RESUMEN
Reversing CD8+ T cell dysfunction is crucial in treating chronic hepatitis B virus (HBV) infection, yet specific molecular targets remain unclear. Our study analyzed co-signaling receptors during hepatocellular priming and traced the trajectory and fate of dysfunctional HBV-specific CD8+ T cells. Early on, these cells upregulate PD-1, CTLA-4, LAG-3, OX40, 4-1BB, and ICOS. While blocking co-inhibitory receptors had minimal effect, activating 4-1BB and OX40 converted them into antiviral effectors. Prolonged stimulation led to a self-renewing, long-lived, heterogeneous population with a unique transcriptional profile. This includes dysfunctional progenitor/stem-like (TSL) cells and two distinct dysfunctional tissue-resident memory (TRM) populations. While 4-1BB expression is ubiquitously maintained, OX40 expression is limited to TSL. In chronic settings, only 4-1BB stimulation conferred antiviral activity. In HBeAg+ chronic patients, 4-1BB activation showed the highest potential to rejuvenate dysfunctional CD8+ T cells. Targeting all dysfunctional T cells, rather than only stem-like precursors, holds promise for treating chronic HBV infection.
Asunto(s)
Linfocitos T CD8-positivos , Virus de la Hepatitis B , Hepatitis B Crónica , Humanos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Hepatitis B Crónica/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal , Animales , Receptores OX40/metabolismo , Ratones , Receptor de Muerte Celular Programada 1/metabolismo , Antígenos CD/metabolismoRESUMEN
Vaccines have reduced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) morbidity and mortality, yet emerging variants challenge their effectiveness. The prevailing approach to updating vaccines targets the antibody response, operating under the presumption that it is the primary defense mechanism following vaccination or infection. This perspective, however, can overlook the role of T cells, particularly when antibody levels are low or absent. Here we show, through studies in mouse models lacking antibodies but maintaining functional B cells and lymphoid organs, that immunity conferred by prior infection or mRNA vaccination can protect against SARS-CoV-2 challenge independently of antibodies. Our findings, using three distinct models inclusive of a novel human/mouse ACE2 hybrid, highlight that CD8+ T cells are essential for combating severe infections, whereas CD4+ T cells contribute to managing milder cases, with interferon-γ having an important function in this antibody-independent defense. These findings highlight the importance of T cell responses in vaccine development, urging a broader perspective on protective immunity beyond just antibodies.
Asunto(s)
COVID-19 , Vacunas , Humanos , Animales , Ratones , SARS-CoV-2 , Linfocitos T CD8-positivos , COVID-19/prevención & control , Anticuerpos , Vacunación , Anticuerpos Antivirales , Anticuerpos NeutralizantesRESUMEN
Differentiation of CD4+ T cells into either follicular helper T (TFH) or type 1 helper T (TH1) cells influences the balance between humoral and cellular adaptive immunity, but the mechanisms whereby pathogens elicit distinct effector cells are incompletely understood. Here we analyzed the spatiotemporal dynamics of CD4+ T cells during infection with recombinant vesicular stomatitis virus (VSV), which induces early, potent neutralizing antibodies, or recombinant lymphocytic choriomeningitis virus (LCMV), which induces a vigorous cellular response but inefficient neutralizing antibodies, expressing the same T cell epitope. Early exposure of dendritic cells to type I interferon (IFN), which occurred during infection with VSV, induced production of the cytokine IL-6 and drove TFH cell polarization, whereas late exposure to type I IFN, which occurred during infection with LCMV, did not induce IL-6 and allowed differentiation into TH1 cells. Thus, tight spatiotemporal regulation of type I IFN shapes antiviral CD4+ T cell differentiation and might instruct vaccine design strategies.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Interferón Tipo I/metabolismo , Inmunidad Adaptativa , Traslado Adoptivo , Animales , Linfocitos T CD4-Positivos/clasificación , Diferenciación Celular/inmunología , Femenino , Interleucina-6/biosíntesis , Virus de la Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/patogenicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Análisis Espacio-Temporal , Linfocitos T Colaboradores-Inductores/inmunología , Células TH1/inmunología , Virus de la Estomatitis Vesicular Indiana/inmunología , Virus de la Estomatitis Vesicular Indiana/patogenicidad , Virus de la Estomatitis Vesicular New Jersey/inmunología , Virus de la Estomatitis Vesicular New Jersey/patogenicidadRESUMEN
Tight control of inflammatory gene expression by antagonistic environmental cues is key to ensure immune protection while preventing tissue damage. Prostaglandin E2 (PGE2) modulates macrophage activation during homeostasis and disease, but the underlying mechanisms remain incompletely characterized. Here we dissected the genomic properties of lipopolysaccharide (LPS)-induced genes whose expression is antagonized by PGE2. The latter molecule targeted a set of inflammatory gene enhancers that, already in unstimulated macrophages, displayed poorly permissive chromatin organization and were marked by the transcription factor myocyte enhancer factor 2A (MEF2A). Deletion of MEF2A phenocopied PGE2 treatment and abolished type I interferon (IFN I) induction upon exposure to innate immune stimuli. Mechanistically, PGE2 interfered with LPS-mediated activation of ERK5, a known transcriptional partner of MEF2. This study highlights principles of plasticity and adaptation in cells exposed to a complex environment and uncovers a transcriptional circuit for IFN I induction with relevance for infectious diseases or cancer.
