RESUMO
Although mRNA vaccine efficacy against severe coronavirus disease 2019 remains high, variant emergence has prompted booster immunizations. However, the effects of repeated exposures to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens on memory T cells are poorly understood. Here, we utilize major histocompatibility complex multimers with single-cell RNA sequencing to profile SARS-CoV-2-responsive T cells ex vivo from humans with one, two or three antigen exposures, including vaccination, primary infection and breakthrough infection. Exposure order determined the distribution between spike-specific and non-spike-specific responses, with vaccination after infection leading to expansion of spike-specific T cells and differentiation to CCR7-CD45RA+ effectors. In contrast, individuals after breakthrough infection mount vigorous non-spike-specific responses. Analysis of over 4,000 epitope-specific T cell antigen receptor (TCR) sequences demonstrates that all exposures elicit diverse repertoires characterized by shared TCR motifs, confirmed by monoclonal TCR characterization, with no evidence for repertoire narrowing from repeated exposure. Our findings suggest that breakthrough infections diversify the T cell memory repertoire and current vaccination protocols continue to expand and differentiate spike-specific memory.
Assuntos
COVID-19 , SARS-CoV-2 , Linfócitos T CD8-Positivos , Humanos , Fenótipo , Receptores de Antígenos de Linfócitos T/genética , Glicoproteína da Espícula de Coronavírus/genética , Vacinas Sintéticas , Vacinas de mRNARESUMO
BACKGROUND AND OBJECTIVE: Both chronic and aggressive periodontal disease are associated with vitamin D deficiency. The active form of vitamin D, 1,25(OH)2 D3 , induces the expression of the antimicrobial peptide LL-37 and innate immune mediators in cultured human gingival epithelial cells (GECs). The aim of this study was to further delineate the mechanism by which vitamin D enhances the innate defense against the development of periodontal disease (PD). MATERIALS AND METHODS: Wild-type C57Bl/6 mice were made deficient in vitamin D by dietary restriction. Cultured primary and immortalized GEC were stimulated with 1,25(OH)2 D3 , followed by infection with Porphyromonas gingivalis, and viable intracellular bacteria were quantified. Conversion of vitamin D3 to 25(OH)D3 and 1,25(OH)2 D3 was quantified by ELISA. Effect of vitamin D on basal IL-1α expression in mice was determined by topical administration to the gingiva of wild-type mice, followed by qRT-PCR. RESULTS: Dietary restriction of vitamin D led to alveolar bone loss and increased inflammation in the gingiva in the mouse model. In primary human GEC and established human cell lines, treatment of GEC with 1,25(OH)2 D3 inhibited the intracellular growth of P. gingivalis. Cultured GEC expressed two 25-hydroxylases (CYP27A1 and CYP2R1), as well as 1-α hydroxylase, enabling conversion of vitamin D to both 25(OH)D3 and 1,25(OH)2 D3 . Topical application of both vitamin D3 and 1,25(OH)2 D3 to the gingiva of mice led to rapid inhibition of IL-1α expression, a prominent pro-inflammatory cytokine associated with inflammation, which also exhibited more than a 2-fold decrease from basal levels in OKF6/TERT1 cells upon 1,25(OH)2 D3 treatment, as determined by RNA-seq. CONCLUSION: Vitamin D deficiency in mice contributes to PD, recapitulating the association seen in humans, and provides a unique model to study the development of PD. Vitamin D increases the activity of GEC against the invasion of periodontal pathogens and inhibits the inflammatory response, both in vitro and in vivo. GEC can convert inactive vitamin D to the active form in situ, supporting the hypothesis that vitamin D can be applied directly to the gingiva to prevent or treat periodontal disease.
