Somatically hypermutated antibodies isolated from SARS-CoV-2 Delta infected patients cross-neutralize heterologous variants.

SARS-CoV-2 Omicron variants feature highly mutated spike proteins with extraordinary abilities in evading antibodies isolated earlier in the pandemic. Investigation of memory B cells from patients primarily with breakthrough infections with the Delta variant enables isolation of a number of neutralizing antibodies cross-reactive to heterologous variants of concern (VOCs) including Omicron variants (BA.1-BA.4). Structural studies identify altered complementarity determining region (CDR) amino acids and highly unusual heavy chain CDR2 insertions respectively in two representative cross-neutralizing antibodies-YB9-258 and YB13-292. These features are putatively introduced by somatic hypermutation and they are heavily involved in epitope recognition to broaden neutralization breadth. Previously, insertions/deletions were rarely reported for antiviral antibodies except for those induced by HIV-1 chronic infections. These data provide molecular mechanisms for cross-neutralization of heterologous SARS-CoV-2 variants by antibodies isolated from Delta variant infected patients with implications for future vaccination strategy.

Immune response and severity of Omicron BA.5 reinfection among individuals previously infected with different SARS-CoV-2 variants.

Introduction: COVID-19 continues to spread worldwide, with an increasing number of individuals experiencing reinfection after recovering from their primary infection. However, the nature and progression of this infection remain poorly understood. We aimed to investigate the immune response, severity and outcomes of Omicron BA.5 reinfection among individuals previously infected with different SARS-CoV-2 variants. Methods: We enrolled 432 COVID-19 cases who had experienced prior infection with the ancestral SARS-CoV-2 virus, Delta variant or Omicron BA.2 variant between January 2020 and May 2022 in Guangzhou, China. All cases underwent follow-up from March to April, 2023 through telephone questionnaires and clinical visits. Nasal lavage fluid and peripheral blood were collected to assess anti-RBD IgA, anti-RBD IgG and virus-specific IFN-γ secreting T cells. Results: Our study shows that 73.1%, 56.7% and 12.5% of individuals with a prior infection of the ancestral virus, Delta or Omicron BA.2 variant experienced reinfection with the BA.5 variant, respectively. Fever, cough and sore throat were the most common symptoms of BA.5 reinfection, with most improving within one week and none progressing to a critical condition. Compared with individuals without reinfection, reinfected patients with a prior Delta infection exhibited elevated levels of nasal anti-RBD IgA, serum anti-RBD IgG and IFN-γ secreting T cells, whereas there was no noticeable change in reinfected individuals with a prior BA.2 infection. Conclusion: These results suggest that BA.5 reinfection is common but severe outcomes are relatively rare. Reinfection with a novel SARS-CoV-2 variant different from the prior infection may induce a more robust immune protection, which should be taken into account during vaccine development.

Extensive neutralization against SARS-CoV-2 variants elicited by Omicron-specific subunit vaccine as a heterologous booster.

To overcome the increased risk of SARS-CoV-2 reinfection or post-vaccination infection caused by the Omicron variant, Omicron-specific vaccines were considered a potential strategy. We reported the increased magnitude and breadth of antibody response against VOCs elicited by post-vaccination Delta and Omicron infection, compared to WT infection without vaccination. Then, in mouse models, three doses of Omicron-RBD immunization elicited comparable neutralizing antibody (NAb) titers with three doses of WT-RBD immunization, but the neutralizing activity was not cross-active. By contrast, a heterologous Omicron-RBD booster following two doses of WT-RBD immunization increased the NAb titers against Omicron by 9-folds than the homologous WT-RBD booster. Moreover, it retains neutralization against both WT and current VOCs. Results suggest that Omicron-specific subunit booster shows its advantages in the immune protection from both WT and current VOCs and that SARS-CoV-2 vaccines including two or more virus lineages might improve the NAb response.

Clinical characteristics and immune profile alterations in vaccinated individuals with breakthrough Delta SARS-CoV-2 infections.

