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AbstractThe evolution of SARS-CoV-2 has led to the emergence of numerous variants of concern (VOCs), marked by changes in the viral spike glycoprotein, the primary target for neutralising antibody (nAb) responses. Emerging VOCs, particularly omicron sub-lineages, show resistance to nAbs induced by prior infection or vaccination. The precise spike protein changes contributing to this resistance remain unclear in infectious cell culture systems. In the present study, a large panel of infectious SARS-CoV-2 mutant viruses, each with spike protein changes found in VOCs, including omicron JN.1 and its derivatives KP.2 and KP.3, was generated using a reverse genetic system. The susceptibility of these viruses to antibody neutralisation was measured using plasma from convalescent and vaccinated individuals. Synergistic roles of combined substitutions in the spike receptor binding domain (RBD) were observed in neutralisation resistance. However, recombinant viruses with the entire spike protein from a specific VOC showed enhanced resistance, indicating that changes outside the RBD are also significant. In silico analyses of spike antibody epitopes suggested changes in neutralisation could be due to altered antibody binding affinities. Assessing ACE2 usage for entry through anti-ACE2 antibody blocking and ACE2 siRNA revealed that omicron BA.2.86 and JN.1 mutant viruses were less dependent on ACE2 for entry. However, surface plasmon resonance analysis showed increased affinity for ACE2 for both BA.2.86 and JN.1 compared to the ancestral spike. This detailed analysis of specific changes in the SARS-CoV-2 spike enhances understanding of coronavirus evolution, particularly regarding neutralising antibody evasion and ACE2 entry receptor dependence.
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Rapidly waning immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires continued global access to affordable vaccines. Globally, inactivated SARS-CoV-2 vaccines have been widely used during the SARS-CoV-2 pandemic. In this proof-of-concept study we adapted an original-D614G SARS-CoV-2 virus to Vero cell culture as a strategy to enhance inactivated vaccine manufacturing productivity. A passage 60 (P60) virus showed enhanced fitness and 50-fold increased virus yield in a bioreactor compared to the original-D614G virus. It further remained susceptible to neutralization by plasma from SARS-CoV-2 vaccinated and convalescent individuals, suggesting exposure of relevant epitopes. Monovalent inactivated P60 and bivalent inactivated P60/omicron BA.1 vaccines induced neutralizing responses against original-D614G and BA.1 viruses in mice and hamsters, demonstrating that the P60 virus is a suitable vaccine antigen. Antibodies further cross-neutralized delta and BA.5 viruses. Importantly, the inactivated P60 vaccine protected hamsters against disease upon challenge with original-D614G or BA.1 virus, with minimal lung pathology and lower virus loads in the upper and lower airways. Antigenicity of the P60 virus was thus retained compared to the original virus despite the acquisition of cell culture adaptive mutations. Consequently, cell culture adaptation may be a useful approach to increase yields in inactivated vaccine antigen production.
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Anticorpos Neutralizantes , Anticorpos Antivirais , Vacinas contra COVID-19 , COVID-19 , SARS-CoV-2 , Vacinas de Produtos Inativados , Animais , Células Vero , Chlorocebus aethiops , SARS-CoV-2/imunologia , Vacinas de Produtos Inativados/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/imunologia , COVID-19/virologia , Camundongos , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Humanos , Proteção Cruzada/imunologia , Cricetinae , FemininoRESUMO
BACKGROUND & AIMS: In individuals highly exposed to HCV, reinfection is common, suggesting that natural development of sterilising immunity is difficult. In those that are reinfected, some will develop a persistent infection, while a small proportion repeatedly clear the virus, suggesting natural protection is possible. The aim of this study was to characterise immune responses associated with rapid natural clearance of HCV reinfection. METHODS: Broad neutralising antibodies (nAbs) and Envelope 2 (E2)-specific memory B cell (MBC) responses were examined longitudinally in 15 individuals with varied reinfection outcomes. RESULTS: Broad nAb responses were associated with MBC recall, but not with clearance of reinfection. Strong evidence of antigen imprinting was found, and the B-cell receptor repertoire showed a high level of clonality with ongoing somatic hypermutation of many clones over subsequent reinfection events. Single-cell transcriptomic analyses showed that cleared reinfections featured an activated transcriptomic profile in HCV-specific B cells that rapidly expanded upon reinfection. CONCLUSIONS: MBC quality, but not necessarily breadth of nAb responses, is important for protection against antigenically diverse variants, which is encouraging for HCV vaccine development. IMPACT AND IMPLICATIONS: HCV continues to have a major health burden globally. Limitations in the health infrastructure for diagnosis and treatment, as well as high rates of reinfection, indicate that a vaccine that can protect against chronic HCV infection will greatly complement current efforts to eliminate HCV-related disease. With alternative approaches to testing vaccines, such as controlled human inoculation trials under consideration, we desperately need to identify the correlates of immune protection. In this study, in a small but rare cohort of high-risk injecting drug users who were reinfected multiple times, breadth of neutralisation was not associated with ultimate clearance of the reinfection event. Alternatively, characteristics of the HCV-specific B-cell response associated with B-cell proliferation were. This study indicates that humoral responses are important for protection and suggests that for genetically very diverse viruses, such as HCV, it may be beneficial to look beyond just antibodies as correlates of protection.