Assuntos
Perda do Osso Alveolar/fisiopatologia , Calcifediol/farmacologia , Gengiva/fisiologia , Inflamação/fisiopatologia , Vitamina D/farmacologia , Perda do Osso Alveolar/imunologia , Animais , Células Cultivadas , Humanos , Inflamação/imunologia , Interleucina-1alfa/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Porphyromonas gingivalis , Vitaminas/farmacologiaRESUMO
Vaccination, especially with multiple doses, provides substantial population-level protection against COVID-19, but emerging variants of concern (VOC) and waning immunity represent significant risks at the individual level. Here we identify correlates of protection (COP) in a multicenter prospective study following 607 healthy individuals who received three doses of the Pfizer-BNT162b2 vaccine approximately six months prior to enrollment. We compared 242 individuals who received a fourth dose to 365 who did not. Within 90 days of enrollment, 239 individuals contracted COVID-19, 45% of the 3-dose group and 30% of the four-dose group. The fourth dose elicited a significant rise in antibody binding and neutralizing titers against multiple VOCs reducing the risk of symptomatic infection by 37% [95%CI, 15%-54%]. However, a group of individuals, characterized by low baseline titers of binding antibodies, remained susceptible to infection despite significantly increased neutralizing antibody titers upon boosting. A combination of reduced IgG levels to RBD mutants and reduced VOC-recognizing IgA antibodies represented the strongest COP in both the 3-dose group (HR = 6.34, p = 0.008) and four-dose group (HR = 8.14, p = 0.018). We validated our findings in an independent second cohort. In summary combination IgA and IgG baseline binding antibody levels may identify individuals most at risk from future infections.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Humanos , Vacina BNT162 , Estudos Prospectivos , COVID-19/prevenção & controle , SARS-CoV-2 , Imunoglobulina A , Imunoglobulina GRESUMO
The marine macroalga, Ulva lactuca, has been exposed for 48 h to different concentrations of Ag added as either silver nanoparticles (AgNP) or aqueous metal (AgNO(3)) and the resulting toxicity, estimated from reductions in quenching of chlorophyll-a fluorescence, and accumulation of Ag measured. Aqueous Ag was toxic at available concentrations as low as about 2.5 µg l(-1) and exhibited considerable accumulation that could be defined by the Langmuir equation. AgNP were not phytotoxic to the macroalga at available Ag concentrations up to at least 15 µg l(-1) and metal measured in U. lactuca was attributed to a physical association of nanoparticles at the algal surface. At higher AgNP concentrations, a dose-response relationship was observed that was similar to that for aqueous Ag recorded at much lower concentrations. These findings suggest that AgNP are only indirectly toxic to marine algae through the dissolution of Ag(+) ions into bulk sea water, albeit at concentrations orders of magnitude greater than those predicted in the environment.
Assuntos
Nanopartículas Metálicas/toxicidade , Alga Marinha/efeitos dos fármacos , Prata/toxicidade , Ulva/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Monitoramento Ambiental/métodos , Alga Marinha/crescimento & desenvolvimento , Testes de Toxicidade , Ulva/crescimento & desenvolvimentoRESUMO
SARS-CoV-2 infection causes diverse outcomes ranging from asymptomatic infection to respiratory distress and death. A major unresolved question is whether prior immunity to endemic, human common cold coronaviruses (hCCCoVs) impacts susceptibility to SARS-CoV-2 infection or immunity following infection and vaccination. Therefore, we analyzed samples from the same individuals before and after SARS-CoV-2 infection or vaccination. We found hCCCoV antibody levels increase after SARS-CoV-2 exposure, demonstrating cross-reactivity. However, a case-control study indicates that baseline hCCCoV antibody levels are not associated with protection against SARS-CoV-2 infection. Rather, higher magnitudes of pre-existing betacoronavirus antibodies correlate with more SARS-CoV-2 antibodies following infection, an indicator of greater disease severity. Additionally, immunization with hCCCoV spike proteins before SARS-CoV-2 immunization impedes the generation of SARS-CoV-2-neutralizing antibodies in mice. Together, these data suggest that pre-existing hCCCoV antibodies hinder SARS-CoV-2 antibody-based immunity following infection and provide insight on how pre-existing coronavirus immunity impacts SARS-CoV-2 infection, which is critical considering emerging variants.