Despite timely immunization programs, and efficacious vaccines conveying protection against SARS-CoV-2 infection, breakthrough infections in vaccinated individuals have been reported. The Delta variant of concern (VOC) outbreak in Guangzhou resulted in local transmission in vaccinated and non-vaccinated residents, providing a unique opportunity to study the protective effects of the inactivated vaccines in breakthrough infection. Here, we find that the 2-dose vaccinated group has similar peak viral titers and comparable speeds of viral RNA clearance to the non-vaccinated group but accelerated viral suppression in the middle course of the disease. We quantitatively demonstrate that peak viral pneumonia is significantly mitigated in the 2-dose vaccine group (median 0.298%) compared with the non-vaccinated (5.77%) and 1-dose vaccine (3.34%) groups. Pneumonia absorbance is approximately 6 days ahead in the 2-dose group (median 10 days) than in the non-vaccinated group (16 days) (p = 0.003). We also observe reduced cytokine inflammation and markedly undisturbed gene transcription profiles of peripheral blood mononuclear cells (PBMCs) in the 2-dose group. In short, our study demonstrates that prior vaccination substantially restrains pneumonia development, reduces cytokine storms, and facilitates clinical recovery.

Correction: Incidence, clinical course and risk factor for recurrent PCR positivity in discharged COVID-19 patients in Guangzhou, China: A prospective cohort study.

[This corrects the article DOI: 10.1371/journal.pntd.0008648.].

Smoking Status Modifies the Relationship between Th2 Biomarkers and Small Airway Obstruction in Asthma.

Background: Cigarette smoking and Th2-inflammation are both crucial in the pathogenesis of asthma. However, it is unknown whether smoking can affect the association between Th2-inflammation and small airway obstruction in adults with asthma. Methods: Adults diagnosed with asthma by a pulmonologist according to Global Initiative for Asthma guidelines were recruited from September 2016 to April 2018 to participate in this study. Participants were divided into two groups, the small airway obstruction group (those with FEF25-75% predicted value ≤ 65%) and the normal small airway function group (those with FEF25-75% predicted value > 65%). Final data analysis included 385 and 93 people in the Obstructive Group and the Normal Group, respectively. Total serum IgE level and blood eosinophil count were used as biomarkers of the Th2 phenotype. Results: The Obstructive Group had a larger fraction of smokers, higher blood eosinophil count, and lower lung function than the Normal Group. Current-smoking status was associated with an increased risk of small airway obstruction (adjusted odds ratio = 4.677, 95% confidence interval [1.593-13.730]); and log-IgE level was associated with a decreased risk of small airway obstruction (0.403 [0.216-0.754]). Smoking status stratified analysis showed an association between log-IgE level and a decreased risk of small airway obstruction only in never-smoker asthmatics (0.487 [0.249-0.954]). Conclusions: Current-smoking status and total serum IgE are, respectively, associated with small airway obstruction. Smoking status modifies the relationship between Th2 biomarkers and small airway function. These findings contribute to the understanding of risk factors associated with asthma endotyping.

Protective humoral and cellular immune responses to SARS-CoV-2 persist up to 1 year after recovery.

SARS-CoV-2 vaccination has been launched worldwide to build effective population-level immunity to curb the spread of this virus. The effectiveness and duration of protective immunity is a critical factor for public health. Here, we report the kinetics of the SARS-CoV-2 specific immune response in 204 individuals up to 1-year after recovery from COVID-19. RBD-IgG and full-length spike-IgG concentrations and serum neutralizing capacity decreases during the first 6-months, but is maintained stably up to 1-year after hospital discharge. Even individuals who had generated high IgG levels during early convalescent stages had IgG levels that had decreased to a similar level one year later. Notably, the RBD-IgG level positively correlates with serum neutralizing capacity, suggesting the representative role of RBD-IgG in predicting serum protection. Moreover, viral-specific cellular immune protection, including spike and nucleoprotein specific, persisted between 6 months and 12 months. Altogether, our study supports the persistence of viral-specific protective immunity over 1 year.

Osthole Attenuates Macrophage Activation in Experimental Asthma by Inhibitingthe NF-ĸB/MIF Signaling Pathway.

Inhibition of activated macrophages is an alternative therapeutic strategy for asthma. We investigated whether a coumarin compound, osthole, isolated from Cnidium monnieri (L.) Cuss, alleviated macrophage activation in vivo and in vitro. Osthole could reduce expression of a marker of activated macrophages, cluster of differentiation (CD)206, in an ovalbumin-challenge model of asthma in mice. Osthole could also inhibit infiltration of inflammatory cells, collagen deposition and production of proinflammatory cytokines [interleukin (IL)-1ß, tumor necrosis factor-ɑ, macrophage migration inhibitory factor (MIF)] in asthmatic mice. In vitro, expression of phosphorylated-IĸBɑ, MIF and M2 cytokines (Ym-1, Fizz-1, arginase-1) in IL-4-induced macrophages decreased upon exposure to the NF-ĸB inhibitor MG-132. In our short hairpin (sh)RNA-MIF-knockdown model, reduced expression of M2 cytokines was detected in the IL-4 + shRNA-MIF group. Osthole could attenuate the proliferation and migration of an IL-4-induced rat alveolar macrophages line (NR8383). Osthole could reduce IL-4-induced translocation of nuclear factor-kappa B (NF-ĸB) in NR8383 cells. Collectively, our results suggest that osthole ameliorates macrophage activation in asthma by suppressing the NF-ĸB/MIF signaling pathway, and might be a potential agent for treating asthma.

