Clinical characteristics and host immunity responses of SARS-CoV-2 Omicron variant BA.2 with deletion of ORF7a, ORF7b and ORF8.

BACKGROUND: The pathogenicity and virulence of the Omicron strain have weakened significantly pathogenesis of Omicron variants. Accumulating data indicated accessory proteins play crucial roles in host immune evasion and virus pathogenesis of SARS-CoV-2. Therefore, the impact of simultaneous deletion of accessory protein ORF7a, ORF7b and ORF8 on the clinical characteristics and specific immunity in Omicron breakthrough infected patients (BIPs) need to be verified. METHODS: Herein, plasma cytokines were identified using a commercial Multi-cytokine detection kit. Enzyme-linked immunosorbent assay and pseudovirus neutralization assays were utilized to determine the titers of SARS-CoV-2 specific binding antibodies and neutralizing antibodies, respectively. In addition, an enzyme-linked immunospot assay was used to quantify SARS-CoV-2 specific T cells and memory B cells. RESULTS: A local COVID-19 outbreak was caused by the Omicron BA.2 variant, which featured a deletion of 871 base pairs (∆871 BA.2), resulting in the removal of ORF7a, ORF7b, and ORF8. We found that hospitalized patients with ∆871 BA.2 had significantly shorter hospital stays than those with wild-type (WT) BA.2. Plasma cytokine levels in both ∆871 BA.2 and WT BA.2 patients were within the normal range of reference, and there was no notable difference in the titers of SARS-CoV-2 ancestor or Omicron-specific binding IgG antibodies, neutralizing antibody titers, effector T cells, and memory B cells frequencies between ∆871 BA.2 and WT BA.2 infected adult patients. However, antibody titers in ∆871 BA.2 infected adolescents were higher than in adults. CONCLUSIONS: The simultaneous deletion of ORF7a, ORF7b, and ORF8 facilitates the rapid clearance of the BA.2 variant, without impacting cytokine levels or affecting SARS-CoV-2 specific humoral and cellular immunity in Omicron-infected individuals.

Emerging triple-reassortant influenza C virus with household-associated infection during an influenza A(H3N2) outbreak, China, 2022.

Recent research have shown that influenza C virus (ICV) has a possible higher clinical impact than previously thought. But knowledge about ICV is limited compared with influenza A and B viruses, due to poor systematic surveillance and inability to propagate. Herein, a case infected with triple reassortant ICV was identified during an influenza A(H3N2) outbreak, which was the first report of ICV infection in mainland China. Phylogenetic analysis showed that this ICV was triple reassortant. Serological evidence revealed that the index case might be related to family-clustering infection. Therefore, it is essential to heighten surveillance for the prevalence and variation of ICV in China, during the COVID-19 pandemic.

Assessment of antibody responses against SARS-CoV-2 in unvaccinated individuals and vaccinees from Omicron-BA.2 infection in Zhaoqing, Guangdong Province, China.

Currently, the majority of the global population has been vaccinated with the COVID-19 vaccine, and characterization studies of antibodies in vivo from Omicron breakthrough infection and naive infection populations are urgently needed to provide pivotal clues about accurate diagnosis, treatment, and next-generation vaccine design against SARS-CoV-2 infection. We showed that after infection with Omicron-BA.2, the antibody levels of specific IgM against the Wuhan strain and specific IgG against Omicron were not significantly elevated within 27 days of onset. Interestingly, in this study, the levels of humoral immunity against Omicron-specific IgM were significantly increased after breakthrough infection, suggesting that the detection of Omicron-specific IgM antibodies can be used as a test criterion of Omicron breakthrough infection. In addition, we observed that serums from unvaccinated individuals and the majority of vaccinated infections possessed only low or no neutralizing activity against Omicron at the onset of Omicron breakthrough infections, and at the later stage of Omicron-BA.2 breakthrough infection, levels of neutralization antibody against the Wuhan and Omicron strains were elevated in infected individuals. The findings of this study provide important clues for the diagnosis of Omicron breakthrough infections, antibody characterization studies and vaccine design against COVID-19.

