Interaction and antiviral treatment of coinfection between SARS-CoV-2 and influenza in vitro.

BACKGROUND: The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has lasted for three years. Coinfection with seasonal influenza may occur resulting in more severe diseases. The interaction between these two viruses for infection and the effect of antiviral treatment remains unclear. METHODS: A SARS-CoV-2 and influenza H1N1 coinfection model on Calu-3 cell line was established, upon which the simultaneous and sequential coinfection was evaluated by comparing the viral load. The efficacy of molnupiravir and baloxavir against individual virus and coinfection were also studied. RESULTS: The replication of SARS-CoV-2 was significantly interfered when the influenza virus was infected simultaneously or in advance (p < 0.05). On the contrary, the replication of the influenza virus was not affected by the SARS-CoV-2. Molnupiravir monotherapy had significant inhibitory effect on SARS-CoV-2 when the concentration reached to 6.25 µM but did not show any significant anti-influenza activity. Baloxavir was effective against influenza within the dosage range and showed significant effect of anti-SARS-CoV-2 at 16 µM. In the treatment of coinfection, molnupiravir had significant effect for SARS-CoV-2 from 6.25 µM to 100 µM and inhibited H1N1 at 100 µM (p < 0.05). The tested dosage range of baloxavir can inhibit H1N1 significantly (p < 0.05), while at the highest concentration of baloxavir did not further inhibit SARS-CoV-2, and the replication of SARS-CoV-2 significantly increased in lower concentrations. Combination treatment can effectively inhibit influenza H1N1 and SARS-CoV-2 replication during coinfection. Compared with molnupiravir or baloxavir monotherapy, combination therapy was more effective in less dosage to inhibit the replication of both viruses. CONCLUSIONS: In coinfection, the replication of SARS-CoV-2 would be interfered by influenza H1N1. Compared with molnupiravir or baloxavir monotherapy, treatment with a combination of molnupiravir and baloxavir should be considered for early treatment in patients with SARS-CoV-2 and influenza coinfection.

Early Treatment of High-Risk Hospitalized Coronavirus Disease 2019 (COVID-19) Patients With a Combination of Interferon Beta-1b and Remdesivir: A Phase 2 Open-label Randomized Controlled Trial.

BACKGROUND: Early antiviral therapy was effective in the treatment of coronavirus disease 2019 (COVID-19). We assessed the efficacy and safety of combined interferon beta-1b and remdesivir treatment in hospitalized COVID-19 patients. METHODS: We conducted a multicentre, prospective open-label, randomized-controlled trial involving high-risk adults hospitalized for COVID-19. Patients were randomly assigned to a 5-day interferon beta-1b 16 million units daily and remdesivir 200 mg loading on day 1 followed by 100 mg daily on day 2 to 5 (combination group), or to remdesivir only of similar regimen (control group) (1:1). The primary endpoint was the time to complete alleviation of symptoms (NEWS2 = 0). RESULTS: Two-hundred and twelve patients were enrolled. The median days of starting treatment from symptom onset was 3 days. The median age was 65 years, and 159 patients (75%) had chronic disease. The baseline demographics were similar. There was no mortality. For the primary endpoint, the combination group was significantly quicker to NEWS2 = 0 (4 vs 6.5 days; hazard ratio [HR], 6.59; 95% confidence interval [CI], 6.1-7.09; P < .0001) when compared to the control group. For the secondary endpoints, the combination group was quicker to negative nasopharyngeal swab (NPS) viral load (VL) (6 vs 8 days; HR, 8.16; 95% CI, 7.79-8.52; P < .0001) and to develop seropositive immunoglobulin G (IgG) (8 vs 10 days; HR, 10.78; 95% CI, 9.98-11.58; P < .0001). All adverse events resolved upon follow-up. Combination group (HR, 4.1 95% CI, 1.9-8.6, P < .0001) was the most significant independent factor associated with NEWS2 = 0 on day 4. CONCLUSIONS: Early treatment with interferon beta-1b and remdesivir was safe and better than remdesivir only in alleviating symptoms, and in shortening viral shedding and hospitalization with earlier seropositivity in high-risk COVID-19 patients. CLINICAL TRIALS REGISTRATION: NCT04647695.

Diagnostic Value of a SARS-CoV-2 Rapid Test Kit for Detection of Neutralizing Antibodies as a Point-of-Care Surveillance Test.