The Role of Macrophage Migration Inhibitory Factor (MIF) in Asthmatic Airway Remodeling.

PURPOSE: Recent studies have demonstrated that macrophage migration inhibitory factor (MIF) is of importance in asthmatic inflammation. The role of MIF in modulating airway remodeling has not yet been thoroughly elucidated to date. In the present study, we hypothesized that MIF promoted airway remodeling by intensifying airway smooth muscle cell (ASMC) autophagy and explored the specific mechanisms. METHODS: MIF knockdown in the lung tissues of C57BL/6 mice was conducted by instilling intratracheally adeno-associated virus (AAV) vectors (MIF-mutant AAV9) into mouse lung tissues. Mice genetically deficient in the autophagy marker ATG5 (ATG5+/-) was used to detect the role of autophagy in ovalbumin (OVA)-asthmatic murine models. Moreover, to block the expression of MIF and CD74 in vitro models, inhibitors, antibodies and lentivirus transfection techniques were employed. RESULTS: First, MIF knockdown in the lung tissues of mice showed markedly reduced airway remodeling in OVA murine mice models. Secondly, ASMC autophagy was increased in the OVA-challenged models. Mice genetically deficient in the autophagy marker ATG5 (ATG5+/-) that were primed and challenged with OVA showed lower airway remodeling than genetically wild-type asthmatic mice. Thirdly, MIF can induce ASMC autophagy in vitro. Moreover, the cellular source of MIF which promoted ASMC autophagy was macrophages. Finally, MIF promoted ASMC autophagy in a CD74-dependent manner. CONCLUSIONS: MIF can increase asthmatic airway remodeling by enhancing ASMC autophagy. Macrophage-derived MIF can promote ASMC autophagy by targeting CD74.

Incidence, clinical course and risk factor for recurrent PCR positivity in discharged COVID-19 patients in Guangzhou, China: A prospective cohort study.

The phenomenon of COVID-19 patients tested positive for SARS-CoV-2 after discharge (redetectable as positive, RP) emerged globally. The data of incidence rate and risk factors for RP event and the clinical features of RP patients may provide recommendations for virus containment and cases management for COVID-19. We prospectively collected and analyzed the epidemiological, clinical and virological data from 285 adult patients with COVID-19 and acquired their definite clinical outcome (getting PCR positive or not during post-discharge surveillance). By March 10, 27 (9.5%) discharged patients had tested positive for SARS-CoV-2 in their nasopharyngeal swab after a median duration of 7·0 days (IQR 5·0-8·0). Compared to first admission, RP patients generally had milder clinical symptoms, lower viral load, shorter length of stay and improved pulmonary conditions at readmission (p<0.05). Elder RP patients (≥ 60 years old) were more likely to be symptomatic compared to younger patients (7/8, 87.5% vs. 3/19, 18.8%, p = 0.001) at readmission. Age, sex, epidemiological history, clinical symptoms and underlying diseases were similar between RP and non-RP patients (p>0.05). A prolonged duration of viral shedding (>10 days) during the first hospitalization [adjusted odds ratio [aOR]: 5.82, 95% confidence interval [CI]: 2.50-13.57 for N gene; aOR: 9.64, 95% CI: 3.91-23.73 for ORF gene] and higher Ct value (ORF) in the third week of the first hospitalization (aOR: 0.69; 95% CI: 0.50-0.95) were associated with RP events. In conclusion, RP events occurred in nearly 10% of COVID-19 patients shortly after the negative tests, were not associated with worsening symptoms and unlikely reflect reinfection. Patients' lack of efficiency in virus clearance was a risk factor for RP result. It is noteworthy that elder RP patients (≥ 60 years old) were more susceptible to clinical symptoms at readmission.

Digestive symptoms of COVID-19 and expression of ACE2 in digestive tract organs.

