the E gene and 0.87 for the N gene. Notably, we assessed on 60 paired saliva and nasopharyngeal samples. The overall agreement was 91.66%, and Kappa value of 0.74 showed a high agreement between two types of samples. When using nasopharyngeal swabs as the reference standard, positive percent agreement, and negative percent agreement were 91.83% and 90.90%, respectively.ConclusionIn the present study, we established and validated an in‐house real‐time RT‐PCR for molecular detection of SARS‐CoV‐2 in a resource‐limited country.

The efficacy, safety and immunogenicity Nanocovax: results of a randomized, double-blind, placebo-controlled Phase 3 trial. (preprint)

Background Nanocovax is a recombinant severe acute respiratory syndrome coronavirus 2 subunit vaccine composed of full-length prefusion stabilized recombinant SARS-CoV-2 spike glycoproteins (S-2P) and aluminum hydroxide adjuvant. In a Phase 1 and 2 studies, (NCT04683484) the vaccine was found to be safe and induce a robust immune response in healthy adult participants. Methods We conducted a multicenter, randomized, double-blind, placebo-controlled study to evaluate the safety, immunogenicity, and protective efficacy of the Nanocovax vaccine against Covid-19 in approximately 13,007 volunteers aged 18 years and over. The immunogenicity was assessed based on Anti-S IgG antibody response, surrogate virus neutralization, wild-type SARS-CoV-2 neutralization and the types of helper T-cell response by intracellular staining (ICS) for interferon gamma (IFNg) and interleukin-4 (IL-4). The vaccine efficacy (VE) was calculated basing on serologically confirmed cases of Covid-19. Findings Up to day 180, incidences of solicited and unsolicited adverse events (AE) were similar between vaccine and placebo groups. 100 serious adverse events (SAE) were observed in both vaccine and placebo groups (out of total 13007 participants). 96 out of these 100 SAEs were determined to be unrelated to the investigational products. 4 SAEs were possibly related, as determined by the Data and Safety Monitoring Board (DSMB) and investigators. Reactogenicity was absent or mild in the majority of participants and of short duration. These findings highlight the excellent safety profile of Nanocovax. Regarding immunogenicity, Nanocovax induced robust IgG and neutralizing antibody responses. Importantly, Anti S-IgG levels and neutralizing antibody titers on day 42 were higher than those of natural infected cases. Nanocovax was found to induce Th2 polarization rather than Th1. Post-hoc analysis showed that the VE against symptomatic disease was 51.5% (95% confidence interval [CI] was [34.4%-64.1%]. VE against severe illness and death were 93.3% [62.2- 98.1]. Notably, the dominant strain during the period of this study was Delta variant. Interpretation Nanocovax 25 microgram (mcg) was found to be safe with the efficacy against symptomatic infection of Delta variant of 51.5%.

Human Brain Organoids as an In Vitro Model System of Viral Infectious Diseases

Brain organoids, or brainoids, have shown great promise in the study of central nervous system (CNS) infection. Modeling Zika virus (ZIKV) infection in brain organoids may help elucidate the relationship between ZIKV infection and microcephaly. Brain organoids have been used to study the pathogenesis of SARS-CoV-2, human immunodeficiency virus (HIV), HSV-1, and other viral infections of the CNS. In this review, we summarize the advances in the development of viral infection models in brain organoids and their potential application for exploring mechanisms of viral infections of the CNS and in new drug development. The existing limitations are further discussed and the prospects for the development and application of brain organs are prospected.

Safety and Immunogenicity of Nanocovax, a SARS-CoV-2 Recombinant Spike Protein Vaccine (preprint)

