The recently emerged SARS-CoV-2 Omicron variant encodes 37 amino acid substitutions in the spike protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the ...effectiveness of available vaccines and antibody-based therapeutics. Here we show that the Omicron RBD binds to human ACE2 with enhanced affinity, relative to the Wuhan-Hu-1 RBD, and binds to mouse ACE2. Marked reductions in neutralizing activity were observed against Omicron compared to the ancestral pseudovirus in plasma from convalescent individuals and from individuals who had been vaccinated against SARS-CoV-2, but this loss was less pronounced after a third dose of vaccine. Most monoclonal antibodies that are directed against the receptor-binding motif lost in vitro neutralizing activity against Omicron, with only 3 out of 29 monoclonal antibodies retaining unaltered potency, including the ACE2-mimicking S2K146 antibody
. Furthermore, a fraction of broadly neutralizing sarbecovirus monoclonal antibodies neutralized Omicron through recognition of antigenic sites outside the receptor-binding motif, including sotrovimab
, S2X259
and S2H97
. The magnitude of Omicron-mediated immune evasion marks a major antigenic shift in SARS-CoV-2. Broadly neutralizing monoclonal antibodies that recognize RBD epitopes that are conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.
Abstract To achieve active tumor targeting and sequential release of 3 drugs to a tumor site in one nanoparticulate system, self-decomposable SiO2 nanoparticles modified by ...3-aminopropyltriethoxysilane (APTS) as their inner structure were used to double load HCPT (in the NP core) and Dox (on the NP surface). Meanwhile, monoclonal antibodies (mAb198.3) against the FAT1 antigen and Bcl-2 siRNA were conjugated onto PEGylated Au-PEG-COOH nanoparticles. The obtained drug-loaded SiO2 nanoparticles were coated with the Au-PEG-mAb.198.3/siRNA nanoparticles through electrostatic interaction to form the SiO2 @AuNP sequential drug delivery system, which featured the controlled and sequential release of siRNA, Dox and HCPT step by step to maximize its anticancer efficacy. The results revealed that the SiO2 @AuNP sequential drug delivery system specifically targeted tumor cells and was internalize rapidly, followed by endosome escape and sequential drug release. Importantly, the sustainable release characteristics of SiO2 made the Tmax difference between HCPT and Dox approximately 8–12 h, and this enhanced the sensitizing efficiency of HCPT on Dox compared with co-administration. The in vivo antitumor results demonstrated that the tumor size after SiO2 @AuNP treatment is 1/400 compared with the saline control group and approximately 1/40 of the HCPT/Dox co-treatment group without any noticeable systemic toxicity.
The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to almost seven million deaths worldwide. SARS-CoV-2 causes infection through respiratory ...transmission and can occur either without any symptoms or with clinical manifestations which can be mild, severe or, in some cases, even fatal. Innate immunity provides the initial defense against the virus by sensing pathogen-associated molecular patterns and triggering signaling pathways that activate the antiviral and inflammatory responses, which limit viral replication and help the identification and removal of infected cells. However, temporally dysregulated and excessive activation of the innate immune response is deleterious for the host and associates with severe COVID-19. In addition to its defensive role, innate immunity is pivotal in priming the adaptive immune response and polarizing its effector function. This capacity is relevant in the context of both SARS-CoV-2 natural infection and COVID-19 vaccination. Here, we provide an overview of the current knowledge of the innate immune responses to SARS-CoV-2 infection and vaccination.
Coronavirus disease 2019 (COVID-19) is an infectious disease caused by beta-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has rapidly spread across the globe starting ...from February 2020. It is well established that during viral infection, extracellular vesicles become delivery/presenting vectors of viral material. However, studies regarding extracellular vesicle function in COVID-19 pathology are still scanty. Here, we performed a comparative study on exosomes recovered from the plasma of either MILD or SEVERE COVID-19 patients. We show that although both types of vesicles efficiently display SARS-CoV-2 spike-derived peptides and carry immunomodulatory molecules, only those of MILD patients are capable of efficiently regulating antigen-specific CD4
T-cell responses. Accordingly, by mass spectrometry, we show that the proteome of exosomes of MILD patients correlates with a proper functioning of the immune system, while that of SEVERE patients is associated with increased and chronic inflammation. Overall, we show that exosomes recovered from the plasma of COVID-19 patients possess SARS-CoV-2-derived protein material, have an active role in enhancing the immune response, and possess a cargo that reflects the pathological state of patients in the acute phase of the disease.
Identifying defined T cell clones within a polyclonal population is key to clarifying their phenotype and function. Here, we present a protocol for detecting specified T cell clones in a ...heterogeneous cell population. We describe steps for stimulating human CD4+ T cells isolated from blood with a protein antigen, sorting antigen-specific cells by fluorescence-activated cell sorting, and detecting among these the presence of predefined T cell clones, based on their T cell receptor (TCR). TCR cDNA is amplified through 5′-RACE (TCR-SMART) and detected by qPCR.
