Linked transmission chains of imported SARS-CoV-2 variant B.1.351 across mainland France, January 2021.

Two cases of confirmed SARS-CoV-2 infection with the B.1.351 variant were reported in France in mid-January, 2020. These cases attended a gathering in Mozambique in mid-December 2020. Investigations led to the identification of five imported cases responsible for 14 transmission chains and a total 36 cases. Epidemiological characteristics seemed comparable to those described before the emergence of the South African variant B.1.351. The lack of tertiary transmission outside of the personal sphere suggests that distancing and barrier measures were effective.

South African (501Y.V2) and the United Kingdom (B.1.1.7) SARS-CoV-2 spike (S) protein variants demonstrate a higher binding affinity to ACE2.

INTRODUCTION: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), affects the lower respiratory tract by binding to angiotensin-converting enzyme 2 (ACE2) via its S-protein. Recent emerging SARS-CoV-2 variants from the United Kingdom (B.1.1.7) and South Africa (501Y.V2) are spreading worldwide at an alarming rate. The new variants have manifested amino acid substitution K417N, E484K and N501Y on the RBD domain that binds to ACE2. As such, these mutations may influence the binding of the S-protein to ACE2 and affect viral entry into the host cell. Methods In this study, we modelled the amino acids substitutions on the S-protein and utilised HADDOCK server to assess the S-protein RBD domain binding with ACE2. Additionally, we calculated the binding affinity of ACE2 to S-protein WT, B.1.1.7 and 501Y.V2 variants using Molecular Mechanics-Generalized Born Surface Area (MM/GBSA). Results We demonstrate that the S-protein of both variants possesses higher binding affinity to ACE2 than WT, with the South African 501Y.V2 is a more infective strain than the B.1.1.7 that originated in the United Kingdom. Conclusion The South African 501Y.V2 variant presents three amino acid substitutions that changed the H-bonding network resulting in a higher affinity to ACE2, indicating that the 501Y.V2 strain is more infective than the B.1.1.7 strain.

Liushen Capsules, a promising clinical candidate for COVID-19, alleviates SARS-CoV-2-induced pulmonary in vivo and inhibits the proliferation of the variant virus strains in vitro.

BACKGROUND: Coronavirus disease 2019 (COVID-19) causes a global pandemic and has devastating effects around the world, however, there are no specific antiviral drugs and vaccines for the constant mutation of SARS-CoV-2. PURPOSE: In this study, we evaluted the antiviral and anti-inflammatory activities of Liushen Capsules (LS) on different novel coronavirus in vitro, studied its therapeutic effects on novel SARS-CoV-2 infected mice and observed the LS's clinical efficacy and safety in COVID-19. METHODS: The antiviral and aiti-inflammatory effects of LS on the 501Y.V2/B.1.35 and G/478K.V1/ B.1.617.2 strains were determined in vitro. A hACE2 mouse model of novel SARS-CoV-2 pneumonia was established. Survival rates, histological changes, inflammatory markers, lung virus titers and the expression of the key proteins in the NF-κB/MAPK signaling pathway was detected by western blotting and immumohistochemical staining in the lungs were measured. Subsequently, the disease duration, prognosis of disease, time of negative nucleic acid and the cytokines levels in serum were used to assess the efficacy of treatment with LS in patients. RESULTS: The results showed that LS (2, 1, 0.5 µg/mL) could significantly inhibit the replication of the two SARS-CoV-2 variants and the expression of pro-inflammatory cytokines (IL-6, IL-8, IP-10, CCL-5, MIP-1α, IL-1α) induced by the virus in vitro. As for the survival experiment in mice, the survival rate of virus group was 20%, while LS-treatment groups (40, 80, 160 mg/kg) could increase the survival rate to 60, 100 and 100%, respectively. LS (40, 80, 160 mg/kg) could significantly decrease the lung titers in mice and it could improve the pathological changes, inhibit the excessive inflammatory mediators (IFN-α, IFN-γ, IP-10, MCP-1) and the protein expression of p-NF-κB p65 in mice. Moreover, LS could significantly decrease SARS-CoV-2-induced activation of p-NF-κB p65, p-IκBα, and p-p38 MAPK and increase the protein expression of the IκBα. In addition, the patient got complete relief of symptoms after being treated with LS for 6 days and was proven with negative PCR test after being treated for 23 days. Finally, treatment with LS could reduce the release of inflammatory cytokines (IL-6, PDGF-AA/BB, Eotaxin, MCP-1, MIP-1α, MIP-1ß, GRO, CCL-5, MCP-3, IP-10, IL-1α). CONCLUSION: LS effectively alleviated novel SARS-CoV-2 or variants induced pneumonia in vitro and in vivo, and improved the prognosis of COVID-19. In light of the efficacy and safety profiles, LS could be considered for the treatment of COVID-19 with a broad-spectrum antiviral and anti-inflammatory agent.

