Interference reduction isothermal nucleic acid amplification strategy for COVID-19 variant detection.

Common reference methods for COVID-19 variant diagnosis include viral sequencing and PCR-based methods. However, sequencing is tedious, expensive, and time-consuming, while PCR-based methods have high risk of insensitive detection in variant-prone regions and are susceptible to potential background signal interference in biological samples. Here, we report a loop-mediated interference reduction isothermal nucleic acid amplification (LM-IR-INA) strategy for highly sensitive single-base mutation detection in viral variants. This strategy exploits the advantages of nicking endonuclease-mediated isothermal amplification, luminescent iridium(III) probes, and time-resolved emission spectroscopy (TRES). Using the LM-IR-INA strategy, we established a luminescence platform for diagnosing COVID-19 D796Y single-base substitution detection with a detection limit of 2.01 × 105 copies/µL in a linear range of 6.01 × 105 to 3.76 × 108 copies/µL and an excellent specificity with a variant/wild-type ratio of significantly less than 0.0625%. The developed TRES-based method was also successfully applied to detect D796Y single-base substitution sequence in complicated biological samples, including throat and blood, and was a superior to steady-state technique. LM-IR-INA was also demonstrated for detecting the single-base substitution D614G as well as the multiple-base mutation H69/V70del without mutual interference, indicating that this approach has the potential to be used as a universal viral variant detection strategy.

Lineage-mosaic and mutation-patched spike proteins for broad-spectrum COVID-19 vaccine.

SARS-CoV-2 spread in humans results in continuous emergence of new variants, highlighting the need for vaccines with broad-spectrum antigenic coverage. Using inter-lineage chimera and mutation-patch strategies, we engineered a recombinant monomeric spike variant (STFK1628x) that contains key regions and residues across multiple SAR-CoV-2 variants. STFK1628x demonstrated high immunogenicity and mutually complementary antigenicity to its prototypic form (STFK). In hamsters, a bivalent vaccine composed of STFK and STFK1628x elicited high titers of broad-spectrum neutralizing antibodies to 19 circulating SARS-CoV-2 variants, including Omicron sublineages BA.1, BA.1.1, BA.2, BA.2.12.1, BA.2.75, and BA.4/5. Furthermore, this vaccine conferred robust protection against intranasal challenges by either SARS-CoV-2 ancestral strain or immune-evasive Beta and Omicron BA.1. Strikingly, vaccination with the bivalent vaccine in hamsters effectively blocked within-cage virus transmission of ancestral SARS-CoV-2, Beta variant, and Omicron BA.1 to unvaccinated sentinels. Thus, our study provided insight and antigen candidates for the development of next-generation COVID-19 vaccines.

Interference reduction isothermal nucleic acid amplification strategy for COVID-19 variant detection

Interference reduction isothermal nucleic acid amplification strategy for COVID-19 variant detection.ga1

High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects.

CRISPR-Cas13 systems have recently been used for targeted RNA degradation in various organisms. However, collateral degradation of bystander RNAs has limited their in vivo applications. Here, we design a dual-fluorescence reporter system for detecting collateral effects and screening Cas13 variants in mammalian cells. Among over 200 engineered variants, several Cas13 variants including Cas13d and Cas13X exhibit efficient on-target activity but markedly reduced collateral activity. Furthermore, transcriptome-wide off-targets and cell growth arrest induced by Cas13 are absent for these variants. High-fidelity Cas13 variants show similar RNA knockdown activity to wild-type Cas13 but no detectable collateral damage in transgenic mice or adeno-associated-virus-mediated somatic cell targeting. Thus, high-fidelity Cas13 variants with minimal collateral effects are now available for targeted degradation of RNAs in basic research and therapeutic applications.

Cross-species tropism and antigenic landscapes of circulating SARS-CoV-2 variants.

Mutations in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (RBD) may alter viral host tropism and affect the activities of neutralizing antibodies. Here, we investigated 153 RBD mutants and 11 globally circulating variants of concern (VOCs) and variants of interest (VOIs) (including Omicron) for their antigenic changes and cross-species tropism in cells expressing 18 ACE2 orthologs. Several RBD mutations strengthened viral infectivity in cells expressing ACE2 orthologs of non-human animals, particularly those less susceptible to the ancestral strain. The mutations surrounding amino acids (aas) 439-448 and aa 484 are more likely to cause neutralization resistance. Strikingly, enhanced cross-species infection potential in the mouse and ferret, instead of the neutralization-escape scores of the mutations, account for the positive correlation with the cumulative prevalence of mutations in humans. These findings present insights for potential drivers of circulating SARS-CoV-2 variants and provide informative parameters for tracking and forecasting spreading mutations.

Cross-species tropism and antigenic landscapes of circulating SARS-CoV-2 variants

Zhang et al. show in vitro cross-species infectivity and neutralization-escape characteristics of 153 SARS-CoV-2 RBD mutants and 11 globally circulating VOC/VOI variants. They reveal an association between enhanced cross-species infection potential and the current cumulative prevalence of mutations, which can inform surveillance and forecasting of SARS-CoV-2 spike mutations.

A recombinant spike protein subunit vaccine confers protective immunity against SARS-CoV-2 infection and transmission in hamsters.

Multiple safe and effective vaccines that elicit immune responses against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are necessary to respond to the ongoing coronavirus disease 2019 (COVID-19) pandemic. Here, we developed a protein subunit vaccine composed of spike ectodomain protein (StriFK) plus a nitrogen bisphosphonate-modified zinc-aluminum hybrid adjuvant (FH002C). StriFK-FH002C generated substantially higher neutralizing antibody titers in mice, hamsters, and cynomolgus monkeys than those observed in plasma isolated from COVID-19 convalescent individuals. StriFK-FH002C also induced both TH1- and TH2-polarized helper T cell responses in mice. In hamsters, StriFK-FH002C immunization protected animals against SARS-CoV-2 challenge, as shown by the absence of virus-induced weight loss, fewer symptoms of disease, and reduced lung pathology. Vaccination of hamsters with StriFK-FH002C also reduced within-cage virus transmission to unvaccinated, cohoused hamsters. In summary, StriFK-FH002C represents an effective, protein subunit-based SARS-CoV-2 vaccine candidate.

Programmable RNA editing with compact CRISPR-Cas13 systems from uncultivated microbes.

Competitive coevolution between microbes and viruses has led to the diversification of CRISPR-Cas defense systems against infectious agents. By analyzing metagenomic terabase datasets, we identified two compact families (775 to 803 amino acids (aa)) of CRISPR-Cas ribonucleases from hypersaline samples, named Cas13X and Cas13Y. We engineered Cas13X.1 (775 aa) for RNA interference experiments in mammalian cell lines. We found Cas13X.1 could tolerate single-nucleotide mismatches in RNA recognition, facilitating prophylactic RNA virus inhibition. Moreover, a minimal RNA base editor, composed of engineered deaminase (385 aa) and truncated Cas13X.1 (445 aa), exhibited robust editing efficiency and high specificity to induce RNA base conversions. Our results suggest that there exist untapped bacterial defense systems in natural microbes that can function efficiently in mammalian cells, and thus potentially are useful for RNA-editing-based research.