Background: Nanocovax is a recombinant severe acute respiratory syndrome coronavirus 2 subunit vaccine composed of full-length prefusion stabilized recombinant SARS-CoV-2 spike glycoproteins (S-2P) and aluminum hydroxide adjuvant.Methods: We conducted a dose-escalation, open label trial (phase 1) and a randomized, double-blind, placebo-controlled trial (phase 2) to evaluate the safety and immunogenicity of the Nanocovax vaccine (in 25 microgram (mcg), 50 mcg, and 75 mcg doses, aluminum hydroxide adjuvanted). In phase 1, 60 participants received two intramuscular injection of the vaccine following dose-escalation procedure. The primary outcomes were reactogenicity and laboratory tests to evaluate the vaccine safety. In phase 2 which involved in 560 healthy adults, the primary outcomes are vaccine safety, and anti-S IgG antibody response. Secondary outcomes were surrogate virus neutralization, wild-type SARS-CoV-2 neutralization, and T-cell responses by intracellular staining (ICS) for interferon gamma (IFNg). Anti-S IgG and neutralizing antibody levels were compared with convalescent serum samples from symptomatic Covid-19 patients.Findings: For phase 1 study, no serious adverse events (SAE) were observed for all 60 participants. Most adverse events (AE) were grade 1 and disappeared shortly after injection. For phase 2 study, after randomization, 480 participants were assigned to receive the vaccine with adjuvant, and 80 participants were assigned to receive placebo. Reactogenicity was absent or mild in the majority of participants and of short duration (mean ≤3 days). Unsolicited adverse events were mild in most participants. There were no serious adverse events related to Nanocovax. Regarding the immunogenicity, Nanocovax induced robust anti-S antibody responses. In general, there humoral responses were similar among vaccine groups up to day 90. Anti S-IgG levels and neutralizing antibody titers at the peak response on day 42 were all higher than those of convalescent sera.Interpretation: Up to day 90, Nanocovax was found to be safe, well tolerated, and induced robust immune responses. 25 mcg was selected for a phase 3 trial to evaluate the vaccine efficacy.Trial Registration: ClinicalTrials.gov number, NCT04683484. NCT04683484, registration date in clinicaltrial.gov is Dec24, 2020, We has started our Phase 1 clinical trial in Vietnam on Dec 17, 2020,Funding: Research funded by Nanogen Pharmaceutical Biotechnology JSC., and the Ministry of Science and Technology of VietnamDeclaration of Interest: The following authors Thuy Phuong Nguyen, Hiep Khong, Tri Minh Le, Tuyen Thi Ngoc Trang, Thanh Thi Dinh, Thuong Van Vo, Thao Thi Thu Vu, Quynh Bao Phuong Nguyen, Vuong Tan Phan, Vinh The Tran, Mai Thi Nhu Tran, Truc Thi Thanh Nguyen, Phat Tan Ha, Hieu Trong Huynh, Khanh Duy Nguyen, Chung Chinh Doan, Thuan Trong Ung, Si Minh Do are employees of Nanogen Pharmaceutical Biotechnology JSC. All other authors declare no competing interests.Ethical Approval: The trials were designed and funded by Nanogen Pharmaceutical Biotechnology JSC and the Ministry of Science and Technology (MOST) of Vietnam. The trial protocol was approved by the Ethics Committee/Protocol Review Board of the Ministry of Health (Vietnam)

Pain Symptoms in Patients with Coronavirus Disease (COVID-19): A Literature Review.

PURPOSE: On 11 March, 2020, the coronavirus disease (COVID-19) outbreak was declared as a global pandemic by the World Health Organization. It brought substantial physical and psychological burden on individuals and financial loss across countries. Patients with COVID-19 may exhibit various symptoms, such as fever, cough, dyspnea, muscle pain, sore throat, headache, chest pain, and abdominal pain, at 2-14 days after exposure to the novel coronavirus (severe acute respiratory syndrome [SARS]-CoV-2). Pain symptoms present important challenge to clinicians' diagnosis when treating COVID-19 patients with mild symptoms. Considering the increasing number of confirmed COVID-19 cases, the pain symptoms should be systematically summarized. RESULTS: The virus can invade different tissues of the body and cause different pain manifestations. SARS-CoV-2 primarily invades the respiratory system, and patients develop sore throat, fever, cough, and other pneumonia-associated symptoms. Moreover, it infects the nervous system (eg, headache, dizziness, and confusion), digestive system (eg, abdominal pain, diarrhea), and cardiovascular system (eg, chest pain, palmus, and cardiac injury). The incidence rate is 1.7-33.9% for headache, 0.7-47.1% for sore throat, 1.5-61.0% for myalgia/arthralgia, 1.6-17.7% for chest pain, and 1.9-14.5% for abdominal pain. In comparison with chest and abdominal pain, COVID-19 patients are more likely to develop headache, sore throat, and myalgia/arthralgia. CONCLUSION: Different pain reflects the damage of different body systems. Therefore, the summary of pain symptoms for COVID-19 patients can help doctors improve the accuracy and efficiency of diagnosis when treating COVID-19 patients with atypical or mild symptoms and adopt more targeted treatment methods.

Association between ageing population, median age, life expectancy and mortality in coronavirus disease (COVID-19).

As of May 5, 2020, the number of confirmed coronavirus disease (COVID-19) cases has been more than 3.5 million with 243,540 deaths. We aimed to determine the associations between ageing population, median age, life expectancy at birth and COVID-19 mortality. The numbers of COVID-19 cases and deaths in the European region were obtained from the World Health Organization database. The data on percentage of the population aged 65 and over, median age and life expectancy at birth were extracted from the World Factbook of Central Intelligence Agency. A total of 56 countries/areas in the Europe reported COVID-19 cases and deaths (1,121,853 cases and 100,938 deaths) on April 20, 2020. The results showed significant positive associations between COVID-19 mortality and ageing population (r =0.274; P =0.021), median age (r =0.255; P=0.029), male median age (r =0.284; P =0.017), female median age (r =0.224; P=0.049), life expectancy at birth (r =0.336; P=0.006), male life expectancy at birth (r =0.342; P=0.005), female life expectancy at birth (r =0.312; P=0.01) in the 56 European countries/areas. This study illustrated that COVID-19 mortality was positively associated with ageing population, median age, and life expectancy at birth.