COVID-19 vaccine responsiveness in patients with multiple myeloma and waldenström macroglobulinemia

BACKGROUND: Multiple myeloma (MM) and Waldenström macroglobulinemia (WM) are associated with significant immunoparesis. Based on the ongoing COVID-19 pandemic, there is an urgent need to understand whether patients are able to mount a sufficient response to COVID-19 vaccines. METHODS: MM and WM patients are vaccinated with mRNA-1273 (Moderna), BNT162b2 mRNA (Pfizer/BioNTech), or JNJ-78436735 (Johnson & Johnson) in a prospective clinical trial. Primary endpoint is SARS-CoV-2 spike protein (S) antibody (Ab) detection 28 days after final vaccination. Secondary endpoints include functional serologic assessments and T-cell responses at 28 days, 6 months, 9 months, and 12 months following vaccination. S Abs were detected by Elecsys assay (Roche Diagnostics), with 3 0.80 U/mL defined as positive and titers > 250 U/mL considered stronger correlates of neutralization. SARS-CoV-2 wildtype and variant S-specific Ab isotypes and FcγR binding profiles were quantified by custom Luminex assay. Antibody-dependent neutrophil and cellular phagocytosis (ADNP and ADCP) were assessed using flow cytometry. RESULTS: To date 141 patients have been enrolled, 137 (91 MM and 46 WM) of whom had an S Ab assessment. Median Ab titer was 178.0 (IQR, 16.10-1166.0) for MM and 3.92 (IQR, 0-278.9) for WM. S Ab response rate was 91% (83/91) in MM and 56% (27/46) in WM. However, responses achieving S Ab >250 U/mL were 47.3% (43/91) in MM and 26.1% (12/46) in WM. In patients 375 years, responses >250 u/mL were 13.3% (2/15;p<0.05). Vaccine-specific S Ab responses >250 u/mL following mRNA-1273, BNT162b2, and JNJ-78436735 were 67.6% (23/34;p<0.05), 38.3% (18/47;p=NS), and 20% (2/10;p=NS) in MM and 50.0% (8/16;p<0.05), 14.8% (4/27;p<0.05), and 0% (0/3;p=NS) in WM. Among MM patients with progressive disease, S Ab response >250 u/mL occurred in 30% (6/20) as opposed to 55.6% (30/54) for VGPR+ (p<0.05). MM patients having autologous stem cell transplant within 12 months demonstrated 100% (5/5;p<0.05) S Ab responses. For MM patients actively receiving an anti-CD38 monoclonal Ab or an immunomodulatory drug, S Ab response occurred in 38.9% (14/36;p=NS) and 50.9% (28/55;p<0.05). Among WM patients, S Ab responses >250 U/mL occurred in 63.6% (7/11;p<0.05) previously untreated;0% (0/9;p<0.05) who received rituximab within 12 months;10% (2/20);p<0.05) on an active Bruton Tyrosine Kinase (BTK) inhibitor;and 20% (3/15;p=NS) who received other therapies. Functional Ab studies were performed on 14 MM patients, 14 WM, patients, and 14 healthy donors (HD) (Figure 1). All patients were assessed 28 days following their final vaccination and myeloma patients had an additional assessment 28 days following initial vaccination. MM and WM patients demonstrated less IGG1 and IGG3 S Ab production than HD. MM patients showed increased IgA and IgM S Ab production as well as increased FcgR2A binding following a second vaccine in contrast to HD. Both ADNP and ADCP were reduced in MM and WM patients. MM patients demonstrated improved ADCP in SARS-CoV-2 variants B.1.351, B.1.117, and P.1 versus wildtype (p<0.05). CONCLUSIONS: We report the first known evidence of impaired functional humoral responses following COVID-19 vaccines in patients with MM and WM. Overall, WM patients showed more severe impairment of COVID-19 S Ab responses. Most previously untreated WM patients achieved S Ab responses, however the most significant reduction in S Ab responses were seen in WM patients who received rituximab within 12 months or active BTK inhibitors. For MM patients, being in disease remission associated with improved S Ab response. Among MM and WM patients, age 375 years associated with significantly lower rates and vaccination with MRNA-1273 (Moderna) elicited significantly higher S Ab response rates than other vaccines. A defect in ADNP and more profound defect in ADCP suggests overall compromised opsinophagocytic activity among MM and WM patients. Data comparing first and second vaccine responses in MM patients, suggest less efficient class switching to IGG as well as incomple e maturation of their FcgR2A binding profiles but normal maturation of FcgR3A. Interestingly, ADCP was improved in several emerging SARS-CoV-2 variants. T-cell studies are pending and will be updated. Further understanding of the immunological response to COVID19 vaccination is needed to clarify patients risks, and necessity for booster or alternative protective measures against COVID-19. (Figure Presented).