Detection and tracking of antibodies play an increasingly prominent role in population surveillance and implementation of public health measures to combat the current coronavirus disease 2019 (COVID-19) pandemic, with much attention placed on developing commercial serological assays as point-of-care diagnostic tools. While many rapid diagnostic tests (RDTs) that detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG and IgM antibodies have been evaluated, there is currently limited insight into detection of neutralizing antibodies (nAbs) by such modalities. Here, we evaluate performance characteristics of an RDT that detects SARS-CoV-2 IgG antibodies and, importantly, nAbs based on both infection- and vaccine-immunized cohorts by direct comparison to known antibody titers obtained from live virus microneutralization (VMN) assays. We further contextualize interpretations of band intensity of the RDT with reference to the World Health Organization (WHO) International Standard. We report a sensitivity of 94.37% and specificity of 92.50% for SARS-CoV-2 IgG detection and a sensitivity of 94.37% and specificity of 92.68% for nAbs. A limit of detection was determined as 3.125 IU/mL and 25.00 IU/mL, respectively, with reference to the WHO International Standard. We confirm that indication of nAb concentration, as elucidated by band intensity on the RDT, correlated with nAb titers defined by VMN assays and surrogate nAb assays. We additionally observe no cross-reactivity of the nAb test line to SARS-CoV-1 but report display of weak seropositivity for one sample on the SARS-CoV-2 IgG test line. Our study reveals promising performance characteristics of the assessed RDT, which implicates its usefulness in a wide range of diagnostic and epidemiological settings. IMPORTANCE In the ongoing coronavirus disease 2019 (COVID-19) pandemic, antibody tests play an increasingly important role in detecting previous infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and monitoring of response to vaccinations. In particular, neutralizing antibodies have recently been demonstrated to be highly predictive of immune protection against symptomatic infection. Our study is the first to evaluate a rapid diagnostic test based on samples acquired from both recovered COVID-19 patients and individuals vaccinated for SARS-CoV-2, which detects neutralizing antibodies in addition to SARS-CoV-2 IgG. We report promising sensitivity, specificity, and cross-reactivity profiles, which implicate its usefulness in a wide range of settings as a diagnostic point-of-care tool to aid in curbing transmission and reducing mortality caused by COVID-19 symptoms.

Correlation of Immunogenicity and Reactogenicity of BNT162b2 and CoronaVac SARS-CoV-2 Vaccines.

COVID-19 infection is a global health issue, and vaccination is the main strategy to control this pandemic. In this study, 189 participants received BNT162b2 or CoronaVac vaccine, and 133 of them recorded adverse events (AEs) daily for 4 weeks after vaccination. Their neutralizing antibody against SARS-CoV-2 was determined with live virus microneutralization (vMN) assay. The vMN geometric mean titer (GMT) on day 56 was 129.9 (95% confidence interval [CI],108.6 to 155.2) in the BNT162b2 group and 13.1 (95% CI, 11.2 to 15.3) in the CoronaVac group. Day 56 vMN GMT was 147.9 (95% CI, 118.9 to 184.1) in females and 129.9 (95% CI, 108.6 to 155.2) in males receiving BNT162b2, while it was 14.0 (95% CI, 11.6 to 17.0) in females and 11.4 (95% CI, 8.7 to 15.0) in males receiving CoronaVac. Injection site pain (88.8%) and redness (77.5%) were the most commonly BNT162b2-related AEs, and injection site pain (37.7%) and tiredness (26.4%) were more frequent in the CoronaVac group. Women showed a higher frequency of headache (45.7% versus 29.4%) and joint pain (26.1 versus 14.7%) than men in BTN162b2 group. Headache (26.5% versus 0%) and tiredness (38.2% versus 5.3%) were more common in women than in men vaccinated with CoronaVac. No correlation between any AE and antibody response was observed in BNT162b2 or CoronaVac platforms. After taking the gender factor into account, in the BNT162b2 group, a low correlation between day 21 vMN titer and redness (rho = 0.34) or itching (rho = 0.32) was presented in females, and a low correlation between day 56 vMN titer and fever (rho = 0.35) was presented in males. Taken together, AEs could have a low correlation with BNT162b2 vaccine response. IMPORTANCE Effective vaccines against SARS-CoV-2 are vital tools for containing the COVID-19 pandemic by increasing population immunity. While currently available vaccines can elicit antibody response against SARS-CoV-2 with high efficacy, the associated side effects may cause vaccine hesitancy. Our work is important in that we have thoroughly analyzed the correlation between immunogenicity and reactogenicity of two COVID-19 vaccines (BNT162b2 and CoronaVac) in the study. Our results showed that women had higher levels of neutralizing antibodies than men after receiving BNT162b2 or CoronaVac. Furthermore, a low correlation was observed between day 21 vMN titer and local reactions (redness and itching) in females, as well as between day 56 vMN titer and fever in males receiving BNT162b2. Thus, common side effects are not always a negative impact of vaccination but may serve as an indicator of immunogenicity of vaccines. Our study may help in increasing the public's acceptance and confidence over COVID-19 vaccination and ultimately achieving the goal of containing COVID-19 pandemic.