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Hepacivirus , Reinfecção , Humanos , Reinfecção/imunologia , Hepacivirus/imunologia , Hepacivirus/genética , Hepatite C/imunologia , Masculino , Feminino , Células B de Memória/imunologia , Anticorpos Anti-Hepatite C/sangue , Anticorpos Anti-Hepatite C/imunologia , Linfócitos B/imunologia , Adulto , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Pessoa de Meia-IdadeRESUMO
As severe acute respiratory coronavirus 2 (SARS-CoV-2) variants continue to emerge, it is important to characterize immune responses against variants which can inform on protection efficacies following booster vaccination. In this study, neutralizing breadth and antigen-specific CD8+ T cell responses were analyzed in both infection-naïve and infection-experienced individuals following administration of a booster bivalent Wuhan-Hu-1+BA.4/5 Comirnaty® mRNA vaccine. Significantly higher neutralizing titers were found after this vaccination compared to the pre-third booster vaccination time point. Further, neutralizing breadth to omicron variants, including BA.1, BA.2, BA.5, BQ.1 and XBB.1, was found to be boosted following bivalent vaccination. SARS-CoV-2-specific CD8+ T cells were identified, but with no evidence that frequencies were increased following booster vaccinations. Spike protein-specific CD8+ T cells were the only responses detected after vaccination and non-spike-specific CD8+ T cells were only detected after infection. Both spike-specific and non-spike-specific CD8+ T cells were found at much lower frequencies than CD8+ T cells specific to cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza (Flu). Taken together, these results show that the bivalent Wuhan-Hu-1+BA.4/5 Comirnaty® mRNA vaccine boosted the breadth of neutralization to newer SARS-CoV-2 variants and that vaccination is able to induce spike protein-specific CD8+ T cell responses, which are maintained longitudinally.
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COVID-19 , Infecções por Vírus Epstein-Barr , Adulto , Humanos , Anticorpos Neutralizantes , Vacina BNT162 , Linfócitos T CD8-Positivos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Vacinas de mRNA , COVID-19/prevenção & controle , Herpesvirus Humano 4RESUMO
BACKGROUND: Given the importance of vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the prevention of severe coronavirus disease 2019 (COVID-19), detailed long-term analyses of neutralising antibody responses are required to inform immunisation strategies. METHODS: In this study, longitudinal neutralising antibody titres to an ancestral SARS-CoV-2 isolate and cross-neutralisation to delta and omicron isolates were analysed in individuals previously infected with SARS-CoV-2, vaccinated against COVID-19, or a complex mix thereof with up to two years of follow-up. FINDINGS: Both infection-induced and vaccine-induced neutralising responses against SARS-CoV-2 appeared to follow similar decay patterns. Following vaccination in previously infected individuals, neutralising antibody responses were more durable than prior to vaccination. Further, this study shows that vaccination after infection, as well as booster vaccination, increases the cross-neutralising potential to both delta and omicron SARS-CoV-2 variants. INTERPRETATION: Taken together, these results suggest that neither type of antigen exposure is superior for neutralising antibody durability. However, these results support vaccination to increase the durability and cross-neutralisation potential of neutralising responses, thereby enhancing protection against severe COVID-19. FUNDING: This work was supported by grants from The Capital Region of Denmark's Research Foundation, the Novo Nordisk Foundation, the Independent Research Fund Denmark, the Candys Foundation, and the Danish Agency for Science and Higher Education.