Antiviral NL-CVX1 Efficiently Blocks Infection of SARS-CoV-2 Viral Variants of Concern (VOC)

Background. Using a computational approach, NL-CVX1 was developed by Neoleukin Therapeutics, Inc. to create a de novo protein that both blocks SARS-CoV-2 infection and is highly resilient to viral escape. In this study we evaluated the efficacy of NL-CVX1 against variants of the original SARS-CoV-2 strain, including important viral variants of concern (VOC) such as B.1.1.7, B.1.351, and P.1. Methods. The relative binding affinity of NL-CVX1 to the SARS-CoV-2 viral spike protein of VOC was measured using biolayer interferometry (Octet). A competitive ELISA measured the ability of NL-CVX1 to compete with hACE2 for binding to the receptor binding domain (RBD) of the SARS-CoV-2 S protein from the original strain and VOC. The activity of NL-CVX1 in preventing viral infection was assessed by evaluating the cytopathic effects (CPE) of SARS-CoV-2 in a transmembrane protease, serine 2-expressing Vero E6 cell line (Vero E6/TMPRSS2) and determining the viral load using quantitative real-time reverse transcriptase polymerase chain reaction in infected cells. A K18hACE2 mouse model of SARS CoV-2 infection was used to study the dose-response of NL-CVX1 anti-viral activity in vivo. Results. NL-CVX1 binds the RBD of different VOC of SARS-CoV-2 at low nanomolar concentrations (Fig 1;Kd < 1-~5 nM). When competing with hACE2, NL-CVX1 achieved 100% inhibition against hACE2 binding to the RBD of different VOC with IC50s values ranging from 0.7-53 nM (Fig 2). NL-CVX1 neutralized the B.1.1.7 variant as efficiently as the original strain in Vero E6/TMPRSS2 cells, with EC50 values of 16 nM and 101. 2 nM, respectively (Fig 3). In mice, we found that a single intranasal dose of 100 μg NL-CVX1 prevented clinically significant SARS-CoV-2 infection and protected mice from succumbing to infection. Results from additional in vitro and in vivo experiments to be conducted this summer will be presented. Figure 1. NL-CVX1 binds the RBD from multiple strains of SARS-CoV-2 at low nanomolar concentrations. Figure 2. NL-CVX1 achieves 100% inhibition against all strains tested, including SARS-CoV-2 VOC. Figure 3. NL-CVX1 neutralizes the B.1.1.7 variant as efficiently as the original SARSCoV-2 strain. Conclusion. In vitro and in vivo data (Fig 4) demonstrate that NL-CVX1 is a promising drug candidate for the prevention and treatment of COVID-19. As a hACE2 mimetic, it is resilient to antibody escape mutations found in SARS-CoV-2 VOC. NL-CVX1 further demonstrates the power and utility of de novo protein design for developing proteins as human therapeutics. Figure 4. NL-CVX1 is effective in preventing clinically significant SARS-CoV-2 viral infection in a K18hACE2 mouse model.

Extraction-free RT-PCR to Detect SARS-CoV-2 Variants of Concern

Background. SARS-CoV-2 variants of concern (VOC) have challenged real-time reverse transcriptase polymerase chain reaction (RT-PCR) methods for the diagnosis of COVID-19. Methods. The CDC 2019-Novel Coronavirus real-time RT-PCR panel was modified to create a single-plex extraction-free proxy RT-PCR assay, VOCFast™. This assay uses the nucleocapsid N1 as well as novel primer/probe pairs to target VOC mutations in the Orf1a and spike (S) genes. For analytical validation of VOCFast, synthetic controls for the Wuhan, alpha/B.1.1.7, beta/B.1.351, and gamma/P.1 strains were tested at various concentrations. Clinical validation was performed using patient anterior nares swab and saliva specimens collected in the Denver, CO area between Nov 2020 and Feb 2021 or in March 2021. Orthogonal next-generation sequencing (NGS) was also performed. Results. Similar N1 quantification cycle (Cq) values corresponding to viral load were observed for all strains, suggesting that VOC mutations do not affect performance of the N1 primer/probe. Orf1a-mut and S1-mut primer/probes generated a stable high Cq value for the Wuhan strain. Conversely, Orf1a-mut Cq values were inversely correlated with viral load for all VOC. The S1-mut Cq was inversely correlated with viral load of the alpha strain, but did not reliably amplify beta/gamma VOC. The limit of detection was 8 copies/uL. The first set of COVID-19 patient specimens revealed no amplification using Orf1amut whereas 53% of specimens collected in Mar 2021 demonstrated amplification by Orf-1a. Orthogonal testing by the SARS-CoV-2 NGS Assay and COVID-DX software demonstrated that 12/12 alpha strains, 2/2 beta/gamma strains, and 33/33 Wuhan strains were correctly identified by VOCFast. Conclusion. The combination of the N1, Orf1a-mut, and S1-mut primers/probes in VOCFast can distinguish the Wuhan, alpha, and beta/gamma strains and it consistent with NGS results. Testing of clinical samples revealed that VOC emerged in Denver, CO in March 2021. Future work to discriminate beta, gamma, and emerging VOC is ongoing. In summary, VOCFast is an extraction-free RT-PCR assay for nasal swab and saliva specimens that can identify VOC with a turnaround time suitable for clinical testing.

