Detection of antibodies against SARS-CoV-2 Spike protein by screen-printed carbon electrodes modified by colloidal gold nanoparticles.

In this work, electrochemical bioanalytical systems for the determination of antibodies against the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike protein (anti-rS) is reported. Environmentally friendly chemicals were applied in the synthesis of gold nanoparticles (AuNPs). The AuNPs were integrated onto the screen-printed carbon electrodes (SPE), and the biological recognition part was based on recombinant SARS-CoV-2 Spike protein (rS), which during the immobilization was cross-linked by glutaraldehyde. Immobilized rS protein based biological recognition part enabled selective recognition of anti-rS antibodies. The current flux of AuNPs reduction (at +200 mV) in a pure phosphate buffer (PB) was employed as the transduction signal. It has been reported that the formation of anti-rS layers on the surface of AuNPs delays the electrode response time (ts), tracked at the current flux density values of 80 µA cm-2. Using the AuNP-modified SPE, we demonstrated a rapid anti-rS detection within a detection limit of 2 ng mL-1 (0.125 binding antibody units mL-1, 17 pM). This system can be applied to track the response of immune system towards SARS-CoV-2 infection and monitoring of Coronavirus Disease 2019 (COVID-19).

Comparative analysis of clinical and immunological profiles across Omicron BA.5.2 subvariants using next-generation sequencing in a Chinese cohort.

Objective: The Omicron BA.5.2 variant of SARS-CoV-2 has undergone several evolutionary adaptations, leading to multiple subvariants. Rapid and accurate characterization of these subvariants is essential for effective treatment, particularly in critically ill patients. This study leverages Next-Generation Sequencing (NGS) to elucidate the clinical and immunological features across different Omicron BA.5.2 subvariants. Methods: We enrolled 28 patients infected with the Omicron variant, hospitalized in Zhangjiajie People's Hospital, Hunan, China, between January 20, 2023, and March 31, 2023. Throat swabs were collected upon admission for NGS-based identification of Omicron subvariants. Clinical data, including qSOFA scores and key laboratory tests, were collated. A detailed analysis of lymphocyte subsets was conducted to ascertain the extent of immune cell damage and disease severity. Results: Patients were infected with various Omicron subvariants, including BA.5.2.48, BA.5.2.49, BA.5.2.6, BF.7.14, DY.1, DY.2, DY.3, and DY.4. Despite having 43 identical mutation sites, each subvariant exhibited unique marker mutations. Critically ill patients demonstrated significant depletion in total lymphocyte count, T cells, CD4, CD8, B cells, and NK cells (P < 0.05). However, there were no significant differences in clinical and immunological markers across the subvariants. Conclusion: This study reveals that critically ill patients infected with different Omicron BA.5.2 subvariants experience similar levels of cellular immune dysfunction and inflammatory response. Four mutations - ORF1a:K3353R, ORF1a:L3667F, ORF1b:S997P, S:T883I showed correlation with immunological responses although this conclusion suffers from the small sample size. Our findings underscore the utility of NGS in the comprehensive assessment of infectious diseases, contributing to more effective clinical decision-making.

Autoantibodies against type I IFNs in humans with alternative NF-κB pathway deficiency.

Patients with autoimmune polyendocrinopathy syndrome type 1 (APS-1) caused by autosomal recessive AIRE deficiency produce autoantibodies that neutralize type I interferons (IFNs)1,2, conferring a predisposition to life-threatening COVID-19 pneumonia3. Here we report that patients with autosomal recessive NIK or RELB deficiency, or a specific type of autosomal-dominant NF-κB2 deficiency, also have neutralizing autoantibodies against type I IFNs and are at higher risk of getting life-threatening COVID-19 pneumonia. In patients with autosomal-dominant NF-κB2 deficiency, these autoantibodies are found only in individuals who are heterozygous for variants associated with both transcription (p52 activity) loss of function (LOF) due to impaired p100 processing to generate p52, and regulatory (IκBδ activity) gain of function (GOF) due to the accumulation of unprocessed p100, therefore increasing the inhibitory activity of IκBδ (hereafter, p52LOF/IκBδGOF). By contrast, neutralizing autoantibodies against type I IFNs are not found in individuals who are heterozygous for NFKB2 variants causing haploinsufficiency of p100 and p52 (hereafter, p52LOF/IκBδLOF) or gain-of-function of p52 (hereafter, p52GOF/IκBδLOF). In contrast to patients with APS-1, patients with disorders of NIK, RELB or NF-κB2 have very few tissue-specific autoantibodies. However, their thymuses have an abnormal structure, with few AIRE-expressing medullary thymic epithelial cells. Human inborn errors of the alternative NF-κB pathway impair the development of AIRE-expressing medullary thymic epithelial cells, thereby underlying the production of autoantibodies against type I IFNs and predisposition to viral diseases.