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COVID-19 , Humanos , SARS-CoV-2 , Vacinas contra COVID-19 , Vacinação , Imunização Secundária , Anticorpos Neutralizantes , Anticorpos AntiviraisRESUMO
BACKGROUND: Capsid virus-like particles (cVLP) have proven safe and immunogenic and can be a versatile platform to counter pandemics. We aimed to clinically test a modular cVLP COVID-19 vaccine in individuals who were naive to SARS-CoV-2. METHODS: In this phase 1, single-centre, dose-escalation, adjuvant-selection, open-label clinical trial, we recruited participants at the Radboud University Medical Center in Nijmegen, Netherlands, and sequentially assigned them to seven groups. Eligible participants were healthy, aged 18-55 years, and tested negative for SARS-CoV-2 and anti-SARS-CoV-2 antibodies. Participants were vaccinated intramuscularly on days 0 and 28 with 6 µg, 12 µg, 25 µg, 50 µg, or 70 µg of the cVLP-based COVID-19 vaccine (ABNCoV2). A subgroup received MF59-adjuvanted ABNCoV2. Follow-up was for 24 weeks after second vaccination. The primary objectives were to assess the safety and tolerability of ABNCoV2 and to identify a dose that optimises the tolerability-immunogenicity ratio 14 days after the first vaccination. The primary safety endpoint was the number of related grade 3 adverse events and serious adverse events in the intention-to-treat population. The primary immunogenicity endpoint was the concentration of ABNCoV2-specific antibodies. The trial is registered with ClinicalTrials.gov, NCT04839146. FINDINGS: 45 participants (six to nine per group) were enrolled between March 15 and July 15, 2021. Participants had a total of 249 at least possibly related solicited adverse events (185 grade 1, 63 grade 2, and one grade 3) within a week after vaccination. Two serious adverse events occurred; one was classified as a possible adverse reaction. Antibody titres were dose-dependent with levels plateauing at a vaccination dose of 25-70 µg ABNCoV2. After second vaccination, live virus neutralisation activity against major SARS-CoV-2 variants was high but was lower with an omicron (BA.1) variant. Vaccine-specific IFNγ+ CD4+ T cells were induced. INTERPRETATION: Immunisation with ABNCoV2 was well tolerated, safe, and resulted in a functional immune response. The data support the need for additional clinical development of ABNCoV2 as a second-generation SARS-CoV-2 vaccine. The modular cVLP platform will accelerate vaccine development, beyond SARS-CoV-2. FUNDING: EU, Carlsberg Foundation, and the Novo Nordisk Foundation.
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COVID-19 , Vacinas Virais , Humanos , Adjuvantes Imunológicos , Capsídeo , Proteínas do Capsídeo , Vacinas contra COVID-19 , SARS-CoV-2 , Vacinas Virais/efeitos adversosRESUMO
The effects of dexamethasone (DXM) treatment on pulmonary immunity in COVID-19-associated acute respiratory distress syndrome (CARDS) remain insufficiently understood. We performed transcriptomic RNA-seq analysis of bronchoalveolar lavage fluid from 20 mechanically ventilated patients: 12 with CARDS (with or without DXM) and 8 non-COVID-19 critically ill controls. CARDS with DXM was characterized by upregulation of genes related to B-cell and complement pathway activation, antigen presentation, phagocytosis, and FC-γ receptor signaling. Most interferon-stimulated genes were upregulated in CARDS, particularly in CARDS without DXM. In conclusion, DXM treatment was not associated with regulation of proinflammatory pathways in CARDS but with regulation of other local immune responses. Clinical Trials Registration. NCT04354584.