Antibody Response of Combination of BNT162b2 and CoronaVac Platforms of COVID-19 Vaccines against Omicron Variant.

By vaccinating SARS-CoV-2 naïve individuals who have already received two doses of COVID-19 vaccines, we aimed to investigate whether a heterologous prime-boost strategy, using vaccines of different platforms as the booster dose, can enhance the immune response against SARS-CoV-2 virus variants. Participants were assigned into four groups, each receiving different combination of vaccinations: two doses of BNT162b2 followed by one dose of BNT162b2 booster (B-B-B); Combination of BNT162b2 (first dose) and CoronaVac (second dose) followed by one dose of BNT162b2 booster (B-C-B); two doses of CoronaVac followed by one dose of CoronaVac booster (C-C-C); two doses of CoronaVac followed by one dose of BNT162b2 booster (C-C-B). The neutralizing antibody in sera against the virus was determined with live virus microneutralization assay (vMN). The B-B-B group and C-C-B group demonstrated significantly higher immunogenicity against SARS-CoV-2 Wild type (WT), Beta variant (BV) and Delta variant (DV). In addition, the B-B-B group and C-C-B group showed reduced but existing protection against Omicron variant (OV). Moreover, A persistent rise in vMN titre against OV was observed 3 days after booster dose. Regarding safety, a heterologous prime-boost vaccine strategy is well tolerated. In this study, it was demonstrated that using vaccines of different platforms as booster dose can enhance protection against SARS-CoV-2 variants, offering potent neutralizing activity against wild-type virus (WT), Beta variant (BV), Delta variant (DV) and some protection against the Omicron variant (OV). In addition, a booster mRNA vaccine results in a more potent immune response than inactivated vaccine regardless of which platform was used for prime doses.

Immunogenicity of a Heterologous Prime-Boost COVID-19 Vaccination with mRNA and Inactivated Virus Vaccines Compared with Homologous Vaccination Strategy against SARS-CoV-2 Variants.

The emergence of SARS-CoV-2 variants may impact the effectiveness of vaccines, while heterologous vaccine strategy is considered to provide better protection. The immunogenicity of an mRNA-inactivated virus vaccine against the SARS-CoV-2 wild-type (WT) and variants was evaluated in the study. SARS-CoV-2 naïve adults (n = 123) were recruited and placed in the following groups: BNT162b2, CoronaVac or BNT162b2-CoronaVac (Combo) Group. Blood samples were collected to measure neutralization antibodies (NAb) by a live virus microneutralization assay (vMN) and surrogate NAb test. The day 56 vMN geometric mean titre (GMT) was 26.2 [95% confident interval (CI), [22.3-30.9] for Combo, 136.9 (95% CI, 104.2-179.7) for BNT162b2, and 14.7 (95% CI, 11.6-18.6) for CoronaVac groups. At 6 months post-first dose, the GMT declined to 8.0, 28.8 and 7.1 in the Combo, BNT162b2 and CoronaVac groups, respectively. Three groups showed reduced neutralizing activity against D614G, beta, theta and delta variants. At day 56 GMT (74.6) and month 6 GMT (22.7), the delta variant in the BNT162b2 group was higher than that in the Combo (day 56, 7.4; month 6, 5.5) and CoronaVac groups (day 56, 8.0; month 6, 5) (p < 0.0001). Furthermore, the mean surrogate NAb value on day 56 in the BNT162b2 group was 594.7 AU/mL and higher than 40.5 AU/mL in Combo and 38.8 AU/mL in CoronaVac groups (p < 0.0001). None of the participants developed severe adverse events, and all other adverse events were self-limiting. The Combo vaccination strategy was safe. The overall vaccine immunogenicity at day 56 and 6 months were comparable to the homologous CoronaVac group but inferior to the homologous BNT162b2 group, against both the WT and all variants. Furthermore, the antibody response of vaccines waned at 6 months and thereby, a third dose of the vaccine is needed for these vaccines.

In-House Immunofluorescence Assay for Detection of SARS-CoV-2 Antigens in Cells from Nasopharyngeal Swabs as a Diagnostic Method for COVID-19.