Assuntos
Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , COVID-19/imunologia , Resfriado Comum/imunologia , Imunidade Humoral/imunologia , SARS-CoV-2/imunologia , Animais , Infecções Assintomáticas , COVID-19/virologia , Estudos de Casos e Controles , Linhagem Celular , Resfriado Comum/virologia , Reações Cruzadas/imunologia , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína da Espícula de Coronavírus/imunologiaRESUMO
Although mRNA vaccine efficacy against severe COVID-19 remains high, variant emergence and breakthrough infections have changed vaccine policy to include booster immunizations. However, the effect of diverse and repeated antigen exposures on SARS-CoV-2 memory T cells is poorly understood. Here, we utilize DNA-barcoded MHC-multimers combined with scRNAseq and scTCRseq to capture the ex vivo profile of SARS-CoV-2-responsive T cells within a cohort of individuals with one, two, or three antigen exposures, including vaccination, primary infection, and breakthrough infection. We found that the order of exposure determined the relative distribution between spike- and non-spike-specific responses, with vaccination after infection leading to further expansion of spike-specific T cells and differentiation to a CCR7-CD45RA+ effector phenotype. In contrast, individuals experiencing a breakthrough infection mount vigorous non-spike-specific responses. In-depth analysis of over 4,000 epitope-specific T cell receptor sequences demonstrates that all types of exposures elicit diverse repertoires characterized by shared, dominant TCR motifs, with no evidence for repertoire narrowing from repeated exposure. Our findings suggest that breakthrough infections diversify the T cell memory repertoire and that current vaccination protocols continue to expand and differentiate spike-specific memory responses.
RESUMO
Infection and vaccination repeatedly expose individuals to antigens that are conserved between influenza virus subtypes. Nevertheless, antibodies recognizing variable influenza epitopes greatly outnumber antibodies reactive against conserved epitopes. Elucidating factors contributing to the paucity of broadly reactive influenza antibodies remains a major obstacle for developing a universal influenza vaccine. Here, we report that inducing broadly reactive influenza antibodies increases autoreactive antibodies in humans and mice and exacerbates disease in four distinct models of autoimmune disease. Importantly, transferring broadly reactive influenza antibodies augments disease in the presence of inflammation or autoimmune susceptibility. Further, broadly reactive influenza antibodies spontaneously arise in mice with defects in B cell tolerance. Together, these data suggest that self-tolerance mechanisms limit the prevalence of broadly reactive influenza antibodies, which can exacerbate disease in the context of additional risk factors.
Assuntos
Vacinas contra Influenza , Influenza Humana , Infecções por Orthomyxoviridae , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , Autoimunidade , Epitopos , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Humanos , CamundongosRESUMO
Peptides with broad-spectrum antimicrobial activity are found widely expressed throughout nature. As they participate in a number of different aspects of innate immunity in mammals, they have been termed Host Defense Peptides (HDPs). Due to their common structural features, including an amphipathic structure and cationic charge, they have been widely shown to interact with and disrupt microbial membranes. Thus, it is not surprising that human HDPs have activity against enveloped viruses as well as bacteria and fungi. However, these peptides also exhibit activity against a wide range of non-enveloped viruses as well, acting at a number of different steps in viral infection. This review focuses on the activity of human host defense peptides, including alpha- and beta-defensins and the sole human cathelicidin, LL-37, against both enveloped and non-enveloped viruses. The broad spectrum of antiviral activity of these peptides, both in vitro and in vivo suggest that they play an important role in the innate antiviral defense against viral infections. Furthermore, the literature suggests that they may be developed into antiviral therapeutic agents.
