A modified high-resolution melting-based assay (HRM) to identify the SARS-CoV-2 N501Y variant.

High-resolution melting (HRM) analysis is a PCR-based method that can be used as a screening assay to identify SARS-CoV-2 variants. However, conventional HRM assays hardly detect slight melting temperature differences at the A-T to T-A transversion. As the N501Y substitution results from A-T to T-A transversion in A23063, few or no studies have shown that a conventional HRM assay can identify N501Y variants. This study successfully developed an HRM assay for identifying the N501Y mutation. Two HRM assays were used in the N501 site because the discrimination results were affected by the virus copy numbers. One is a conventional HRM assay (detectable at 103-106 copies/mL) and the other is a modified HRM assay by adding the wild-type fragment (detectable at 105-1010 copies/mL). Using viral RNAs from cultured variants (Alpha, Beta, and Gamma), a modified HRM assay correctly identified three N501Y variants because of high-copy-number RNAs in those viral samples. The sensitivity and specificity of the N501Y assay were 93.3% and 100%, respectively, based on 209 clinical samples (105 for N501; 104 for N501Y). These results suggest that our HRM-based assay is a powerful tool for rapidly identifying various SARS-CoV-2 variants.

Rapid Identification of SARS-CoV-2 Omicron BA.5 Spike Mutation F486V in Clinical Specimens Using a High-Resolution Melting-Based Assay.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant BA.5 emerged as of February 2022 and replaced the earlier Omicron subvariants BA.1 and BA.2. COVID-19 genomic surveillance should be continued as new variants seem to subsequently appear, including post-BA.5 subvariants. A rapid assay is needed to differentiate between the currently dominant BA.5 variant and other variants. This study successfully developed a high-resolution melting (HRM)-based assay for BA.4/5-characteristic spike mutation F486V detection and demonstrated that our assay could discriminate between BA.1, BA.2, and BA.5 subvariants in clinical specimens. The mutational spectra at two regions (G446/L452 and F486) for the variant-selective HRM analysis was the focus of our assay. The mutational spectra used as the basis to identify each Omicron subvariant were as follows: BA.1 (G446S/L452/F486), BA.2 (G446/L452/F486), and BA.4/5 (G446/L452R/F486V). Upon mutation-coding RNA fragment analysis, the wild-type fragments melting curves were distinct from those of the mutant fragments. Based on the analysis of 120 clinical samples (40 each of subvariants BA.1, BA.2, and BA.5), this method's sensitivity and specificity were determined to be more than 95% and 100%, respectively. These results clearly demonstrate that this HRM-based assay is a simple screening method for monitoring Omicron subvariant evolution.

Current Vaccine Platforms in Enhancing T-Cell Response.

The induction of T cell-mediated immunity is crucial in vaccine development. The most effective vaccine is likely to employ both cellular and humoral immune responses. The efficacy of a vaccine depends on T cells activated by antigen-presenting cells. T cells also play a critical role in the duration and cross-reactivity of vaccines. Moreover, pre-existing T-cell immunity is associated with a decreased severity of infectious diseases. Many technical and delivery platforms have been designed to induce T cell-mediated vaccine immunity. The immunogenicity of vaccines is enhanced by controlling the kinetics and targeted delivery. Viral vectors are attractive tools that enable the intracellular expression of foreign antigens and induce robust immunity. However, it is necessary to select an appropriate viral vector considering the existing anti-vector immunity that impairs vaccine efficacy. mRNA vaccines have the advantage of rapid and low-cost manufacturing and have been approved for clinical use as COVID-19 vaccines for the first time. mRNA modification and nanomaterial encapsulation can help address mRNA instability and translation efficacy. This review summarizes the T cell responses of vaccines against various infectious diseases based on vaccine technologies and delivery platforms and discusses the future directions of these cutting-edge platforms.

Discrimination of SARS-CoV-2 Omicron Sublineages BA.1 and BA.2 Using a High-Resolution Melting-Based Assay: a Pilot Study.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. As of March 2022, Omicron variant BA.2 is rapidly replacing variant BA.1. As variant BA.2 may cause more severe disease than variant BA.1, variant BA.2 requires continuous monitoring. The current study aimed to develop a novel high-resolution melting (HRM) assay for variants BA.1 and BA.2 and to determine the sensitivity and specificity of our method using clinical samples. Here, we focused on the mutational spectra at three regions in the spike receptor-binding domain (RBD; R408, G446/L452, and S477/T478) for the variant-selective HRM analysis. Each variant was identified based on the mutational spectra as follows: no mutations (Alpha variant); L452R and T478K (Delta variant); G446S and S477N/T478K (Omicron variant BA.1); and R408S and S477N/T478K (Omicron variant BA.2). Upon analysis of mutation-coding RNA fragments, the melting curves of the wild-type fragments were distinct from those of the mutant fragments. The sensitivity and specificity of this method were determined as 100% and more than 97.5%, respectively, based on 128 clinical samples (40 Alpha, 40 Delta, 40 Omicron variant BA.1/BA.1.1, and 8 Omicron variant BA.2). These results suggest that this HRM-based assay is a promising screening method for monitoring the transmission of Omicron variants BA.1 and BA.2. IMPORTANCE This study seeks to apply a novel high-resolution melting (HRM) assay to identify and discriminate BA.1 and BA.2 sublineages of the SARS-CoV-2 Omicron variant. Variant BA.2 may cause more severe disease than variant BA.1, meaning that identifying this variant is an important step toward improving the care of patients suffering from COVID-19. However, screening for these variants remains difficult, as current methods mostly rely on next-generation sequencing, which is significantly costlier and more time-consuming than other methods. We believe that our study makes a significant contribution to the literature because we show that this method was 100% sensitive and over 97.5% specific in our confirmation of 128 clinical samples.

New vaccine production platforms used in developing SARS-CoV-2 vaccine candidates.

The threat of the current coronavirus disease pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is accelerating the development of potential vaccines. Candidate vaccines have been generated using existing technologies that have been applied for developing vaccines against other infectious diseases. Two new types of platforms, mRNA- and viral vector-based vaccines, have been gaining attention owing to the rapid advancement in their methodologies. In clinical trials, setting appropriate immunological endpoints plays a key role in evaluating the efficacy and safety of candidate vaccines. Updated information about immunological features from individuals who have or have not been exposed to SARS-CoV-2 continues to guide effective vaccine development strategies. This review highlights key strategies for generating candidate SARS-CoV-2 vaccines and considerations for vaccine development and clinical trials.