Asunto(s)
Dinoprostona/inmunología , Interferón Tipo I/inmunología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Animales , Línea Celular , Células Cultivadas , Regulación de la Expresión Génica/inmunología , Humanos , Inflamación/genética , Inflamación/inmunología , Interferón Tipo I/biosíntesis , Lipopolisacáridos , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 7 Activada por Mitógenos/metabolismoRESUMEN
Kupffer cells (KCs) are highly abundant, intravascular, liver-resident macrophages known for their scavenger and phagocytic functions. KCs can also present antigens to CD8+ T cells and promote either tolerance or effector differentiation, but the mechanisms underlying these discrepant outcomes are poorly understood. Here, we used a mouse model of hepatitis B virus (HBV) infection, in which HBV-specific naive CD8+ T cells recognizing hepatocellular antigens are driven into a state of immune dysfunction, to identify a subset of KCs (referred to as KC2) that cross-presents hepatocellular antigens upon interleukin-2 (IL-2) administration, thus improving the antiviral function of T cells. Removing MHC-I from all KCs, including KC2, or selectively depleting KC2 impaired the capacity of IL-2 to revert the T cell dysfunction induced by intrahepatic priming. In summary, by sensing IL-2 and cross-presenting hepatocellular antigens, KC2 overcome the tolerogenic potential of the hepatic microenvironment, suggesting new strategies for boosting hepatic T cell immunity.
Asunto(s)
Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/inmunología , Reactividad Cruzada/inmunología , Interleucina-2/inmunología , Macrófagos del Hígado/inmunología , Animales , Hepatitis B/inmunología , Tolerancia Inmunológica/inmunología , Ratones , Ratones TransgénicosRESUMEN
Effector CD8(+) T cells (CD8 TE) play a key role during hepatotropic viral infections. Here, we used advanced imaging in mouse models of hepatitis B virus (HBV) pathogenesis to understand the mechanisms whereby these cells home to the liver, recognize antigens, and deploy effector functions. We show that circulating CD8 TE arrest within liver sinusoids by docking onto platelets previously adhered to sinusoidal hyaluronan via CD44. After the initial arrest, CD8 TE actively crawl along liver sinusoids and probe sub-sinusoidal hepatocytes for the presence of antigens by extending cytoplasmic protrusions through endothelial fenestrae. Hepatocellular antigen recognition triggers effector functions in a diapedesis-independent manner and is inhibited by the processes of sinusoidal defenestration and capillarization that characterize liver fibrosis. These findings reveal the dynamic behavior whereby CD8 TE control hepatotropic pathogens and suggest how liver fibrosis might reduce CD8 TE immune surveillance toward infected or transformed hepatocytes.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Virus de la Hepatitis B/fisiología , Hepatitis B/inmunología , Hígado/inmunología , Monitorización Inmunológica , Animales , Movimiento Celular , Células Endoteliales/metabolismo , Hepatitis B/patología , Hepatocitos/metabolismo , Ácido Hialurónico/metabolismo , Hígado/citología , Cirrosis Hepática , Ratones , Ratones Endogámicos C57BL , Adhesividad Plaquetaria , Organismos Libres de Patógenos EspecíficosRESUMEN
The responses of CD8+ T cells to hepatotropic viruses such as hepatitis B range from dysfunction to differentiation into effector cells, but the mechanisms that underlie these distinct outcomes remain poorly understood. Here we show that priming by Kupffer cells, which are not natural targets of hepatitis B, leads to differentiation of CD8+ T cells into effector cells that form dense, extravascular clusters of immotile cells scattered throughout the liver. By contrast, priming by hepatocytes, which are natural targets of hepatitis B, leads to local activation and proliferation of CD8+ T cells but not to differentiation into effector cells; these cells form loose, intravascular clusters of motile cells that coalesce around portal tracts. Transcriptomic and chromatin accessibility analyses reveal unique features of these dysfunctional CD8+ T cells, with limited overlap with those of exhausted or tolerant T cells; accordingly, CD8+ T cells primed by hepatocytes cannot be rescued by treatment with anti-PD-L1, but instead respond to IL-2. These findings suggest immunotherapeutic strategies against chronic hepatitis B infection.