Serosurvey in SARS-CoV-2 inactivated vaccine-elicited neutralizing antibodies against authentic SARS-CoV-2 and its viral variants.

Various variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been emerging and circulating in different parts of the world. Millions of vaccine doses have been administered globally, which reduces the morbidity and mortality of coronavirus disease-2019 efficiently. Here, we assess the immune responses of individuals after two shots of BBIBP-CorV or CoronaVac inactivated vaccine. We measured neutralizing antibody responses after the second vaccination by using authentic SARS-CoV-2 and its viral variants. All the serum samples efficiently neutralized SARS-CoV-2 wild-type lineage, in contrast, a part of serum samples failed to neutralize Alpha, Beta, Gamma, Delta, or Eta lineages, and only several serum samples were able to neutralize Omicron lineage virus strains (BA.1 and BA.2) with low neutralization titer. As compared with the neutralization of SARS-CoV-2 wild-type lineage, the neutralization of all other SARS-CoV-2 variant lineages was significantly lower. Considering that all the SARS-CoV-2 mutation viruses challenged the antibody neutralization induced by BBIBP-CorV and CoronaVac, it is necessary to carry out a third booster vaccination to increase the humoral immune response against the SARS-CoV-2 mutation viruses.

Combination of Isothermal Recombinase-Aided Amplification and CRISPR-Cas12a-Mediated Assay for Rapid Detection of Major Severe Acute Respiratory Syndrome Coronavirus 2 Variants of Concern.

Coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 variants is a new and unsolved threat; therefore, it is an urgent and unmet need to develop a simple and rapid method for detecting and tracking SARS-CoV-2 variants. The spike gene of SARS-CoV-2 was amplified by isothermal recombinase-aided amplification (RAA) followed by the cleavage of CRISPR-Cas12a in which five allele-specific crRNAs and two Omicron-specific crRNAs were designed to detect and distinguish major SARS-CoV-2 variants of concerns (VOCs), including alpha, beta, delta variants, and Omicron sublineages BA.1 and BA.2. The whole reaction can be carried out in one tube at 39°C within 1.5-2 h, and the results can be read out by a fluorescence meter or naked eyes. Our results show that the RAA/CRISPR-Cas12a-based assay could readily distinguish the signature mutations, i.e., K417N, T478K, E484K, N501Y, and D614G, with a sensitivity of 100.0% and a specificity of 94.9-100.0%, respectively. The assay had a low limit of detection (LOD) of 104 copies/reaction and a concordance of 92.59% with Sanger sequencing results when detecting 54 SARS-CoV-2 positive clinical samples. The two Omicron-specific crRNAs can readily and correctly distinguish Omicron BA.1 and BA.2 sublineages with a LOD of as low as 20 copies/reaction. Furthermore, no cross-reaction was observed for all crRNAs analyzed when detecting clinical samples infected with 11 common respiratory pathogens. The combination of isothermal amplification and CRISPR-Cas12a-mediated assay is suitable for rapid detection of major SARS-CoV-2 variants in point-of-care testing and in resource-limiting settings. This simple assay could be quickly updated for emerging variants and implemented to routinely monitor and track the spread of SARS-CoV-2 variants.

Humoral immune response to authentic circulating severe acute respiratory syndrome coronavirus 2 variants elicited by booster vaccination with distinct receptor-binding domain subunits in mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants could induce immune escape by mutations of the spike protein which are threatening to weaken vaccine efficacy. A booster vaccination is expected to increase the humoral immune response against SARS-CoV-2 variants in the population. We showed that immunization with two doses of wild type receptor-binding domain (RBD) protein, and booster vaccination with wild type or variant RBD protein all significantly increased binding and neutralizing antibody titers against wild type SARS-CoV-2 and its variants in mice. Only the booster immunization by Omicron (BA.1)RBD induced a strong antibody titer against the omicron virus strain and comparable antibody titers against all the other virus strains. These findings might shed the light on coronavirus disease 2019 booster immunogens.