Immunofluorescence is a traditional diagnostic method for respiratory viruses, allowing rapid, simple and accurate diagnosis, with specific benefits of direct visualization of antigens-of-interest and quality assessment. This study aims to evaluate the potential of indirect immunofluorescence as an in-house diagnostic method for SARS-CoV-2 antigens from nasopharyngeal swabs (NPS). Three primary antibodies raised from mice were used for immunofluorescence staining, including monoclonal antibody against SARS-CoV nucleocapsid protein, and polyclonal antibodies against SARS-CoV-2 nucleocapsid protein and receptor-binding domain of SARS-CoV-2 spike protein. Smears of cells from NPS of 29 COVID-19 patients and 20 non-infected individuals, and cells from viral culture were stained by the three antibodies. Immunofluorescence microscopy was used to identify respiratory epithelial cells with positive signals. Polyclonal antibody against SARS-CoV-2 N protein had the highest sensitivity and specificity among the three antibodies tested, detecting 17 out of 29 RT-PCR-confirmed COVID-19 cases and demonstrating no cross-reactivity with other tested viruses except SARS-CoV. Detection of virus-infected cells targeting SARS-CoV-2 N protein allow identification of infected individuals, although accuracy is limited by sample quality and number of respiratory epithelial cells. The potential of immunofluorescence as a simple diagnostic method was demonstrated, which could be applied by incorporating antibodies targeting SARS-CoV-2 into multiplex immunofluorescence panels used clinically, such as for respiratory viruses, thus allowing additional routine testing for diagnosis and surveillance of SARS-CoV-2 even after the epidemic has ended with low prevalence of COVID-19.

Antibody Response of BNT162b2 and CoronaVac Platforms in Recovered Individuals Previously Infected by COVID-19 against SARS-CoV-2 Wild Type and Delta Variant.

Vaccinating recovered patients previously infected by COVID-19 with mRNA vaccines to boost their immune response against wild-type viruses (WT), we aimed to investigate whether vaccine platform and time of vaccination affect immunogenicity against the SARS-CoV-2 WT and Delta variant (DV). Convalescent patients infected by COVID-19 were recruited and received one booster dose of the BNT162b2 (PC-B) or CoronaVac (PC-C) vaccines, while SARS-CoV-2 naïve subjects received two doses of the BNT162b2 (CN-B) or CoronaVac (CN-C) vaccines. The neutralizing antibody in sera against the WT and DV was determined with live virus neutralization assay (vMN). The vMN geometric mean titre (GMT) against WT in recovered individuals previously infected by COVID-19 reduced significantly from 60.0 (95% confidence interval (CI), 46.5-77.4) to 33.9 (95% CI, 26.3-43.7) at 6 months post recovery. In the PC-B group, the BNT162b2 vaccine enhanced antibody response against WT and DV, with 22.3-fold and 20.4-fold increases, respectively. The PC-C group also showed 1.8-fold and 2.2-fold increases for WT and DV, respectively, after receiving the CoronaVac vaccine. There was a 10.6-fold increase in GMT in the CN-B group and a 1.3-fold increase in the CN-C group against DV after full vaccination. In both the PC-B and PC-C groups, there was no difference between GMT against WT and DV after vaccination. Subjects in the CN-B and CN-C groups showed inferior GMT against DV compared with GMT against WT after vaccination. In this study, one booster shot effectively enhanced the pre-existing neutralizing activity against WT and DV in recovered subjects.

Performance of a Surrogate SARS-CoV-2-Neutralizing Antibody Assay in Natural Infection and Vaccination Samples.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing antibody (NAb) production is a crucial humoral response that can reduce re-infection or breakthrough infection. The conventional test used to measure NAb production capacity levels is the live virus-neutralizing assay. However, this test must be conducted under biosafety level-3 containment. Pseudovirus or surrogate NAb tests, such as angiotensin-converting enzyme 2 inhibition tests, can be performed under level-2 containment. The aim of this study was to evaluate the performance of a surrogate SARS-CoV-2 NAb assay (sNAb) using samples from naturally infected individuals and vaccine recipients in comparison with the live virus microneutralization assay (vMN). Three hundred and eighty serum samples which were collected from 197 patients with COVID-19, 96 vaccine recipients and 84 normal individuals were analyzed. Overall, the sensitivity, specificity, positive predictive value, and negative predictive value of the sNAb (iFlash-2019-NAb assay, Shenzhen, China) were 97.9%, 94.9%, 98.2%, and 93.8%, respectively. Agreement for the assay relative to vMN for naturally infected individuals and vaccine recipients were 98.5% and 93.9%, respectively. A correlation analysis between sNAb and the vMN for both of these groups yielded an R2 value of 0.83. The iFlash RBD NAb assay is found to be sensitive and reliable for neutralizing antibody measurement in patients with the 2019 coronavirus disease and those who have been vaccinated against it.