Characteristics of COVID-19 Vaccine Breakthrough Cases in Minnesota, 2021

Background. Over 600,000 COVID-19 cases, including >7000 deaths reported to MN Dept of Health (MDH) by June 1, 2021. Clinical trials demonstrated high effectiveness of COVID vaccines. We assessed COVID-19 cases among fully vaccinated residents [vaccine breakthrough (VB) cases]. Methods. COVID-19 VB cases were MN residents with completed COVID-19 vaccination series ≥14 days prior to symptom onset or positive for SARS-CoV-2 by nucleic acid amplification or antigen test. COVID-19 cases were reported to MDH and COVID-19 vaccinations reported to the MN Immunization Information Connection (MIIC). COVID-19 cases were matched to MIIC to identify VB and interviewed;medical records of hospitalized cases were reviewed. Available VB case specimens underwent whole genome sequencing (WGS) at MDH or collaborating lab. Results. Jan 19 - June 1, 2021, 2765 VB cases were reported among >2.45 million fully vaccinated residents and 147,445 COVID-19 cases. VB case median (MED) age was 52 y (IQR 38, 68), 83% white, 65% female;MED age of fully vaccinated was 55 y (IQR 30, 68), 77% white, 54% female. Of VB cases, 273 (10%) were hospitalized and 32 (1%) died (MED age 74 y;IQR 66, 85). 2212 (80%) VB cases were interviewed;60% reported symptoms;most common were fatigue (53%), rhinorrhea (49%), cough (42%), headache (41%). 35% reported a comorbidity. Of hospitalized VB cases, 120 had completed record reviews. 64 were admitted for COVID-19 related illness (MED age 74 y, IQR:65, 83) including 27 admitted to ICU (MED age 71 y, IQR: 65, 83). 90% (108) reported a comorbidity, most common being chronic metabolic conditions (46%), obesity (45%), renal disease (31%) and chronic lung disease (26%);27 were immunocompromised (not mutually exclusive), including immunosuppressive therapy (15), hematological malignancy (9), other cancer (11), and organ transplant recipients (8). Of 604 VB case specimens, 79% were B.1.1.7, 9% B.1.427/429, 3% P.1, and 2% B.1.351;lineage distribution was similar to overall 24,157 MN SARS-CoV2 WGS data. Conclusion. Identified VB cases were 0.1% of those vaccinated and < 2% of total cases reported in the time period. COVID-19 vaccines are an important tool in preventing COVID-19. Additional surveillance, including WGS and case characteristics will be useful to monitor VB.

Pharmacokinetic and Safety Phase 1 Study and Microneutralization Assay Results with BRII-196/BRII-198, a Novel Antibody Cocktail Active Against a Wide Range of SARS-CoV-2 Variants

Background. BRII-196 and BRII-198 are human monoclonal antibodies (mAb) with an extended half-life targeting distinct epitopes of the spike protein on SARSCoV-2. Mutations in these epitope regions are continuously emerging, potentially conferring resistance to COVID-19 therapeutics in development. Individual phase I studies showed that BRII-196 or BRII-198 alone were safe and well tolerated in healthy subjects. The BRII-196 and BRII-198 cocktail is currently under evaluation in Phase 2/3 studies for the treatment of COVID-19. Methods. Preclinical study: BRII-196 and BRII-198 were evaluated in the microneutralization assay using pseudo-viruses encoding mutations identified in the spike protein of a panel of SARS-CoV-2 variants of concerns, including strains originating in UK, SA, BR, CA, and India. The fold-change in neutralization IC50 titers relative to wild-type virus was calculated. Phase 1 study: healthy adults received sequential IV BRII-196 and BRII-198 (n=9) or placebo (n=3);and were followed for 180 days. Two dose levels (750mg/750mg and 1500mg/1500mg) were evaluated for safety, pharmacokinetics and immunogenicity. Interim analysis results are presented. Results. Preclinical: BRII-196 and BRII-198 exhibited neutralizing activity against pseudo-virus variants that contained spike mutations of a panel of variants including B.1.1.7 (UK), B.1.351(SA), P.1(BR), B.1.427/429 (CA), B.1.526 (NY), and B.1.617 (IN), comparable to that against wild-type virus. Phase I study: BRII-196 plus BRII-198 was well tolerated with no dose-limiting adverse events (AEs), deaths, serious adverse events, or infusion reactions. The majority of AEs were isolated asymptomatic grade 1-2 laboratory abnormalities. (Table 1). Each mAb displayed pharmacokinetic characteristics expected of extended half-life YTE-antibodies. Conclusion. The BRII-196 and BRII-198 cocktail was well-tolerated, and maintains neutralization against currently reported circulating variants of concern. These preclinical and clinical results support further development of BRII-196 and BRII-198 as a therapeutic or prophylactic option for SARS-CoV-2.