Distinct SARS-CoV-2 specific NLRP3 and IL-1ß responses in T cells of aging patients during acute COVID-19 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19) that presents with varied clinical manifestations ranging from asymptomatic or mild infections and pneumonia to severe cases associated with cytokine storm, acute respiratory distress syndrome (ARDS), and even death. The underlying mechanisms contributing to these differences are unclear, although exacerbated inflammatory sequelae resulting from infection have been implicated. While advanced aging is a known risk factor, the precise immune parameters that determine the outcome of SARS-CoV-2 infection in elderly individuals are not understood. Here, we found aging-associated (age ≥61) intrinsic changes in T cell responses when compared to those from individuals aged ≤ 60, even among COVID-positive patients with mild symptoms. Specifically, when stimulated with SARS-CoV-2 peptides in vitro, peripheral blood mononuclear cell (PBMC) CD4+ and CD8+ T cells from individuals aged ≥61 showed a diminished capacity to produce IFN-γ and IL-1ß. Although they did not have severe disease, aged individuals also showed a higher frequency of PD-1+ cells and significantly diminished IFN-γ/PD-1 ratios among T lymphocytes upon SARS-CoV-2 peptide stimulation. Impaired T cell IL-1ß expression coincided with reduced NLRP3 levels in T lymphocytes. However, the expression of these molecules was not affected in the monocytes of individuals aged ≥61. Together, these data reveal SARS-CoV-2-specific CD4+ and CD8+ T-cell intrinsic cytokine alterations in the individuals older than 61 and may provide new insights into dysregulated COVID-directed immune responses in the elderly.

Viral codon optimization on SARS-CoV-2 Spike boosts immunity in the development of COVID-19 mRNA vaccines.

Life-long persistent herpesviruses carry "trans-inducers" to overcome the unusual codon usage of their glycoproteins for efficient expression. Strikingly, this "trans-inducibility" can be achieved by simply changing the codon-usage of acute virus glycoproteins to that of persistent herpesvirus glycoproteins with herpesviral trans-inducer. Here, we apply the "persistent viral codon-usage-trans-inducer" principle to SARS-CoV-2 Spike mRNA vaccine platform, in which the codon-usage of Spike is changed to that of Herpes Simplex Virus-1 (HSV-1) glycoprotein B (gB) with its "trans-inducer" ICP27. The HSVgB-ICP27-codon-optimized Spike mRNA vaccine induced markedly high antigen expression and stability, total IgG, neutralizing antibody, and T cell response, ultimately enhancing protection against lethal SARS-CoV-2 challenge. Moreover, the HSVgB- codon-optimized Delta (B.1.617.2) strain Spike mRNA vaccine provided significant enhancements in antigen expression and long-term protection against SARS-CoV-2 challenge. Thus, we report a novel persistent viral codon-usage-trans-inducer mRNA vaccine platform for enhanced antigen expression and long-term protection against lethal viral infection.

Possible contribution of rare alleles of human ACE2 in the emergence of SARS-CoV-2 variants escaping the immune response.

Since the start of the SARS-CoV-2 pandemic, the rapid replacement of one lineage by another has been observed. Indeed, SARS-CoV-2 is evolving through a quasispecies mechanism leading to post-infection mutation selection under positive evolutionary pressure (host-driven viral evolution). These mutations may reduce the effectiveness of the specific neutralizing immune response against the virus. We provide here evidence that apart from the selection of SARS-CoV-2 variants by the immune system, selection by the cellular receptor can just as well select variants which escape neutralization.