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Reactividad Cruzada/inmunología , Virus de la Hepatitis B/inmunología , Hepatocitos/inmunología , Hepatocitos/virología , Animales , Antígeno B7-H1/antagonistas & inhibidores , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular , Cromatina/metabolismo , Femenino , Hepatitis B/tratamiento farmacológico , Hepatitis B/inmunología , Hepatitis B/virología , Humanos , Tolerancia Inmunológica , Interleucina-2/inmunología , Interleucina-2/uso terapéutico , Macrófagos del Hígado/inmunología , Activación de Linfocitos , Masculino , Ratones , Transcriptoma/genéticaRESUMEN
The COVID-19 pandemic caused by SARS-CoV-2 has made the development of safe and effective vaccines a critical priority. To date, four vaccines have been approved by European and American authorities for preventing COVID-19, but the development of additional vaccine platforms with improved supply and logistics profiles remains a pressing need. Here we report the preclinical evaluation of a novel COVID-19 vaccine candidate based on the electroporation of engineered, synthetic cDNA encoding a viral antigen in the skeletal muscle. We constructed a set of prototype DNA vaccines expressing various forms of the SARS-CoV-2 spike (S) protein and assessed their immunogenicity in animal models. Among them, COVID-eVax-a DNA plasmid encoding a secreted monomeric form of SARS-CoV-2 S protein receptor-binding domain (RBD)-induced the most potent anti-SARS-CoV-2 neutralizing antibody responses (including against the current most common variants of concern) and a robust T cell response. Upon challenge with SARS-CoV-2, immunized K18-hACE2 transgenic mice showed reduced weight loss, improved pulmonary function, and lower viral replication in the lungs and brain. COVID-eVax conferred significant protection to ferrets upon SARS-CoV-2 challenge. In summary, this study identifies COVID-eVax as an ideal COVID-19 vaccine candidate suitable for clinical development. Accordingly, a combined phase I-II trial has recently started.
Asunto(s)
Vacunas contra la COVID-19/administración & dosificación , COVID-19/prevención & control , Inmunización/métodos , Modelos Animales , SARS-CoV-2/aislamiento & purificación , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas de ADN/administración & dosificación , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/genética , COVID-19/virología , Femenino , Hurones , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Dominios Proteicos , Ratas Sprague-DawleyRESUMEN
Chronic hepatitis B (CHB) is a major worldwide public health problem and novel anti-HBV therapies preventing liver disease progression to cirrhosis and hepatocellular carcinoma are urgently needed. Over the last several years, capsid assembly modulators (CAM) have emerged as clinically effective anti-HBV agents which can inhibit HBV replication in CHB patients. As part of a drug discovery program aimed at obtaining novel CAM endowed with high in vitro and in vivo antiviral activity, we identified a novel series of sulfamoylbenzamide (SBA) derivatives. Compound 10, one of the most in vitro potent SBA-derived CAM discovered to date, showed excellent pharmacokinetics in mice suitable for oral dosing. When studied in a transgenic mouse model of hepatic HBV replication, it was considerably more potent than NVR 3-778, the first sulfamoylbenzamide (SBA) CAM that entered clinical trials for CHB, at reducing viral replication in a dose-dependent fashion. We present herein the discovery process, the SAR analysis and the pre-clinical profile of this novel SBA CAM.