COVI-VAC™, a Live Attenuated COVID-19 Vaccine, Provides Single Dose Protection Against Heterologous Challenge with SARS-CoV-2 Beta (B.1.351) in the Syrian Golden Hamster Model

Background. Although multiple COVID-19 vaccines are currently in use, emergence of novel SARS-CoV-2 variants with reduced neutralization raises concern of future vaccine escape. COVI-VAC™ is a live attenuated SARS-CoV-2 strain based on WA/1 being developed as an intranasal COVID-19 vaccine. COVI-VAC is attenuated through removal of the furin cleavage site and introduction of 283 silent, deoptimizing mutations that maintain viral amino acid sequence but slow viral replication in vivo by up to 5 logs. Notably, COVI-VAC presents all viral antigens in their native conformation and is not limited to spike. COVI-VAC demonstrated attenuation, immunogenicity and single dose protection in both the Syrian golden hamster and non-human primate models and currently in Phase 1 clinical trials. In this study, we evaluated efficacy of COVI-VAC against challenge with the Beta/B.1.351 variant in Syrian golden hamsters. Methods. Syrian golden hamsters, 7-10 weeks of age were, vaccinated intranasally with 8.25x104 PFU COVI-VAC (n=28) or vehicle control (n=16). Twenty seven days post-vaccination, animals were challenged intranasally with 3x104 PFU of wildtype (WT) SARS-CoV-2 Beta. Animals were weighed daily. Further analysis is being conducted with serum and key tissues from pre and post challenge timepoints to include neutralizing antibody, biodistribution (subgenomic qPCR) and histopathology. Results. COVI-VAC prevented weight loss following challenge with the heterologous variant of SARS-CoV-2, B.1.351/Beta (Figure). Results of additional analyses will be available before the IDWeek meeting. Change in Weight following SARS-CoV-2 Beta Challenge Conclusion. COVI-VAC is protective against heterologous challenge with SARSCoV-2 Beta. By presenting all viral antigens, COVI-VAC may be less affected by viral evolution than spike-based vaccines.

INO-4800 DNA Vaccine Induces Neutralizing Antibodies and T cell Activity Against Global SARS-CoV-2 Variants

Background. Global surveillance has identified emerging SARS-CoV-2 variants of concern (VOC) associated with increased transmissibility, disease severity, and resistance to neutralization by current vaccines under emergency use authorization (EUA). Here we assessed cross-immune responses of INO-4800 vaccinated subjects against SARS-CoV-2 VOCs. Methods. We used a SARS-CoV-2 IgG ELISA and a pseudo neutralization assay to assess humoral responses, and an IFNγ ELISpot to measure cellular responses against SARS-CoV-2 VOC in subjects immunized with the DNA vaccine, INO-4800. Results. IgG binding titers were not impacted between wild-type (WT) and B.1.1.7 or B.1.351 variants. An average 1.9-fold reduction was observed for the P.1 variant in subjects tested at week 8 after receiving two doses of INO-4800 (Figure 1a). We performed a SARS-CoV-2 pseudovirus neutralization assay using sera collected from 13 subjects two weeks after administration of a third dose of either 0.5 mg, 1 mg, or 2 mg of INO-4800. Neutralization was detected against WT and the emerging variants in all samples tested. The mean ID50 titers for the WT, B.1.1.7, B.1.351 and P.1. were 643 (range: 70-729), 295 (range: 46-886), 105 (range: 25-309), and 664 (range: 25-2087), respectively. Compared to WT, there was a 2.1 and 6.9-fold reduction for B.1.1.7 and B.1.351, respectively, while there was no difference between WT and the P.1 variant (Figure 1b). Next, we compared cellular immune responses to WT and SARS-CoV-2 Spike variants elicited by INO-4800 vaccination. We observed similar cellular responses to WT (median = 82.2 IQR = 58.9-205.3), B.1.1.7 (79.4, IQR = 38.9- 179.7), B.1.351 (80, IQR = 40.0-208.6) and P.1 (78.3, IQR = 53.1-177.8) Spike peptides (Figure 2). Conclusion. INO-4800 vaccination induced neutralizing antibodies against all variants tested, with reduced levels detected against B.1.351. IFNγ T cell responses were fully maintained against all variants tested.