SARS-COV-2 protein NSP9 promotes cytokine production by targeting TBK1.

SARS-COV-2 infection-induced excessive or uncontrolled cytokine storm may cause injury of host tissue or even death. However, the mechanism by which SARS-COV-2 causes the cytokine storm is unknown. Here, we demonstrated that SARS-COV-2 protein NSP9 promoted cytokine production by interacting with and activating TANK-binding kinase-1 (TBK1). With an rVSV-NSP9 virus infection model, we discovered that an NSP9-induced cytokine storm exacerbated tissue damage and death in mice. Mechanistically, NSP9 promoted the K63-linked ubiquitination and phosphorylation of TBK1, which induced the activation and translocation of IRF3, thereby increasing downstream cytokine production. Moreover, the E3 ubiquitin ligase Midline 1 (MID1) facilitated the K48-linked ubiquitination and degradation of NSP9, whereas virus infection inhibited the interaction between MID1 and NSP9, thereby inhibiting NSP9 degradation. Additionally, we identified Lys59 of NSP9 as a critical ubiquitin site involved in the degradation. These findings elucidate a previously unknown mechanism by which a SARS-COV-2 protein promotes cytokine storm and identifies a novel target for COVID-19 treatment.

Prior SARS-CoV-2 Infection Enhances Initial mRNA Vaccine Response with a Lower Impact on Long-Term Immunity.

Spike-encoding mRNA vaccines in early 2021 effectively reduced SARS-CoV-2-associated morbidity and mortality. New booster regimens were introduced due to successive waves of distinct viral variants. Therefore, people now have a diverse immune memory resulting from multiple SARS-CoV-2 Ag exposures, from infection to following vaccination. This level of community-wide immunity can induce immunological protection from SARS-CoV-2; however, questions about the trajectory of the adaptive immune responses and long-term immunity with respect to priming and repeated Ag exposure remain poorly explored. In this study, we examined the trajectory of adaptive immune responses following three doses of monovalent Pfizer BNT162b2 mRNA vaccination in immunologically naive and SARS-CoV-2 preimmune individuals without the occurrence of breakthrough infection. The IgG, B cell, and T cell Spike-specific responses were assessed in human blood samples collected at six time points between a moment before vaccination and up to 6 mo after the third immunization. Overall, the impact of repeated Spike exposures had a lower improvement on T cell frequency and longevity compared with IgG responses. Natural infection shaped the responses following the initial vaccination by significantly increasing neutralizing Abs and specific CD4+ T cell subsets (circulating T follicular helper, effector memory, and Th1-producing cells), but it had a small benefit at long-term immunity. At the end of the three-dose vaccination regimen, both SARS-CoV-2-naive and preimmune individuals had similar immune memory quality and quantity. This study provides insights into the durability of mRNA vaccine-induced immunological memory and the effects of preimmunity on long-term responses.

Convalescent Plasma for Covid-19-Induced ARDS in Mechanically Ventilated Patients.

BACKGROUND: Passive immunization with plasma collected from convalescent patients has been regularly used to treat coronavirus disease 2019 (Covid-19). Minimal data are available regarding the use of convalescent plasma in patients with Covid-19-induced acute respiratory distress syndrome (ARDS). METHODS: In this open-label trial, we randomly assigned adult patients with Covid-19-induced ARDS who had been receiving invasive mechanical ventilation for less than 5 days in a 1:1 ratio to receive either convalescent plasma with a neutralizing antibody titer of at least 1:320 or standard care alone. Randomization was stratified according to the time from tracheal intubation to inclusion. The primary outcome was death by day 28. RESULTS: A total of 475 patients underwent randomization from September 2020 through March 2022. Overall, 237 patients were assigned to receive convalescent plasma and 238 to receive standard care. Owing to a shortage of convalescent plasma, a neutralizing antibody titer of 1:160 was administered to 17.7% of the patients in the convalescent-plasma group. Glucocorticoids were administered to 466 patients (98.1%). At day 28, mortality was 35.4% in the convalescent-plasma group and 45.0% in the standard-care group (P = 0.03). In a prespecified analysis, this effect was observed mainly in patients who underwent randomization 48 hours or less after the initiation of invasive mechanical ventilation. Serious adverse events did not differ substantially between the two groups. CONCLUSIONS: The administration of plasma collected from convalescent donors with a neutralizing antibody titer of at least 1:160 to patients with Covid-19-induced ARDS within 5 days after the initiation of invasive mechanical ventilation significantly reduced mortality at day 28. This effect was mainly observed in patients who underwent randomization 48 hours or less after ventilation initiation. (Funded by the Belgian Health Care Knowledge Center; ClinicalTrials.gov number, NCT04558476.).