Asunto(s)
Antivirales , Cápside , Animales , Antivirales/farmacocinética , Proteínas de la Cápside , Virus de la Hepatitis B , Ratones , Ensamble de Virus , Replicación ViralRESUMEN
BACKGROUND & AIMS: Besides secreting pro-inflammatory cytokines, chemokines and effector molecules, effector CD8+ T cells that arise upon acute infection with certain viruses have been shown to produce the regulatory cytokine interleukin (IL)-10 and, therefore, contain immunopathology. Whether the same occurs during acute hepatitis B virus (HBV) infection and role that IL-10 might play in liver disease is currently unknown. METHODS: Mouse models of acute HBV pathogenesis, as well as chimpanzees and patients acutely infected with HBV, were used to analyse the role of CD8+ T cell-derived IL-10 in liver immunopathology. RESULTS: Mouse HBV-specific effector CD8+ T cells produce significant amounts of IL-10 upon in vivo antigen encounter. This is corroborated by longitudinal data in a chimpanzee acutely infected with HBV, where serum IL-10 was readily detectable and correlated with intrahepatic CD8+ T cell infiltration and liver disease severity. Unexpectedly, mouse and human CD8+ T cell-derived IL-10 was found to act in an autocrine/paracrine fashion to enhance IL-2 responsiveness, thus preventing antigen-induced HBV-specific effector CD8+ T cell apoptosis. Accordingly, the use of mouse models of HBV pathogenesis revealed that the IL-10 produced by effector CD8+ T cells promoted their own intrahepatic survival and, thus supported, rather than suppressed liver immunopathology. CONCLUSION: Effector CD8+ T cell-derived IL-10 enhances acute liver immunopathology. Altogether, these results extend our understanding of the cell- and tissue-specific role that IL-10 exerts in immune regulation. Lay summary: Interleukin-10 is mostly regarded as an immunosuppressive cytokine. We show here that HBV-specific CD8+ T cells produce IL-10 upon antigen recognition and that this cytokine enhances CD8+ T cell survival. As such, IL-10 paradoxically promotes rather than suppresses liver disease.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Interleucina-10/fisiología , Hígado/inmunología , Enfermedad Aguda , Animales , Apoptosis , Virus de la Hepatitis B/inmunología , Humanos , Interleucina-2/farmacología , Hígado/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Pan troglodytesRESUMEN
Chronic infection with hepatitis B virus (HBV) is a major risk factor for the development of hepatocellular carcinoma (HCC). The pathogenesis of HBV-associated HCC involves both viral and host factors. The latter include a functionally inefficient CD8(+) T-cell response that fails to clear the infection from the liver but sustains a chronic necroinflammatory process that contributes to the development of HCC. According to this scenario, amelioration of immune-mediated chronic liver injury may prevent HCC. Because platelets facilitate immune-mediated liver injury by promoting the hepatic accumulation of virus-specific CD8(+) T cells, we evaluated the long-term consequences of antiplatelet therapy in an HBV transgenic mouse model of chronic immune-mediated necroinflammatory liver disease that progresses to HCC. Treatment with aspirin and clopidogrel during the chronic phase of the disease diminished the number of intrahepatic HBV-specific CD8(+) T cells and HBV-nonspecific inflammatory cells, the severity of liver fibrosis, and the development of HCC. Antiplatelet therapy improved overall survival without causing significant side effects. In contrast, the same antiplatelet regimen had no antitumor effect when HCC was induced nonimmunologically by chronic exposure to a hepatotoxic chemical. The unprecedented observation that antiplatelet therapy inhibits or delays immune-mediated hepatocarcinogenesis suggests that platelets may be key players in the pathogenesis of HBV-associated liver cancer and supports the notion that immune-mediated necroinflammatory reactions are an important cause of hepatocellular transformation during chronic hepatitis.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Carcinoma Hepatocelular/prevención & control , Hepatitis B Crónica/complicaciones , Hepatitis B Crónica/inmunología , Neoplasias Hepáticas/prevención & control , Inhibidores de Agregación Plaquetaria/farmacología , Análisis de Varianza , Animales , Aspirina , Linfocitos T CD8-positivos/efectos de los fármacos , Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Clopidogrel , Ciclooxigenasa 1/metabolismo , Ciclooxigenasa 2/metabolismo , Hepatitis B Crónica/patología , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Ticlopidina/análogos & derivadosRESUMEN