Immunogenicity and safety of an inactivated SARS-CoV-2 vaccine in people living with HIV: A cross-sectional study.

We aimed to analyze the efficacy and safety of an inactivated SARS-CoV-2 vaccine in people living with HIV (PLWH). A total of 143 PLWH and 50 healthy individuals were included in this study. A commercially available magnetic chemiluminescence enzyme immunoassay kit was used to detect serum IgG and IgM antibodies against SARS-CoV-2. Serum levels of SARS-CoV-2-specific IgG were significantly higher in the control group than in the PLWH group (p = 0.001). Overall, 76% of individuals in the control group were detected with seropositivity IgG against SARS-CoV-2 compared to 58% in the PLWH group (p = 0.024). In PLWH with IgG seropositivity, CD4+ T-cell counts before antiretroviral therapy (ART) was higher (p = 0.015). Multivariable analysis indicated that CD4+ T cells at IgG detection (odds ratio [OR] = 1.004, p = 0.006) and time after vaccination (OR = 0.977, p = 0.014) were independently associated with seropositivity IgG against SARS-CoV-2 in PLWH. Neutralizing antibody (nAb) titers in PLWH against wild-type SARS-CoV-2 were similar to those in the control group (p = 0.160). The proportion of seropositive nAbs against wild-type SARS-CoV-2 was also similar (95% in the control group vs. 97% in the PLWH group, p = 0.665). Similar results were obtained when nAb was detected against the delta variants with similar titers (p = 0.355) and a similar proportion of seropositive nAbs were observed (p = 0.588). All the side effects observed in our study were mild and self-limiting. The inactivated COVID-19 vaccine appears to be safe with good immunogenicity in Chinese PLWH.

Detection of Major SARS-CoV-2 Variants of Concern in Clinical Samples via CRISPR-Cas12a-Mediated Mutation-Specific Assay.

Objectives: Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants pose a great threat and burden to global public health. Here, we evaluated a clustered regularly interspaced short palindromic repeat-associated enzyme 12a (CRISPR-Cas12a)-based method for detecting major SARS-CoV-2 variants of concern (VOCs) in SARS-CoV-2 positive clinical samples. Methods: Allele-specific CRISPR RNAs (crRNAs) targeting the signature mutations in the spike protein of SARS-CoV-2 are designed. A total of 59 SARS-CoV-2 positive oropharyngeal swab specimens were used to evaluate the performance of the CRISPR-Cas12a-mediated assay to identify major SARS-CoV-2 VOCs. Results: Compared with Sanger sequencing, the eight allele-specific crRNAs analyzed can specifically identify the corresponding mutations with a positive predictive value of 83.3-100% and a negative predictive value of 85.7-100%. Our CRISPR-Cas12a-mediated assay distinguished wild-type and four major VOCs (Alpha, Beta, Delta, and Omicron) of SARS-CoV-2 with a sensitivity of 93.8-100.0% and a specificity of 100.0%. The two methods showed a concordance of 98.3% (58/59) with a κ value of 0.956-1.000, while seven (11.9%) samples were found to be positive for extra mutations by the CRISPR-based assay. Furthermore, neither virus titers nor the sequences adjacent to the signature mutations were associated with the variation of fluorescence intensity detected or the false-positive reaction observed when testing clinical samples. In addition, there was no cross-reaction observed when detecting 33 SARS-CoV-2 negative clinical samples infected with common respiratory pathogens. Conclusions: The CRISPR-Cas12a-based genotyping assay is highly sensitive and specific when detecting both the SARS-CoV-2 wild-type strain and major VOCs. It is a simple and rapid assay that can monitor and track the circulating SARS-CoV-2 variants and the dynamics of the coronavirus disease 2019 (COVID-19) pandemic and can be easily implemented in resource-limited settings.

Viral infection and transmission in a large, well-traced outbreak caused by the SARS-CoV-2 Delta variant.