Assuntos
Antivirais/farmacologia , Animais , Bactérias , Fungos , Humanos , Imunidade Inata , Peptídeos , VirosesRESUMO
Enhancing the generation of broadly reactive antibodies against influenza A virus (IAV) is a pertinent goal toward developing a universal IAV vaccine. While antibodies that bind conserved IAV epitopes have been identified in humans, antibodies specific for the variable epitopes are much more prevalent than antibodies recognizing conserved epitopes. It is important to define the factors that limit the generation of broadly reactive IAV antibodies in order to develop an effective universal IAV vaccine. The predominant theory is that competition within germinal centers favors the synthesis of high-affinity antibodies specific for the variable region of the virus, and limits antibodies specific for conserved IAV epitopes. Here, we show that reducing germinal center formation and removing competition with high-affinity antibodies was not sufficient to increase broadly reactive IAV antibodies or enhance protection against distinct IAV subtypes. These data disprove the prevailing hypothesis that broadly reactive IAV antibodies are rare due to competition within germinal centers, and reveal the critical need to further investigate factors that limit broadly reactive IAV antibodies. Additionally, our data show that IAV-specific IgM antibodies persist in mice in the absence of germinal centers, highlighting the protective capacity of germinal center-independent IgM antibodies, which are not typically considered when testing correlates of protection, and offer an alternate target for delivering a universal IAV vaccine.IMPORTANCE It is estimated that 250,000 to 650,000 individuals worldwide die each year from seasonal influenza A virus (IAV) infections. Current vaccines provide little protection against newly emerging strains. Thus, considerable effort is focused on enhancing the generation of broadly reactive IAV antibodies in order to develop a universal IAV vaccine. However, broadly reactive IAV antibodies are rare and the factors that limit their generation are not completely understood. Our data disprove the prevailing hypothesis that broadly reactive IAV antibodies are uncommon due to competition in the germinal centers with antibodies specific for the variable, hemagglutinin (HA) head. Understanding the factors that constrain development of antibodies specific for conserved regions of IAV is imperative for developing an effective universal IAV vaccine, which could potentially circumvent a catastrophic pandemic. These findings are significant as they highlight the importance of investigating other mechanisms that contribute to the paucity of broadly reactive IAV antibodies.
Assuntos
Anticorpos Antivirais/imunologia , Afinidade de Anticorpos/imunologia , Centro Germinativo/imunologia , Vírus da Influenza A/imunologia , Influenza Humana/imunologia , Infecções por Orthomyxoviridae/imunologia , Animais , Anticorpos Neutralizantes , Especificidade de Anticorpos/imunologia , Reações Cruzadas/imunologia , Modelos Animais de Doenças , Relação Dose-Resposta Imunológica , Feminino , Humanos , Imunização Secundária , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Imuno-Histoquímica , Vacinas contra Influenza/imunologia , Influenza Humana/virologia , Camundongos , Camundongos Transgênicos , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologiaRESUMO
Laser Engineered Net Shaping (LENS™) was used to produce a compositionally graded Ti-xMo (0 ≤ x ≤ 12 wt %) specimen and nine Ti-15Mo (fixed composition) specimens at different energy densities to understand the composition-processing-microstructure relationships operating using additive manufacturing. The gradient was used to evaluate the effect of composition on the prior-beta grain size. The specimens deposited using different energy densities were used to assess the processing parameters influence the microstructure evolutions. The gradient specimen did not show beta grain size reduction with the Mo content. The analysis from the perspective of the two grain refinement mechanisms based on a model known as the Easton & St. John, which was originally developed for aluminum and magnesium alloys shows the lower bound in prior-beta grain refinement with the Ti-Mo system. The low growth restriction factor for the Ti-Mo system of Q = 6,5C0 explains the unsuccessful refinement from the solute-based mechanism. The energy density and the grain size are proportional according to the results of the nine fixed composition specimens at different energy densities. More energy absorption from the material represents bigger molten pools, which in turn relates to lower cooling rates.