CD8+ T cells are key antiviral effectors against hepatitis B virus (HBV), yet their number and function can be compromised in chronic infections. Preclinical HBV models displaying CD8+ T cell dysfunction showed that interleukin-2 (IL-2)-based treatment, unlike programmed cell death ligand 1 (PD-L1) checkpoint blockade, could reverse this defect, suggesting its therapeutic potential against HBV. However, IL-2's effectiveness is hindered by its pleiotropic nature, because its receptor is found on various immune cells, including regulatory T (Treg) cells and natural killer (NK) cells, which can counteract antiviral responses or contribute to toxicity, respectively. To address this, we developed a cis-targeted CD8-IL2 fusion protein, aiming to selectively stimulate dysfunctional CD8+ T cells in chronic HBV. In a mouse model, CD8-IL2 boosted the number of HBV-reactive CD8+ T cells in the liver without substantially altering Treg or NK cell counts. These expanded CD8+ T cells exhibited increased interferon-γ and granzyme B production, demonstrating enhanced functionality. CD8-IL2 treatment resulted in substantial antiviral effects, evidenced by marked reductions in viremia and antigenemia and HBV core antigen-positive hepatocytes. In contrast, an untargeted CTRL-IL2 led to predominant NK cell expansion, minimal CD8+ T cell expansion, negligible changes in effector molecules, and minimal antiviral activity. Human CD8-IL2 trials in cynomolgus monkeys mirrored these results, achieving a roughly 20-fold increase in peripheral blood CD8+ T cells without affecting NK or Treg cell numbers. These data support the development of CD8-IL2 as a therapy for chronic HBV infection.
Asunto(s)
Hepatitis B Crónica , Interleucina-2 , Humanos , Animales , Ratones , Virus de la Hepatitis B , Linfocitos T CD8-positivos , Hepatitis B Crónica/tratamiento farmacológico , Antivirales/farmacología , Antivirales/uso terapéuticoRESUMEN
Background & Aims: Liver diseases resulting from chronic HBV infection are a significant cause of morbidity and mortality. Vaccines that elicit T-cell responses capable of controlling the virus represent a treatment strategy with potential for long-term effects. Here, we evaluated vaccines that induce the activity of type I natural killer T (NKT) cells to limit viral replication and license stimulation of conventional antiviral T-cells. Methods: Vaccines were prepared by conjugating peptide epitopes to an NKT-cell agonist to promote co-delivery to antigen-presenting cells, encouraging NKT-cell licensing and stimulation of T cells. Activity of the conjugate vaccines was assessed in transgenic mice expressing the complete HBV genome, administered intravenously to maximise access to NKT cell-rich tissues. Results: The vaccines induced only limited antiviral activity in unmanipulated transgenic hosts, likely attributable to NKT-cell activation as T-cell tolerance to viral antigens is strong. However, in a model of chronic hepatitis B involving transfer of naive HBcAg-specific CD8+ T cells into the transgenic mice, which typically results in specific T-cell dysfunction without virus control, vaccines containing the targeted HBcAg epitope induced prolonged antiviral activity because of qualitatively improved T-cell stimulation. In a step towards a clinical product, vaccines were prepared using synthetic long peptides covering clusters of known HLA-binding epitopes and shown to be immunogenic in HLA transgenic mice. Predictions based on HLA distribution suggest a product containing three selected SLP-based vaccines could give >90 % worldwide coverage, with an average of 3.38 epitopes targeted per individual. Conclusions: The novel vaccines described show promise for further clinical development as a treatment for chronic hepatitis B. Impact and Implications: Although there are effective prophylactic vaccines for HBV infection, it is estimated that 350-400 million people worldwide have chronic hepatitis B, putting these individuals at significant risk of life-threatening liver diseases. Therapeutic vaccination aimed at activating or boosting HBV-specific T-cell responses holds potential as a strategy for treating chronic infection, but has so far met with limited success. Here, we show that a glycolipid-peptide conjugate vaccine designed to coordinate activity of type I NKT cells alongside conventional antiviral T cells has antiviral activity in a mouse model of chronic infection. It is anticipated that a product based on a combination of three such conjugates, each prepared using long peptides covering clusters of known HLA-binding epitopes, could be developed further as a treatment for chronic hepatitis B with broad global HLA coverage.