For complete details on the use and execution of this protocol, please refer to Notarbartolo et al. (2021).1
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•In vitro stimulation of human CD4+ T cells with a protein antigen•Antigen-specific human CD4+ T cells identification and isolation by FACS•Unbiased targeted amplification of TCR cDNA from low input samples•Detection of defined T cell clones within a heterogeneous population by qPCR
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
Identifying defined T cell clones within a polyclonal population is key to clarifying their phenotype and function. Here, we present a protocol for detecting specified T cell clones in a heterogeneous cell population. We describe steps for stimulating human CD4+ T cells isolated from blood with a protein antigen, sorting antigen-specific cells by fluorescence-activated cell sorting, and detecting among these the presence of predefined T cell clones, based on their T cell receptor (TCR). TCR cDNA is amplified through 5′-RACE (TCR-SMART) and detected by qPCR.
Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection leads to a wide range of clinical manifestations and determines the need for personalized and precision medicine. To better ...understand the biological determinants of this heterogeneity, we explored the plasma proteome of 43 COVID-19 patients with different outcomes by an untargeted liquid chromatography-mass spectrometry approach. The comparison between asymptomatic or pauci-symptomatic subjects (MILDs), and hospitalised patients in need of oxygen support therapy (SEVEREs) highlighted 29 proteins emerged as differentially expressed: 12 overexpressed in MILDs and 17 in SEVEREs. Moreover, a supervised analysis based on a decision-tree recognised three proteins (Fetuin-A, Ig lambda-2chain-C-region, Vitronectin) that are able to robustly discriminate between the two classes independently from the infection stage. In silico functional annotation of the 29 deregulated proteins pinpointed several functions possibly related to the severity; no pathway was associated exclusively to MILDs, while several only to SEVEREs, and some associated to both MILDs and SEVEREs; SARS-CoV-2 signalling pathway was significantly enriched by proteins up-expressed in SEVEREs (
,
,
,
) and in MILDs (
,
). In conclusion, our analysis could provide key information for 'proteomically' defining possible upstream mechanisms and mediators triggering or limiting the domino effect of the immune-related response and characterizing severe exacerbations.
We have characterized group A Streptococcus (GAS) genome-wide responses to hydrogen peroxide and assessed the role of the peroxide response regulator (PerR) in GAS under oxidative stress. Comparison ...of transcriptome changes elicited by peroxide in wild-type bacteria with those in a perR deletion mutant showed that 76 out of 237 peroxide-regulated genes are PerR dependent. Unlike the PerR-mediated upregulation of peroxidases and other peroxide stress defense mechanisms previously reported in Gram-positive species, PerR-dependent genes in GAS were almost exclusively downregulated and encoded proteins involved in purine and deoxyribonucleotide biosynthesis, heme uptake, and amino acid/peptide transport, but they also included a strongly activated putative transcriptional regulator (SPy1198). Of the 161 PerR-independent loci, repressed genes (86 of 161) encoded proteins with functions similar to those coordinated by PerR, in contrast to upregulated loci that encoded proteins that function in DNA damage repair, cofactor metabolism, reactive oxygen species detoxification, pilus biosynthesis, and hypothetical proteins. Complementation of the perR deletion mutant with wild-type PerR restored PerR-dependent regulation, whereas complementation with either one of two PerR variants carrying single mutations in two predicted metal-binding sites did not rescue the mutant phenotype. Metal content analyses of the recombinant wild type and respective PerR mutants, in addition to regulation studies in metal-supplemented and iron-depleted media, showed binding of zinc and iron by PerR and an iron requirement for optimal responses to peroxide. Our findings reveal a novel physiological contribution of PerR in coordinating DNA and protein metabolic functions in peroxide and identify GAS adaptive responses that may serve to enhance oxidative stress resistance and virulence in the host.