SARS-CoV-2 has resulted in numerous cases of Coronavirus Disease 2019 (COVID-19) worldwide. In addition to fever and respiratory symptoms, digestive symptoms also are observed in some patients with COVID-19. Angiotensin-converting enzyme 2 (ACE2) was reported to be the receptor for SARS-CoV-2. The aim of this study was to comprehensively investigate the digestive symptoms that occur in COVID-19 patients, and the potential pathogenic route of the SARS-CoV-2 infection in digestive tract organs (from the oral cavity to the gastrointestinal tract). We investigated the digestive symptoms of 48 patients with COVID-19 and explored ACE2 expression in digestive tract and lung cancers, based on a series of bulk and single-cell RNA sequencing data obtained from public databases. We found that 25% (12/48) of the patients with COVID-19 suffered from digestive symptoms, among which pharyngalgia (7/48) was the most common manifestation, followed by diarrhea (3/48), anorexia (3/48), and nausea (1/48). The bulk tissue RNA sequencing analysis indicated that digestive tract organs had higher ACE2 expression levels compared to the lung, and the expression of ACE2 in the lung increased with age. Single-cell RNA-Seq results showed that the ACE2-positive-cell ratio in digestive tract organs was significantly higher compared to the lung. ACE2 expression was higher in tumor cells compared to normal control (NC) tissues. While in gastric tissues, ACE2 expression gradually increased from chronic gastritis to metaplasia, to early cancer. Our data might provide a theoretical basis for screening the SARS-CoV-2 susceptible population and for the clinical classification of treatment of patients with COVID-19.

Detectable 2019-nCoV viral RNA in blood is a strong indicator for the further clinical severity.

The novel coronavirus (2019-nCoV) infection caused pneumonia. we retrospectively analyzed the virus presence in the pharyngeal swab, blood, and the anal swab detected by real-time PCR in the clinical lab. Unexpectedly, the 2109-nCoV RNA was readily detected in the blood (6 of 57 patients) and the anal swabs (11 of 28 patients). Importantly, all of the 6 patients with detectable viral RNA in the blood cohort progressed to severe symptom stage, indicating a strong correlation of serum viral RNA with the disease severity (p-value = 0.0001). Meanwhile, 8 of the 11 patients with annal swab virus-positive was in severe clinical stage. However, the concentration of viral RNA in the anal swab (Ct value = 24 + 39) was higher than in the blood (Ct value = 34 + 39) from patient 2, suggesting that the virus might replicate in the digestive tract. Altogether, our results confirmed the presence of virus RNA in extra-pulmonary sites.

patD, a Gene Regulated by NtcA, Is Involved in the Optimization of Heterocyst Frequency in the Cyanobacterium Anabaena sp. Strain PCC 7120.

In the filamentous multicellular cyanobacterium Anabaena sp. strain PCC 7120, 5 to 10% of the cells differentiate into heterocysts, which are specialized in N2 fixation. Heterocysts and vegetative cells are mutually dependent for filament growth through nutrient exchange. Thus, the heterocyst frequency should be optimized to maintain the cellular carbon and nitrogen (C/N) balance for filament fitness in the environment. Here, we report the identification of patD, whose expression is directly activated in developing cells by the transcription factor NtcA. The inactivation of patD increases heterocyst frequency and promotes the upregulation of the positive regulator of heterocyst development hetR, whereas its overexpression decreases the heterocyst frequency. The change in heterocyst frequency resulting from the inactivation of patD leads to the reduction in competitiveness of the filaments under combined-nitrogen-depleted conditions. These results indicate that patD regulates heterocyst frequency in Anabaena sp. PCC 7120, ensuring its optimal filament growth.IMPORTANCE Microorganisms have evolved various strategies in order to adapt to the environment and compete with other organisms. Heterocyst differentiation is a prokaryotic model for studying complex cellular regulation. The NtcA-regulated gene patD controls the ratio of heterocysts relative to vegetative cells on the filaments of Anabaena sp. strain PCC 7120. Such a regulation provides a mechanism through which carbon fixation by vegetative cells and nitrogen fixation by heterocysts are properly balanced to ensure optimal growth and keep a competitive edge for long-term survival.

Implementation and preliminary evaluation of 'C-tone': A novel algorithm to improve lexical tone recognition in Mandarin-speaking cochlear implant users.