RESUMEN
Protection from viral infections depends on immunoglobulin isotype switching, which endows antibodies with effector functions. Here, we find that the protein kinase DYRK1A is essential for B cell-mediated protection from viral infection and effective vaccination through regulation of class switch recombination (CSR). Dyrk1a-deficient B cells are impaired in CSR activity in vivo and in vitro. Phosphoproteomic screens and kinase-activity assays identify MSH6, a DNA mismatch repair protein, as a direct substrate for DYRK1A, and deletion of a single phosphorylation site impaired CSR. After CSR and germinal center (GC) seeding, DYRK1A is required for attenuation of B cell proliferation. These findings demonstrate DYRK1A-mediated biological mechanisms of B cell immune responses that may be used for therapeutic manipulation in antibody-mediated autoimmunity.
Asunto(s)
Linfocitos B , Cambio de Clase de Inmunoglobulina , Fosforilación , Cambio de Clase de Inmunoglobulina/genética , Centro Germinal , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismoRESUMEN
Alongside vaccines, antiviral drugs are becoming an integral part of our response to the SARS-CoV-2 pandemic. Nirmatrelvir-an orally available inhibitor of the 3-chymotrypsin-like cysteine protease-has been shown to reduce the risk of progression to severe COVID-19. However, the impact of nirmatrelvir treatment on the development of SARS-CoV-2-specific adaptive immune responses is unknown. Here, by using mouse models of SARS-CoV-2 infection, we show that nirmatrelvir administration blunts the development of SARS-CoV-2-specific antibody and T cell responses. Accordingly, upon secondary challenge, nirmatrelvir-treated mice recruited significantly fewer memory T and B cells to the infected lungs and mediastinal lymph nodes, respectively. Together, the data highlight a potential negative impact of nirmatrelvir treatment with important implications for clinical management and might help explain the virological and/or symptomatic relapse after treatment completion reported in some individuals.
Asunto(s)
Inmunidad Adaptativa , Antivirales , Tratamiento Farmacológico de COVID-19 , Lactamas , Animales , Ratones , COVID-19/inmunología , SARS-CoV-2 , Antivirales/administración & dosificación , Inmunidad Adaptativa/efectos de los fármacos , Lactamas/administración & dosificación , Células T de Memoria/inmunología , Linfocitos B/inmunología , Ratones Endogámicos C57BLRESUMEN
The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespective of economical and climatic conditions. Outer membrane vesicles (OMVs) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMVs can be used as vaccines to induce potent immune responses against the associated proteins. Here, we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in vaccinated mice, resulting in the production of neutralizing antibodies (nAbs) with a titre higher than 1:300. The immunity induced by the vaccine is sufficient to protect the animals from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with the RBM of the Omicron BA.1 variant and that such engineered OMVs induce nAbs against Omicron BA.1 and BA.5, as measured using the pseudovirus neutralization infectivity assay. Importantly, we show that the RBM438-509 ancestral-OMVs elicited antibodies which efficiently neutralize in vitro both the homologous ancestral strain, the Omicron BA.1 and BA.5 variants with a neutralization titre ranging from 1:100 to 1:1500, suggesting its potential use as a vaccine targeting diverse SARS-CoV-2 variants. Altogether, given the convenience associated with the ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.
RESUMEN
BACKGROUND: Endometriosis, which is characterized by the growth of endometrial tissue at ectopic locations as well as vascular development and inflammation, is still an unmet clinical need since an optimal drug that allows for both pain and infertility management does not exist. Since both the eutopic and the ectopic endometrium express the vitamin D receptor (VDR), and VDR agonists are endowed with anti-proliferative and anti-inflammatory properties, we evaluated the effect of elocalcitol, a VDR agonist with low calcaemic liability, in a mouse model of experimentally induced endometriosis. METHODS AND RESULTS: Endometriosis was induced by injection of syngeneic endometrial tissue fragments into adult Balb/c female mice. After having confirmed by immunohistochemistry that endometriotic lesions developing in mice expressed VDR, the mice were administered with elocalcitol (100 µg/kg) or vehicle orally, once a day, for various durations of time. In this model, elocalcitol was able to reduce total lesion weight up to 70% upon treatment for 1 week before and 2 weeks after disease induction. Interestingly, a therapeutic effect was also observed on already established lesions. Elocalcitol was shown to reduce the capacity of mouse endometrial cells to adhere to collagen. In addition in treated mice, a decreased state of peritoneal inflammation was demonstrated by the inhibition of macrophage recruitment and inflammatory cytokine secretion. CONCLUSIONS: The VDR agonist elocalcitol inhibits lesion development in a validated mouse model of endometriosis, and exerts a protective effect on both the implantation and organization of transferred endometrial tissue. These preliminary data in mice provide a sound rationale for further testing in primate models and eventually in humans.