Risk factors and titers of COVID-19 infection in a longitudinal statewide seroepidemiology cohort.

BACKGROUND: Virginia is a large state in the USA, yet it remains unclear what percentage of the population has had natural COVID-19 infection and whether risk factors for infection have changed over time. METHODS: Using a longitudinal cohort, from December 2021-July 2022 we performed follow up serology and a questionnaire on 784 individuals from across Virginia who had previously participated in a statewide COVID-19 seroepidemiology study in 2020. Children were also invited to participate and an additional 62 children also completed the study. Serology was performed using Roche nucleocapsid and spike serological assays. RESULTS: The majority of participants were white (78.6%), over 50 years old (60.9%), and reported having received COVID-19 vaccine (93.4%). 28.6% had evidence of prior COVID-19 infection (nucleocapsid positive). Reweighted by region, age, and sex to match the Virginia census data, the seroprevalence of nucleocapsid antibodies was estimated to be 30.6% (95% CI: 24.7, 36.6). We estimated that 25-53% of COVID-19 infections were asymptomatic. Infection rates were lower in individuals > 60 years old and were higher in Blacks and Hispanics. Infection rates were also higher in those without health insurance, in those with greater numbers of household children, and in those that reported a close contact or having undergone quarantine for COVID-19. Participants from Southwest Virginia had lower seropositivity (16.2%, 95% CI 6.5, 26.0) than other geographic regions. Boosted vaccinees had lower infection rates than non-boosted vaccinees. Frequenting indoor bars was a risk factor for infection, while frequently wearing an N95 mask was protective, though the estimates of association were imprecise. Infection rates were higher in children than adults (56.5% vs. 28.6%). Infection in the parent was a risk factor for child infection. Spike antibody levels declined with time since last vaccination, particularly in those that were vaccinated but not previously infected. Neutralizing antibody positivity was high (97-99%) for wild type, alpha, beta, gamma, delta, and omicron variants. Neutralizing antibody levels were higher in the follow-up survey compared to the first survey in 2020 and among individuals with evidence of natural infection compared to those without. CONCLUSIONS: In this longitudinal statewide cohort we observed a lower-than-expected COVID-19 infection rate as of August 2022. Boosted vaccinees had lower infection rates. Children had higher infection rates and infections tracked within households. Previously identified demographic risk factors for infection tended to persist. Even after the omicron peak, a large number of Virginians remain uninfected with COVID-19, underscoring the need for ongoing vaccination strategies.

SARS-CoV-2 Infection and Development of Islet Autoimmunity in Early Childhood.