Design and Immunogenicity of a Pan-SARS-CoV-2 Synthetic DNA Vaccine

Background. First-generation COVID-19 vaccines are matched to spike protein of the Wuhan-H1 (WT) strain. Convalescent and vaccinee samples show reduced neutralization of SARS-CoV-2 variants of concern (VOC). Next generation DNA vaccines could be matched to single variants or synthetically designed for broader coverage of multiple VOCs. Methods. The synthetic consensus (SynCon®) sequence for INO-4802 SARSCoV-2 spike with focused RBD changes and dual proline mutations was codon-optimized (Figure 1). Sequences for wild-type (pWT) and B.1.351 (pB.1.351) were similarly optimized. Immunogenicity was evaluated in BALB/c mice. Pre-clinical efficacy was assessed in the Syrian Hamster model. Figure 1. Design Strategy for INO-4802 Results. INO-4802 induced potent neutralizing antibody responses against WT, B.1.1.7, P.1, and B.1.351 VOC in a murine model. pWT vaccinated animals showed a 3-fold reduction in mean neutralizing ID50 for the B.1.351 pseudotyped virus. INO-4802 immunized animals had significantly higher (p = 0.0408) neutralizing capacity (mean ID50 816.16). ID50 of pB.1.351 serum was reduced 7-fold for B.1.1.7 and significantly lower (p = 0.0068) than INO-4802 (317.44). INO-4802 neutralized WT (548.28) comparable to pWT. INO-4802 also neutralized P.1 (1026.6) (Figure 2). pWT, pB.1.351 or INO-4802 induced similar T-cell responses against all variants. INO-4802 skewed towards a TH1-response. All hamsters vaccinated with INO-4802 or pB.1.351 were protected from weight loss after B.1.351 live virus challenge. 4/6 pWT immunized hamsters were completely protected. pWT immunized hamsters neutralized WT (1090) but not B.1.351 (39.16). INO-4802 neutralized both WT (672.2) and B.1.351 (1121) (Figure 3). We observed higher increase of binding titers following heterologous boost with INO-4802 (3.6 - 4.4 log2-fold change) than homologous boost with pWT (2.0 - 2.4 log2 fold change) (Figure 4). Conclusion. Vaccines matching single VOCs, like pB.1.351 and pWT, elicit responses against the matched antigen but have reduced cross-reactivity. Presenting a pan-SARS-CoV-2 approach, INO-4802 may offer substantial advantages in terms of cross-strain protection, reduced susceptibility to escape mutants and non-restricted geographical use.

Torus Synestia Nucleic Acid Analysis Platform for Fast, High Multiplex Analysis of Nucleic Acids with Single-Nucleotide Discrimination Level

Background. Nucleic acid amplification testing (NAAT) is an essential tool both for biomedical research and for clinical molecular diagnostics. Currently, there are multiple NAAT platforms available, each offering certain performance and utility advantages and disadvantages as compared to each other. Next generation NAAT platforms aim to deliver increased target detection sensitivity and specificity, low limits of target detection, quantitative high multiplex target capacity, rapid time to results, and simple sample-to-answer workflow. Methods. Here we describe the Torus Synestia System, a NAAT platform capable of rapid, highly multiplexed amplification and detection of both DNA and RNA targets. The platform comprises a small, portable (~ 2kg) amplification and detection device and a disposable single-use cartridge housing a PCR amplification chamber with an integrated label-free microarray for real-time data acquisition and interpretation. The platform offers a 30-min turnaround time with a detection limit of 10 DNA/RNA molecules per assay and single nucleotide discrimination. Results. We demonstrate the Synestia System performance and utility with three distinct molecular applications: 1) detection of 20 genetic loci and 30 single nucleotide polymorphisms in human genomic DNA;2) detection and genotyping of 43 unique bacterial species associated with human urinary tract infections;and 3) detection and profiling human respiratory viral pathogens including SARS-CoV-1/2, seasonal coronaviruses, Influenza A/B, and human respiratory syncytial viruses. In addition, the single-nucleotide specificity of our label-free microarray probes allowed for robust identification and discrimination of newly emerging SARS-CoV-2 lineages, such as B.1.1.7 (a.k.a. UK), B.1.351 (a.k.a. South African), P.1 (a.k.a. Brazilian), and B.1.617 (a.k.a. Indian). Conclusion. The Torus Synestia System has broad applicability in both clinical and research environments. We are confident that the Torus Synestia System will revolutionize syndromic diagnostics at the point of care (PoC) and lead to improved response times during future epidemic and pandemic pathogen outbreaks.