A molecular mimicry between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human proteins supports the possibility that autoimmunity takes place during coronavirus disease 2019 ...(COVID-19) contributing to tissue damage. For example, anti-phospholipid antibodies (aPL) have been reported in COVID-19 as a result of such mimicry and thought to contribute to the immunothrombosis characteristic of the disease. Consistently, active immunization with the virus spike protein may elicit the production of cross-reactive autoantibodies, including aPL. We prospectively looked at the aPL production in healthcare workers vaccinated with RNA- (BNT162b2, n. 100) or adenovirus-based vaccines (ChAdOx1, n. 50). Anti-cardiolipin, anti-beta2 glycoprotein I, anti-phosphatidylserine/prothrombin immunoglobulin G (IgG), IgA, and IgM before and after vaccination were investigated. Anti-platelet factor 4 immunoglobulins were also investigated as autoantibodies associated with COVID-19 vaccination. Additional organ (anti-thyroid) and non-organ (anti-nuclear) autoantibodies and IgG against human proteome were tested as further post-vaccination autoimmunity markers. The antibodies were tested one or three months after the first injection of ChAdOx1 and BNT162b2, respectively; a 12-month clinical follow-up was also performed. Vaccination occasionally induced low titers of aPL and other autoantibodies but did not affect the titer of pre-existing autoantibodies. No significant reactivities against a microarray of approximately 20,000 human proteins were found in a subgroup of ChAdOx1-vaccinees. Consistently, we did not record any clinical manifestation theoretically associated with an underlying autoimmune disorder. The data obtained after the vaccination (two doses for the RNA-based and one dose for the adenovirus-based vaccines), and the clinical follow-up are not supporting the occurrence of an early autoimmune response in this cohort of healthcare workers.
Vaccines against COVID-19 are a powerful tool to control the current SARS-CoV-2 pandemic. A thorough description of their immunogenicity among people living with HIV (PLWHIV) is necessary. We aimed ...to assess the immunogenicity of the mRNA-1273 vaccine among PLWHIV.
In this prospective cohort, adult PLWHIV outpatients were enrolled during the Italian vaccination campaign. Enrolment was allowed irrespective of ongoing combination antiretroviral therapy (ART), plasma HIV viral load and CD4+ T cell count. A two-dose regimen of mRNA-1273, with administrations performed 28 days apart, was employed. The primary outcomes were anti-spike (anti-S) antibody titres and neutralising antibody activity, assessed 28 days after completing the vaccination schedule. A convenient sample of individuals not affected by HIV was also collected to serve as control (referred as healthy-donors, HDs).
We enrolled 71 PLWHIV, mostly male (84·5%), with a mean age of 47 years, a median CD4+ T cell count of 747·0 cells per µL and a median HIV viral load <50 copies/mL. COVID-19-experienced PLWHIV displayed higher anti-S antibody titres (p=0·0007) and neutralising antibody activity in sera (p=0·0007) than COVID-19-naïve PLWHIV. When stratified according to CD4+ T cell count (<350 cells/μL, 350-500 cells/μL, >500 cells/μL), anti-S antibody titres (6/71, median 2173 U/mL IQR 987-4109; 7/71, 5763 IU/mL IQR 4801->12500; 58/71, 2449 U/mL IQR 1524-5704) were not lower to those observed among HDs (10, median 1425 U/mL IQR 599-6131). In addition, neutralising antibody activity, stratified according to the CD4+ T cell count (6/71, median 1314 IQR 606-2477; 7/71, 3329 IU/mL IQR 1905-10508; 58/71, 1227 U/mL IQR 761-3032), was like those displayed by HDs (10, median 2112 U/mL IQR 719-8889).
In our cohort of PLWHIV with well-controlled ART, stable viral suppression and robust CD4+ T cell count, inoculation with mRNA-1273 vaccine given 4 weeks apart produced detectable humoral immune response, similar to individuals without HIV infection, supporting vaccination in PLWHIV.
This study was partially supported by Italian Ministry of Health Ricerca Corrente 2021, by Intesa San Paolo COVID-19 emergency 2020 funds, and by Fondazione Cariplo Grant (INNATE-CoV).
To date there has been limited head-to-head evaluation of immune responses to different types of COVID-19 vaccines. A real-world population-based longitudinal study was designed with the aim to ...define the magnitude and duration of immunity induced by each of four different COVID-19 vaccines available in Italy at the time of this study. Overall, 2497 individuals were enrolled at time of their first vaccination (T0). Vaccine-specific antibody responses induced over time by Comirnaty, Spikevax, Vaxzevria, Janssen Ad26.COV2.S and heterologous vaccination were compared up to six months after immunization. On a subset of Comirnaty vaccinees, serology data were correlated with the ability to neutralize a reference SARS-CoV-2 B strain, as well as Delta AY.4 and Omicron BA.1. The frequency of SARS-CoV-2-specific CD4+ T cells, CD8+ T cells, and memory B cells induced by the four different vaccines was assessed six months after the immunization. We found that mRNA vaccines are stronger inducer of anti-Spike IgG and B-memory cell responses. Humoral immune responses are lower in frail elderly subjects. Neutralization of the Delta AY.4 and Omicron BA.1 variants is severely impaired, especially in older individuals. Most vaccinees display a vaccine-specific T-cell memory six months after the vaccination. By describing the immunological response during the first phase of COVID-19 vaccination campaign in different cohorts and considering several aspects of the immunological response, this study allowed to collect key information that could facilitate the implementation of effective prevention and control measures against SARS-CoV-2.
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