Asunto(s)
Calcitriol/análogos & derivados , Endometriosis/prevención & control , Inflamación/prevención & control , Peritoneo/patología , Receptores de Calcitriol/agonistas , Animales , Calcitriol/farmacología , Adhesión Celular , Proliferación Celular , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Hormona Liberadora de Gonadotropina/análogos & derivados , Humanos , Macrófagos Peritoneales/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Calcitriol/biosíntesis , Factores de TiempoRESUMEN
The development of a tractable small animal model faithfully reproducing human coronavirus disease 2019 pathogenesis would arguably meet a pressing need in biomedical research. Thus far, most investigators have used transgenic mice expressing the human ACE2 in epithelial cells (K18-hACE2 transgenic mice) that are intranasally instilled with a liquid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suspension under deep anesthesia. Unfortunately, this experimental approach results in disproportionate high central nervous system infection leading to fatal encephalitis, which is rarely observed in humans and severely limits this model's usefulness. Here, we describe the use of an inhalation tower system that allows exposure of unanesthetized mice to aerosolized virus under controlled conditions. Aerosol exposure of K18-hACE2 transgenic mice to SARS-CoV-2 resulted in robust viral replication in the respiratory tract, anosmia, and airway obstruction but did not lead to fatal viral neuroinvasion. When compared with intranasal inoculation, aerosol infection resulted in a more pronounced lung pathology including increased immune infiltration, fibrin deposition, and a transcriptional signature comparable to that observed in SARS-CoV-2infected patients. This model may prove useful for studies of viral transmission, disease pathogenesis (including long-term consequences of SARS-CoV-2 infection), and therapeutic interventions.
Asunto(s)
Enzima Convertidora de Angiotensina 2/genética , COVID-19/fisiopatología , Modelos Animales de Enfermedad , Encefalitis Viral/prevención & control , Queratina-18/genética , Rociadores Nasales , SARS-CoV-2/fisiología , Administración por Inhalación , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , COVID-19/inmunología , COVID-19/virología , Encefalitis Viral/mortalidad , Células Epiteliales/metabolismo , Femenino , Humanos , Queratina-18/metabolismo , Pulmón/inmunología , Pulmón/patología , Pulmón/fisiopatología , Masculino , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas/genética , Transcriptoma , Replicación ViralRESUMEN
Group 1 innate lymphoid cells (ILCs), which comprise both natural killer (NK) cells and ILC1s, are important innate effectors that can also positively and negatively influence adaptive immune responses. The latter function is generally ascribed to the ability of NK cells to recognize and kill activated T cells. Here, we used multiphoton intravital microscopy in mouse models of hepatitis B to study the intrahepatic behavior of group 1 ILCs and their cross-talk with hepatitis B virus (HBV)-specific CD8+ T cells. We found that hepatocellular antigen recognition by effector CD8+ T cells triggered a prominent increase in the number of hepatic NK cells and ILC1s. Group 1 ILCs colocalized and engaged in prolonged interactions with effector CD8+ T cells undergoing hepatocellular antigen recognition; however, they did not induce T cell apoptosis. Rather, group 1 ILCs constrained CD8+ T cell proliferation by controlling local interleukin-2 (IL-2) availability. Accordingly, group 1 ILC depletion, or genetic removal of their IL-2 receptor a chain, considerably increased the number of intrahepatic HBV-specific effector CD8+ T cells and the attendant immunopathology. Together, these results reveal a role for group 1 ILCs in controlling T cell-mediated liver immunopathology by limiting local IL-2 concentration and have implications for the treatment of chronic HBV infection.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Inmunidad Innata/inmunología , Interleucina-2/inmunología , Linfocitos/inmunología , Animales , Células Asesinas Naturales/inmunología , Ratones , Ratones Congénicos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Here, we provide detailed protocols for the isolation of mouse Kupffer cells - the liver-resident macrophages - for phenotypic (e.g., via flow cytometry, mass cytometry, or RNA-sequencing) analyses or for functional experiments involving cell culture. The procedures presented can be adapted for the isolation of other hepatic cell populations. For complete details on the use and execution of this protocol, please refer to De Simone et al. (2021).