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COVID-19 , Pneumonia , Síndrome do Desconforto Respiratório , Humanos , Líquido da Lavagem Broncoalveolar , COVID-19/genética , Dexametasona/farmacologia , Dexametasona/uso terapêutico , Pulmão , Síndrome do Desconforto Respiratório/tratamento farmacológico , TranscriptomaRESUMO
With increasing numbers of vaccine-breakthrough infections worldwide, assessing the immunogenicity of vaccinated health-care workers that are frequently exposed to SARS-CoV-2-infected individuals is important. In this study, neutralization titers against SARS-CoV-2 were assessed one month after completed prime-boost vaccine regimens in health-care workers vaccinated with either mRNA-mRNA (Comirnaty®, BioNTech-Pfzier, Mainz, Germany/New York, NY, USA, n = 98) or vector-based (Vaxzevria®, Oxford-AstraZeneca, Cambridge, UK) followed by mRNA-based (Comirnaty® or Spikevax®, Moderna, Cambridge, MA, USA) vaccines (n = 16). Vaccine-induced neutralization titers were compared to time-matched, unvaccinated individuals that were infected with SARS-CoV-2 and presented with mild symptoms (n = 38). Significantly higher neutralizing titers were found in both the mRNA-mRNA (ID50: 2525, IQR: 1667-4313) and vector-mRNA (ID50: 4978, IQR: 3364-7508) prime-boost vaccine regimens when compared to SARS-CoV-2 infection (ID50: 401, IQR: 271-792) (p < 0.0001). However, infection with SARS-CoV-2 induced higher titers when compared to a single dose of Vaxzevria® (p = 0.0072). Between mRNA-mRNA and vector-mRNA prime-boost regimens, the vector-mRNA vaccine regimen induced higher neutralization titers (p = 0.0054). Demographically, both age and time between vaccination doses were associated with vaccine-induced neutralization titers (p = 0.02 and p = 0.03, respectively). This warrants further investigation into the optimal time to administer booster vaccination for optimized induction of neutralizing responses. Although anecdotal (n = 3), those with exposure to SARS-CoV-2, either before or after vaccination, demonstrated superior neutralizing titers, which is suggestive of further boosting through viral exposure.
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BACKGROUND: Given the importance of neutralising antibodies in protection against SARS-CoV-2 infection, it is critical to assess neutralisation persistence long-term following recovery. This study investigated neutralisation titres against SARS-CoV-2 up to 6 months post-symptom onset in individuals with mild COVID-19. METHODS: Plasma neutralisation titres in convalescent COVID-19 individuals were determined at baseline and 6 months post-symptom onset using a cell culture infectious SARS-CoV-2 assay. Total SARS-CoV-2 spike-specific IgG and IgA binding was measured using a lectin capture ELISA and compared between timepoints and correlated to neutralising titres. FINDINGS: All 48 convalescent COVID-19 individuals were found to have detectable SARS-CoV-2 50% inhibitory dilution neutralisation titres (ID50) at baseline and 6 months post-symptom onset with mean ID50 of 1/943 and 1/411, respectively. SARS-CoV-2 neutralisation titres peaked within 1-2 months post-symptom onset. However, 50% of individuals showed comparable ID50 at baseline and 6 months post-symptom onset. Both SARS-CoV-2 spike-specific IgG and IgA levels correlated well with neutralising titres. IgG binding was found to be sustained up to 6 months post-symptom onset, whereas IgA levels declined. INTERPRETATION: This study demonstrates durability of SARS-CoV-2 spike-specific IgG and neutralisation responses following recovery from mild COVID-19. Thus, all subjects included in this study might potentially have protective levels of neutralising antibodies 6 months post-symptom onset. This study also demonstrates a relationship between spike-specific IgA and neutralisation decline, with implications for long-term protection against SARS-CoV-2 infection. FUNDING: Novo Nordisk Foundation, Independent Research Fund Denmark and Danish Agency for Science and Higher Education.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , SARS-CoV-2/patogenicidade , Adulto , COVID-19/epidemiologia , COVID-19/virologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
Despite recent advances in curative therapy, hepatitis C virus (HCV) still remains a global threat. In order to achieve global elimination, a prophylactic vaccine should be considered high priority. Previous immunogens used to induce broad neutralising antibodies (BnAbs) have been met with limited success. To improve immunogen design, factors associated with the early development of BnAbs in natural infection must first be understood. In this study, 43 subjects identified with acute HCV were analysed longitudinally using a panel of heterogeneous HCV pseudoparticles (HCVpp), to understand the emergence of BnAbs. Compared to those infected with a single genotype, early BnAb development was associated with subjects co-infected with at least 2 HCV subtypes during acute infection. In those that were mono-infected, BnAbs were seen to emerge with increasing viral persistence. If subjects acquired a secondary infection, nAb breadth was seen to boost upon viral re-exposure. Importantly, this data highlights the potential for multivalent and prime-boost vaccine strategies to induce BnAbs against HCV in humans. However, the data also indicate that the infecting genotype may influence the development of BnAbs. Therefore, the choice of antigen will need to be carefully considered in future vaccine trials.