Ad26.COV2.S-Elicted Neutralizing Activities Against SARS-CoV-2 Variants of Concern in Phase 1/2a and Phase 3 Clinical Trials

Background. In a Phase 3 trial, the Janssen COVID-19 vaccine, Ad26.COV2.S, showed robust efficacy against severe-critical COVID-19 in countries where different SARS-CoV-2 variants were circulating. We evaluated Ad26.COV2.S-elicited antibody neutralizing activity against variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), and B.1.617.2 (Delta) in sera from participants in clinical trials following a single dose of Ad26.COV2.S. Methods. Neutralizing activities of Ad26.COV2.S (given at a dose level of 5 x 1010 viral particles [vp]) against VOC were assessed by wild-type virus neutralizing (wtVNA) and pseudovirion neutralization (psVNA) assays in sera from participants in Phase 1/2a and Phase 3 clinical trials, respectively. Geometric mean titers (GMTs) were determined at Days 29 and 71 after vaccination. Results. In serum samples from Phase 1/2a participants (n = 6), at Day 29 after 1 dose of Ad26.COV2.S, wtVNA titers against VOC were lower than for the original strain (GMT = 573), with GMT = 65, 14, and 15 for Alpha, Beta, and Delta, respectively, representing 8.8-, 40.9-, and 37.7-fold decreases. By Day 71 after vaccination (n = 14), fold differences between the original strain (GMT = 375) and VOC (GMT = 113, 27, and 28) were smaller (3.3-, 13.9-, and 13.4-fold) than at Day 29, suggestive of B-cell maturation (Figure 1). Day 71 titers against the Delta variant were maintained for at least 8 months following a single dose of Ad26.COV2.S (5 x 1010 vp). In serum samples from Phase 3 participants (n = 8), psVNA titers against VOC were lower than the original strain at Day 71 after vaccination, with the lowest titers observed for the Beta variant (3.6-fold decrease vs original strain). Smaller reductions in Nab titers for VOC were observed in the psVNA assay compared to wtVNA. Conclusion. Ad26.COV2.S-elicited serum neutralizing activity against VOC showed an overall decrease in titers relative to the original strain that was largest for the Beta variant, even though vaccine efficacy against severe-critical COVID-19 was maintained in countries where these variants were circulating versus in countries where they were not circulating. Over time, titers against variants increased, suggesting B-cell affinity maturation leading to increasing coverage of VOC.

Detection of SARS-CoV-2 variant 501Y.V2 in Comoros Islands in January 2021.

Background. Genomic data is key in understanding the spread and evolution of SARS-CoV-2 pandemic and informing the design and evaluation of interventions. However, SARS-CoV-2 genomic data remains scarce across Africa, with no reports yet from the Indian Ocean islands. Methods. We genome sequenced six SARS-CoV-2 positive samples from the first major infection wave in the Union of Comoros in January 2021 and undertook detailed phylogenetic analysis. Results. All the recovered six genomes classified within the 501Y.V2 variant of concern (also known as lineage B.1.351) and appeared to be from 2 sub-clusters with the most recent common ancestor dated 30 th Oct-2020 (95% Credibility Interval: 06 th Sep-2020 to 10 th Dec-2020). Comparison of the Comoros genomes with those of 501Y.V2 variant of concern from other countries deposited into the GISAID database revealed their close association with viruses identified in France and Mayotte (part of the Comoros archipelago and a France, Overseas Department). Conclusions. The recovered genomes, albeit few, confirmed local transmission following probably multiple introductions of the SARS-CoV-2 501Y.V2 variant of concern during the Comoros's first major COVID-19 wave. These findings demonstrate the importance of genomic surveillance and have implications for ongoing control strategies on the islands.

501Y.V2 spike protein resists the neutralizing antibody in atomistic simulations.

SARS-CoV-2 outbreaks worldwide caused COVID-19 pandemic, which is related to several million deaths. In particular, SARS-CoV-2 Spike (S) protein is a major biological target for COVID-19 vaccine design. Unfortunately, recent reports indicated that Spike (S) protein mutations can lead to antibody resistance. However, understanding the process is limited, especially at the atomic scale. The structural change of S protein and neutralizing antibody fragment (FAb) complexes was thus probed using molecular dynamics (MD) simulations. In particular, the backbone RMSD of the 501Y.V2 complex was significantly larger than that of the wild-type one implying a large structural change of the mutation system. Moreover, the mean of Rg, CCS, and SASA are almost the same when compared two complexes, but the distributions of these values are absolutely different. Furthermore, the free energy landscape of the complexes was significantly changed when the 501Y.V2 variant was induced. The binding pose between S protein and FAb was thus altered. The FAb-binding affinity to S protein was thus reduced due to revealing over steered-MD (SMD) simulations. The observation is in good agreement with the respective experiment that the 501Y.V2 SARS-CoV-2 variant can escape from neutralizing antibody (NAb).