The SARS-CoV-2 Delta variant has spread rapidly worldwide. To provide data on its virological profile, we here report the first local transmission of Delta in mainland China. All 167 infections could be traced back to the first index case. Daily sequential PCR testing of quarantined individuals indicated that the viral loads of Delta infections, when they first become PCR-positive, were on average ~1000 times greater compared to lineage A/B infections during the first epidemic wave in China in early 2020, suggesting potentially faster viral replication and greater infectiousness of Delta during early infection. The estimated transmission bottleneck size of the Delta variant was generally narrow, with 1-3 virions in 29 donor-recipient transmission pairs. However, the transmission of minor iSNVs resulted in at least 3 of the 34 substitutions that were identified in the outbreak, highlighting the contribution of intra-host variants to population-level viral diversity during rapid spread.

CRISPR-Cas12a-Based Detection for the Major SARS-CoV-2 Variants of Concern.

A big challenge for the control of COVID-19 pandemic is the emergence of variants of concern (VOCs) or variants of interest (VOIs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may be more transmissible and/or more virulent and could escape immunity obtained through infection or vaccination. A simple and rapid test for SARS-CoV-2 variants is an unmet need and is of great public health importance. In this study, we designed and analytically validated a CRISPR-Cas12a system for direct detection of SARS-CoV-2 VOCs. We further evaluated the combination of ordinary reverse transcription-PCR (RT-PCR) and CRISPR-Cas12a to improve the detection sensitivity and developed a universal system by introducing a protospacer adjacent motif (PAM) near the target mutation sites through PCR primer design to detect mutations without PAM. Our results indicated that the CRISPR-Cas12a assay could readily detect the signature spike protein mutations (K417N/T, L452R/Q, T478K, E484K/Q, N501Y, and D614G) to distinguish alpha, beta, gamma, delta, kappa, lambda, and epsilon variants of SARS-CoV-2. In addition, the open reading frame 8 (ORF8) mutations (T/C substitution at nt28144 and the corresponding change of amino acid L/S) could differentiate L and S lineages of SARS-CoV-2. The low limit of detection could reach 10 copies/reaction. Our assay successfully distinguished 4 SARS-CoV-2 strains of wild type and alpha (B.1.1.7), beta (B.1.351), and delta (B.1.617.2) variants. By testing 32 SARS-CoV-2-positive clinical samples infected with the wild type (n = 5) and alpha (n = 11), beta (n = 8), and delta variants (n = 8), the concordance between our assay and sequencing was 100%. The CRISPR-based approach is rapid and robust and can be adapted for screening the emerging mutations and immediately implemented in laboratories already performing nucleic acid amplification tests or in resource-limited settings. IMPORTANCE We described CRISPR-Cas12-based multiplex allele-specific assay for rapid SARS-CoV-2 variant genotyping. The new system has the potential to be quickly developed, continuously updated, and easily implemented for screening of SARS-CoV-2 variants in resource-limited settings. This approach can be adapted for emerging mutations and implemented in laboratories already conducting SARS-CoV-2 nucleic acid amplification tests using existing resources and extracted nucleic acid.

Seropositive Reaction Rates of 9 B-Cell Epitopes of the SARS-CoV-2 Spike Protein and the Relationship between the Epitopes and Neutralizing Antibody.