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Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Hepacivirus/imunologia , Hepatite C/imunologia , Vacinas Virais/imunologia , Adolescente , Adulto , Portadores de Fármacos , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos , Genótipo , Humanos , Lentivirus/genética , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Testes de Neutralização , Vacinas Virais/administração & dosagem , Adulto JovemRESUMO
The morbidity and mortality resulting from acute gastroenteritis and associated chronic sequelae represent a substantial burden on health care systems worldwide. Few studies have investigated changes in the gut microbiome following an episode of acute gastroenteritis. By using nondirected 16S rRNA gene amplicon sequencing, the fecal microbiota of 475 patients with acute gastroenteritis was examined. Patient age was correlated with the overall microbial composition, with a decrease in the abundance of Faecalibacterium being observed in older patients. We observed the emergence of a potential Escherichia-Shigella-dominated enterotype in a subset of patients, and this enterotype was predicted to be more proinflammatory than the other common enterotypes, with the latter being dominated by Bacteroides or Faecalibacterium The increased abundance of Escherichia-Shigella did not appear to be associated with infection with an agent of a similar sequence similarity. Stool color and consistency were associated with the diversity and composition of the microbiome, with deviations from the norm (not brown and solid) showing increases in the abundances of bacteria such as Escherichia-Shigella and Veillonella Analysis of enriched outliers within the data identified a range of genera previously associated with gastrointestinal diseases, including Treponema, Proteus, Capnocytophaga, Arcobacter, Campylobacter, Haemophilus, Aeromonas, and Pseudomonas Our data represent the first in-depth analysis of gut microbiota in acute gastroenteritis. Phenotypic changes in stool color and consistency were associated with specific changes in the microbiota. Enriched bacterial taxa were detected in cases where no causative agent was identified by using routine diagnostic tests, suggesting that in the future, microbiome analyses may be utilized to improve diagnostics.
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Bactérias/isolamento & purificação , Gastroenterite/etiologia , Microbioma Gastrointestinal , Doença Aguda , Adolescente , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Bactérias/classificação , Criança , Pré-Escolar , Fezes , Gastroenterite/microbiologia , Humanos , Lactente , Pessoa de Meia-Idade , Adulto JovemRESUMO
Given that Campylobacter jejuni is recognized as the most common cause of bacterial gastroenteritis worldwide, recent findings showing comparable levels of Campylobacter concisus in patients with gastroenteritis would suggest that this bacterium is clinically important. The prevalence and abundance of Campylobacter concisus in stool samples collected from patients with acute gastroenteritis was examined using quantitative real-time PCR. The associated virulence determinants exotoxin 9 and zonula occludens toxin DNA were detected for Campylobacter concisus-infected samples using real-time PCR. Campylobacter concisus was detected at high prevalence in patients with gastroenteritis (49.7â%), higher than that observed for Campylobacter jejuni (â¼5â%). The levels of Campylobacter concisus were putatively classified into clinically relevant and potentially transient subgroups based on a threshold developed using Campylobacter jejuni levels, as the highly sensitive real-time PCR probably detected transient passage of the bacterium from the oral cavity. A total of 18â% of patients were found to have clinically relevant levels of Campylobacter concisus, a significant number of which also had high levels of one of the virulence determinants. Of these patients, 78â% were found to have no other gastrointestinal pathogen identified in the stool, which strongly suggests a role for Campylobacter concisus in the aetiology of gastroenteritis in these patients. These results emphasize the need for diagnostic laboratories to employ identification protocols for emerging Campylobacter species. Clinical follow-up in patients presenting with high levels of Campylobacter concisus in the intestinal tract is needed, given that it has been associated with more chronic sequelae.