The New Variants of Corona-Virus Mutations: An Understanding to Their Infectivity, Immunological Tolerance and Vaccine Efficacy

The new COVID-19 variants are triggering a fresh panic all around. Scientists still have not found any closely related identification of these variants in the context of their evolution. The scrupulous deletion/mutations recognized inside the spike protein of the variants are emerging with an amplified pace and are observed to be associated with the alterations in the receptor-binding region (RBD) of the spike protein. This paper highlights the reported mechanistic studies conducted on SARS-CoV-2 mutant variants;the mutant virus's ability in response to the antibody recognition to evade the immune system in humans. The role of cellular immunity in response to its interaction with SARS-CoV-2 variants and importantly the discussion on the antibody-dependent enhancement (ADE) of SARS-CoV-2 disease with therapeutic antibodies and vaccines has been highlighted. It is expected that this may likely be interesting and helpful for readers in clearing all their presumptions related to the spreading severity of the mutant virus strains and more importantly the effectiveness of current and upcoming vaccines for its possible control.

Corrigendum: Characterization of SARS-CoV-2 Variants N501Y.V1 and N501Y.V2 Spike on Viral Infectivity.

[This corrects the article DOI: 10.3389/fcimb.2021.720357.].

Characterization of SARS-CoV-2 Variants N501Y.V1 and N501Y.V2 Spike on Viral Infectivity.

SARS-coronavirus 2 (SARS-CoV-2), pathogen of coronavirus disease 2019 (COVID-19), is constantly evolving to adapt to the host and evade antiviral immunity. The newly emerging variants N501Y.V1 (B.1.1.7) and N501Y.V2 (B.1.351), first reported in the United Kingdom and South Africa respectively, raised concerns due to the unusually rapid global spread. The mutations in spike (S) protein may contribute to the rapid spread of these variants. Here, with a vesicular stomatitis virus (VSV)-based pseudotype system, we demonstrated that the pseudovirus bearing N501Y.V2 S protein has higher infection efficiency than pseudovirus with wildtype (WT) and D614G S protein. Moreover, pseudovirus with N501Y.V1 or N501Y.V2 S protein has better thermal stability than WT and D614G, suggesting these mutations of variants may increase the stability of SARS-CoV-2 S protein and virion. However, the pseudovirus bearing N501Y.V1 or N501Y.V2 S protein has similar sensitivity to inhibitors of protease and endocytosis with WT and D614G. These findings could be of value in preventing the spread of virus and developing drugs for emerging SARS-CoV-2 variants.

Hesperidin Is a Potential Inhibitor against SARS-CoV-2 Infection.

Hesperidin (HD) is a common flavanone glycoside isolated from citrus fruits and possesses great potential for cardiovascular protection. Hesperetin (HT) is an aglycone metabolite of HD with high bioavailability. Through the docking simulation, HD and HT have shown their potential to bind to two cellular proteins: transmembrane serine protease 2 (TMPRSS2) and angiotensin-converting enzyme 2 (ACE2), which are required for the cellular entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results further found that HT and HD suppressed the infection of VeroE6 cells using lentiviral-based pseudo-particles with wild types and variants of SARS-CoV-2 with spike (S) proteins, by blocking the interaction between the S protein and cellular receptor ACE2 and reducing ACE2 and TMPRSS2 expression. In summary, hesperidin is a potential TMPRSS2 inhibitor for the reduction of the SARS-CoV-2 infection.

Sensitive Immunodetection of Severe Acute Respiratory Syndrome Coronavirus 2 Variants of Concern 501Y.V2 and 501Y.V1.

Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants may influence the effectiveness of existing laboratory diagnostics. In the current study we determined whether the British (20I/501Y.V1) and South African (20H/501Y.V2) SARS-CoV-2 variants of concern are detected with an in-house S1-based antigen detection assay, analyzing spiked pools of quantitative reverse-transcription polymerase chain reaction-negative nasopharyngeal swab specimens. The assay, combining 4 monoclonal antibodies, allowed sensitive detection of both the wild type and the variants of concern, despite accumulation of several mutations in the variants' S1 region-results suggesting that this combination, targeting distinct epitopes, enables both specificity and the universality.