Importance: The incidence of diabetes in childhood has increased during the COVID-19 pandemic. Elucidating whether SARS-CoV-2 infection is associated with islet autoimmunity, which precedes type 1 diabetes onset, is relevant to disease etiology and future childhood diabetes trends. Objective: To determine whether there is a temporal relationship between SARS-CoV-2 infection and the development of islet autoimmunity in early childhood. Design, Setting, and Participants: Between February 2018 and March 2021, the Primary Oral Insulin Trial, a European multicenter study, enrolled 1050 infants (517 girls) aged 4 to 7 months with a more than 10% genetically defined risk of type 1 diabetes. Children were followed up through September 2022. Exposure: SARS-CoV-2 infection identified by SARS-CoV-2 antibody development in follow-up visits conducted at 2- to 6-month intervals until age 2 years from April 2018 through June 2022. Main Outcomes and Measures: The development of multiple (≥2) islet autoantibodies in follow-up in consecutive samples or single islet antibodies and type 1 diabetes. Antibody incidence rates and risk of developing islet autoantibodies were analyzed. Results: Consent was obtained for 885 (441 girls) children who were included in follow-up antibody measurements from age 6 months. SARS-CoV-2 antibodies developed in 170 children at a median age of 18 months (range, 6-25 months). Islet autoantibodies developed in 60 children. Six of these children tested positive for islet autoantibodies at the same time as they tested positive for SARS-CoV-2 antibodies and 6 at the visit after having tested positive for SARS-CoV-2 antibodies. The sex-, age-, and country-adjusted hazard ratio for developing islet autoantibodies when the children tested positive for SARS-CoV-2 antibodies was 3.5 (95% CI, 1.6-7.7; P = .002). The incidence rate of islet autoantibodies was 3.5 (95% CI, 2.2-5.1) per 100 person-years in children without SARS-CoV-2 antibodies and 7.8 (95% CI, 5.3-19.0) per 100 person-years in children with SARS-CoV-2 antibodies (P = .02). Islet autoantibody risk in children with SARS-CoV-2 antibodies was associated with younger age (<18 months) of SARS-CoV-2 antibody development (HR, 5.3; 95% CI, 1.5-18.3; P = .009). Conclusion and relevance: In young children with high genetic risk of type 1 diabetes, SARS-CoV-2 infection was temporally associated with the development of islet autoantibodies.

Study of efficacy and antibody duration to fourth-dose booster of Ad5-nCoV or inactivated SARS-CoV-2 vaccine in Chinese adults: a prospective cohort study.

Introduction: China experienced a record surge of coronavirus disease 2019 cases in December 2022, during the pandemic. Methods: We conducted a randomized, parallel-controlled prospective cohort study to evaluate efficacy and antibody duration after a fourth-dose booster with Ad5-nCoV or inactivated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine. Results: A total of 191 participants aged ≥18 years who had completed a three-dose regimen of the inactivated SARS-CoV-2 vaccine 6 months earlier were recruited to receive the intramuscular Ad5-nCoV booster or the inactivated SARS-CoV-2 vaccine. The Ad5-nCoV group had significantly higher antibody levels compared with the inactivated vaccine group at 6 months after the fourth vaccination dose. After the pandemic, the breakthrough infection rate for the Ad5-nCoV and the inactivated vaccine groups was 77.89% and 78.13%, respectively. Survival curve analysis (p = 0.872) and multivariable logistic regression analysis (p = 0.956) showed no statistically significant differences in breakthrough infection between the two groups. Discussion: Compared with a homologous fourth dose, a heterologous fourth dose of Ad5-nCoV elicited a higher immunogenic response in healthy adults who had been immunized with three doses of inactivated vaccine. Nevertheless, the efficacy of the two vaccine types was equivalent after the pandemic.

Acquisition of Humoral Immune Responses in Convalescent Japanese People with SARS-CoV-2 (COVID-19) Infection in 2021.

We investigated humoral immune responses in 222 unvaccinated Japanese people after recovery from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in 2021. Anti-spike-protein IgG antibody levels and neutralizing antibody titers were measured in serum samples obtained within 20-180 days after diagnosis. The geometric mean of antibody titers was 1555 ELU/mL (95% confidence interval (CI) = 1257-1923), and the neutralizing activity (50% inhibitory dilution) was 253 (95% CI = 204-313). The antibody titer and neutralizing activity both increased with increasing disease severity, and both values were approximately fourfold higher for hospitalized patients than for non-hospitalized patients. However, these differences were smaller in older patients. The humoral immune response, which increased with increasing disease severity, gradually decreased over time after SARS-CoV-2 infection. Most patients with mild or moderate symptoms sustained neutralizing activity for up to 180 days after the infection; the decay of the neutralizing activity in the asymptomatic patients was rather faster than in the other groups. Around 11.7% (26/222) of patients had very low neutralizing activity, and half of these were aged in their 20s. Our study's results show the importance of measuring the neutralizing activity to confirm the immune status and to estimate the timing of vaccines.