OBJECTIVE: The aim of the study was to analyze the relationship between serum antibody and neutralizing antibody titers in convalescent coronavirus disease 2019 (COVID-19) patients with different disease severities, and the seropositive reaction rates of 9 reported B-cell epitopes of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: Serum IgG and total antibody titers of 165 convalescent COVID-19 patients were determined by chemiluminescence, the serum neutralization antibody titers were determined by microneutralization assay, and the S/CO values of 9 peptides were detected by indirect enzyme-linked immunosorbent assay. Correlations between the aforementioned indexes were statistically analyzed, and differences in patients with different diseases severities were evaluated. RESULTS: IgG, total antibody, and neutralizing antibody titers increased with disease severity. The positive rate of the receptor-binding region (RBD) was 100%, and the average positive rate for all the 9 peptides was above 50% in 165 patients. IDf showed the highest rate of positivity (86.06%), with a rate of 95% for the (IDf + IDa) pattern. Moreover, S/CO values of RBD and mix (IDh) were significantly correlated with IgG, total antibody titers, and neutralizing antibody titers (p < 0.001), whereas the S/CO values for other 8 peptides showed no obvious correlation. CONCLUSION: In this study, a large sample was used to confirm that the peptide IDf had a high positive reaction rate for all patients (86.06%) and also had the highest detection rate in asymptomatic patients (86.67%). Only long peptide and mixed peptide showed correlation with neutralizing antibody titers, suggesting that the ability of SARS-CoV-2 antibody to neutralize virus infectivity may require the interaction of multiple sites.

A newly identified linear epitope on non-RBD region of SARS-CoV-2 spike protein improves the serological detection rate of COVID-19 patients.

BACKGROUND: Serological test is helpful in confirming and tracking infectious diseases in large population with the advantage of fast and convenience. Using the specific epitope peptides identified from the whole antigen as the detection antigen is sensitive and relatively economical. The development of epitope peptide-based detection kits for COVID-19 patients requires comprehensive information about epitope peptides. But the data on B cell epitope of SARS-CoV-2 spike protein is still limited. More importantly, there is a lack of serological data on the peptides in the population. In this study, we aimed to identify the B cell epitope peptides of spike protein and detect the reactivity in serum samples, for further providing data support for their subsequent serological applications. RESULTS: Two B cell linear epitopes, P104 and P82, located in non-RBD region of SARS-CoV-2 S protein were identified by indirect ELISA screening of an overlapping peptide library of the S protein with COVID-19 patients' convalescent serum. And the peptides were verified by testing with 165 serum samples. P104 has not been reported previously; P82 is contained in peptide S21P2 reported before. The positive reaction rates of epitope peptides S14P5 and S21P2, the two non-RBD region epitopes identified by Poh et al., and P82 and P104 were 77.0%, 73.9%, 61.2% and 30.3%, respectively, for 165 convalescent sera, including 30 asymptomatic patients. Although P104 had the lowest positive rate for total patients (30.3%), it exhibited slight advantage for detection of asymptomatic infections (36.7%). Combination of epitopes significantly improved the positive reaction rate. Among all combination patterns, (S14P5 + S21P2 + P104) pattern exhibited the highest positive reaction rate for all patients (92.7%), as well as for asymptomatic infections (86.7%), confirming the feasibility of P104 as supplementary antigen for serological detection. In addition, we analyzed the correlation between epitopes with neutralizing antibody, but only S14P5 had a medium positive correlation with neutralizing antibody titre (rs = 0.510, P < 0.01). CONCLUSION: Our research proved that epitopes on non-RBD region are of value in serological detection especially when combination more than one epitope, thus providing serological reaction information about the four epitopes, which has valuable references for their usage.

Genomic Evolution and Variation of SARS-CoV-2 in the Early Phase of COVID-19 Pandemic in Guangdong Province, China.

Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) with unknown origin spread rapidly to 222 countries, areas or territories. To investigate the genomic evolution and variation in the early phase of COVID-19 pandemic in Guangdong, 60 specimens of SARS-CoV-2 were used to perform whole genome sequencing, and genomics, amino acid variation and Spike protein structure modeling analyses. Phylogenetic analysis suggested that the early variation in the SARS-CoV-2 genome was still intra-species, with no evolution to other coronaviruses. There were one to seven nucleotide variations (SNVs) in each genome and all SNVs were distributed in various fragments of the genome. The Spike protein bound with human receptor, an amino acid salt bridge and a potential furin cleavage site were found in the SARS-CoV-2 using molecular modeling. Our study clarified the characteristics of SARS-CoV-2 genomic evolution, variation and Spike protein structure in the early phase of local cases in Guangdong, which provided reference for generating prevention and control strategies and tracing the source of new outbreaks.