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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).
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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.