RESUMO
Oral epithelial cells (OECs) represent the first line of defense against viruses that are spread via saliva, including Kaposi's sarcoma-associated herpesvirus (KSHV). Infection of humans by KSHV and viral pathogenesis begins by infecting OECs. One method OECs use to limit viral infections in the oral cavity is the production of antimicrobial peptides (AMPs), or host defense peptides (HDPs). However, no studies have investigated the antiviral activities of any HDP against KSHV. The goal of this study was to determine the antiviral activity of one HDP, LL-37, against KSHV in the context of infecting OECs. Our results show that LL-37 significantly decreased KSHV's ability to infect OECs in both a structure- and dose-dependent manner. However, this activity does not stem from affecting OECs, but instead the virions themselves. We found that LL-37 exerts its antiviral activity against KSHV by disrupting the viral envelope, which can inhibit viral entry into OECs. Our data suggest that LL-37 exhibits a marked antiviral activity against KSHV during infection of oral epithelial cells, which can play an important role in host defense against oral KSHV infection. Thus, we propose that inducing LL-37 expression endogenously in oral epithelial cells, or potentially introducing as a therapy, may help restrict oral KSHV infection and ultimately KSHV-associated diseases.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antivirais/farmacologia , Células Epiteliais/virologia , Infecções por Herpesviridae/tratamento farmacológico , Herpesvirus Humano 8/efeitos dos fármacos , Linhagem Celular , Herpesvirus Humano 8/patogenicidade , Humanos , Queratinócitos , Boca/virologia , Vírion/efeitos dos fármacos , Vírion/metabolismo , Internalização do Vírus/efeitos dos fármacos , CatelicidinasRESUMO
Lethal systemic fungal infections of Candida species are increasingly common, especially in immune compromised patients. By in vitro screening of small molecule mimics of naturally occurring host defense peptides (HDP), we have identified several active antifungal molecules, which also exhibited potent activity in two mouse models of oral candidiasis. Here we show that one such compound, C4, exhibits a mechanism of action that is similar to the parent HDP upon which it was designed. Specifically, its initial interaction with the anionic microbial membrane is electrostatic, as its fungicidal activity is inhibited by cations. We observed rapid membrane permeabilization to propidium iodide and ATP efflux in response to C4. Unlike the antifungal peptide histatin 5, it did not require energy-dependent transport across the membrane. Rapid membrane disruption was observed by both fluorescence and electron microscopy. The compound was highly active in vitro against numerous fluconazole-resistant clinical isolates of C. albicans and non-albicans species, and it exhibited potent, dose-dependent activity in a mouse model of invasive candidiasis, reducing kidney burden by three logs after 24 hours, and preventing mortality for up to 17 days. Together the results support the development of this class of antifungal drug to treat invasive candidiasis.
Assuntos
Antifúngicos/farmacologia , Fatores Celulares Derivados do Hospedeiro/farmacologia , Interações Hospedeiro-Patógeno , Membranas/efeitos dos fármacos , Peptídeos/farmacologia , Antifúngicos/química , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/metabolismo , Candida albicans/ultraestrutura , Complemento C4/imunologia , Resistência à Doença , Farmacorresistência Fúngica , Fatores Celulares Derivados do Hospedeiro/química , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Testes de Sensibilidade Microbiana , Peptídeos/químicaRESUMO
In common with several other respiratory and photosynthetic enzymes, a sub-population of cytosolic phosphoglycerate kinase (PGK) occurs in the nucleus in pea leaves and shoots. The full-length cDNA encoding pea cytosolic PGK has been cloned and sequenced, revealing not only the PGK 'signature' but also a nuclear localization signal (NLS). A translational fusion of PGK and GFP was used to transform tobacco BY-2 cells resulting in GFP locating to the cell nuclei.
Assuntos
Fosfoglicerato Quinase/genética , Pisum sativum/enzimologia , Pisum sativum/genética , Sequência de Aminoácidos , Clonagem Molecular , Citosol/enzimologia , Primers do DNA , Dados de Sequência Molecular , Fosfoglicerato Quinase/química , Fosfoglicerato Quinase/metabolismoRESUMO
Homologues of the yeast ubiquitin ligase-associated protein SGT1 are required for disease resistance in plants mediated by nucleotide-binding site/leucine-rich repeat (NBS-LRR) proteins. Here, by silencing SGT1 in Nicotiana benthamiana, we extend these findings and demonstrate that SGT1 has an unexpectedly general role in disease resistance. It is required for resistance responses mediated by NBS-LRR and other R proteins in which pathogen-derived elicitors are recognized either inside or outside the host plant cell. A requirement also exists for SGT1 in nonhost resistance in which all known members of a host species are resistant against every characterized isolate of a pathogen. Our findings show that silencing SGT1 affects diverse types of disease resistance in plants and support the idea that R protein-mediated and nonhost resistance may involve similar mechanisms.