OBJECTIVES: Because of limited spectral resolution, Mandarin-speaking cochlear implant (CI) users have difficulty perceiving fundamental frequency (F0) cues that are important to lexical tone recognition. To improve Mandarin tone recognition in CI users, we implemented and evaluated a novel real-time algorithm (C-tone) to enhance the amplitude contour, which is strongly correlated with the F0 contour. METHODS: The C-tone algorithm was implemented in clinical processors and evaluated in eight users of the Nurotron NSP-60 CI system. Subjects were given 2 weeks of experience with C-tone. Recognition of Chinese tones, monosyllables, and disyllables in quiet was measured with and without the C-tone algorithm. Subjective quality ratings were also obtained for C-tone. RESULTS: After 2 weeks of experience with C-tone, there were small but significant improvements in recognition of lexical tones, monosyllables, and disyllables (P < 0.05 in all cases). Among lexical tones, the largest improvements were observed for Tone 3 (falling-rising) and the smallest for Tone 4 (falling). Improvements with C-tone were greater for disyllables than for monosyllables. Subjective quality ratings showed no strong preference for or against C-tone, except for perception of own voice, where C-tone was preferred. DISCUSSION: The real-time C-tone algorithm provided small but significant improvements for speech performance in quiet with no change in sound quality. Pre-processing algorithms to reduce noise and better real-time F0 extraction would improve the benefits of C-tone in complex listening environments. CONCLUSIONS: Chinese CI users' speech recognition in quiet can be significantly improved by modifying the amplitude contour to better resemble the F0 contour.

Utility of multiple-locus variant-repeat analysis method for the outbreak of the Pseudomonas aeruginosa isolates.

BACKGROUND: Pseudomonas aeruginosa (P. aeruginosa) predominated in hospitals. METHODS: In order to determine the source of the outbreak and take effective measures to prevent the spread, we tested their relationships between the strains. 97 P. aeruginosa samples were analyzed by multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) method. In order to identify a minimal subset that could provide high discrimination, we evaluated the ability of various VNTR sets. RESULTS: The result showed all of the 11 loci displayed high discrimination, and the lowest loci was ms223 (h = 0.59). The 97 strains were all discriminated (HGDI = 1.0000). The top 7-locus set produced a HGDI value of 1.0000, which was the same as the 11-locus set. The 4-locus set had a HGDI value of 0.9972 with a clustering rate of 11.3%. The strains were divided into four groups based on the phylogenetic clustering and genotypic characteristics. There were obvious differences among the four groups regarding the drug-resistance patterns of Imipenem, Ciprofloxacin, Ceftazidime, Levofloxacin, Meropenem, Piperacillin, Cefepime, Aztreonam (p < 0.05). CONCLUSIONS: In conclusion, the transmission of the strains was not found in this study. The 7-locus set yielded a high discrimination, while for an easier and more robust MLVA scheme, the number of markers can be reduced to 4 loci of ms212, ms211, ms213, and ms142. These four strains from four inpatients in the same ward displayed the same drug resistance spectrum. The MLVA genotype results showed the four strains had the same gene structures. The four patients were from the same treatment group. They showed the IMP-1 allele and belonged to the aac (6')-I type, and there was a deletion of the OprD2 gene in four strains, supporting the MLVA results in suggesting that they are similar.

[Isolation of chlamydophila psittaci from avian samples and establishment of a respiratory infection murine model].

OBJECTIVE: To optimize the isolation and culture technique of Chlamydophila psittaci avian strains and to establish an animal model infected with C. psittaci. METHODS: C. psittaci ompA gene was amplified from DNA extracted from bird livers by polymerase chain reactions (PCR). For the PCR positive avian samples, the liver tissues were homogenized and used to incubated with HeLa or Vero cell monolayers for 72 h in different dilutions, and chlamydia inclusion bodies were detected by immunofluorescence or Giemsa staining. Different dose of the avian strains (2 x 10(4), 2 x 10(5), 2 x 10(6) IFUs) were used to attack C57BL/6 mice by intranasal injection,mice were sacrificed on day 5 or day 10 after infection, and the histopathology changes were analyzed by H&E and immunohistochemistry staining in different organs. RESULTS: Six of one hundred avian samples were positive by C. psittaci ompA gene amplification,and three were positive by cell culture. The C. psittaci avian strains were cultured in Vero or HeLa cells. Vero cells showed stronger tolerance of cytolysis after chlamydia infection and chlamydia inclusion bodies were larger and more dense. Successfully establish a murine model of intranasal infection with C. psittaci, and 2 x 10(5) IFU is the suitable amount of organisms to induce respiratory chlamydia infection. CONCLUSION: The isolation and culture condition was optimized for C. psittaci avian strains, and a murine model of respiratory tract infection by C. psittaci was successfully established, which can be applied to the clincal diagnosis of C. psittaci and epidemiological or pathogenetic study.