RESUMO
Cytotoxic T lymphocytes (CTLs) mediate host defense against viral and intracellular bacterial infections and tumors. However, the magnitude of CTL response and their function needed to confer heterosubtypic immunity against influenza virus infection are unknown. We addressed the role of CD8+ T cells in the absence of any cross-reactive antibody responses to influenza viral proteins using an adenoviral vector expressing a 9mer amino acid sequence recognized by CD8+ T cells. Our results indicate that both CD8+ T cell frequency and function are crucial for heterosubtypic immunity. Low morbidity, lower viral lung titers, low to minimal lung pathology, and better survival upon heterosubtypic virus challenge correlated with the increased frequency of NP-specific CTLs. NP-CD8+ T cells induced by differential infection doses displayed distinct RNA transcriptome profiles and functional properties. CD8+ T cells induced by a high dose of influenza virus secreted significantly higher levels of IFN-γ and exhibited higher levels of cytotoxic function. The mice that received NP-CD8+ T cells from the high-dose virus recipients through adoptive transfer had lower viral titers following viral challenge than those induced by the low dose of virus, suggesting differential cellular programming by antigen dose. Enhanced NP-CD8+ T-cell functions induced by a higher dose of influenza virus strongly correlated with the increased expression of cellular and metabolic genes, indicating a shift to a more glycolytic metabolic phenotype. These findings have implications for developing effective T cell vaccines against infectious diseases and cancer. IMPORTANCE: Cytotoxic T lymphocytes (CTLs) are an important component of the adaptive immune system that clears virus-infected cells or tumor cells. Hence, developing next-generation vaccines that induce or recall CTL responses against cancer and infectious diseases is crucial. However, it is not clear if the frequency, function, or both are essential in conferring protection, as in the case of influenza. In this study, we demonstrate that both CTL frequency and function are crucial for providing heterosubtypic immunity to influenza by utilizing an Ad-viral vector expressing a CD8 epitope only to rule out the role of antibodies, single-cell RNA-seq analysis, as well as adoptive transfer experiments. Our findings have implications for developing T cell vaccines against infectious diseases and cancer.
Assuntos
Linfócitos T CD8-Positivos , Infecções por Orthomyxoviridae , Linfócitos T Citotóxicos , Animais , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Camundongos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T Citotóxicos/imunologia , Camundongos Endogâmicos C57BL , Feminino , Transferência Adotiva , Interferon gama/imunologia , Interferon gama/metabolismo , Proteínas do Nucleocapsídeo/imunologia , Pulmão/imunologia , Pulmão/virologia , Proteínas de Ligação a RNA/imunologia , Proteínas de Ligação a RNA/genética , Nucleoproteínas/imunologia , Nucleoproteínas/genética , Proteínas do Core Viral/imunologia , Proteínas do Core Viral/genéticaRESUMO
Point-of-care antigen tests are an important tool for SARS-CoV-2 detection. Antigen tests are less sensitive than real-time reverse transcriptase PCR (rRT-PCR). Data on the performance of the BinaxNOW antigen test compared to rRT-PCR and viral culture by symptom and known exposure status, timing during disease, or exposure period and demographic variables are limited. During 3 to 17 November 2020, we collected paired upper respiratory swab specimens to test for SARS-CoV-2 by rRT-PCR and Abbott BinaxNOW antigen test at two community testing sites in Pima County, Arizona. We administered a questionnaire to capture symptoms, known exposure status, and previous SARS-CoV-2 test results. Specimens positive by either test were analyzed by viral culture. Previously we showed overall BinaxNOW sensitivity was 52.5%. Here, we showed BinaxNOW sensitivity increased to 65.7% among currently symptomatic individuals reporting a known exposure. BinaxNOW sensitivity was lower among participants with a known exposure and previously symptomatic (32.4%) or never symptomatic (47.1%) within 14 days of testing. Sensitivity was 71.1% in participants within a week of symptom onset. In participants with a known exposure, sensitivity was highest 8 to 10 days postexposure (75%). The positive predictive value for recovery of virus in cell culture was 56.7% for BinaxNOW-positive and 35.4% for rRT-PCR-positive specimens. Result reporting time was 2.5 h for BinaxNOW and 26 h for rRT-PCR. Point-of-care antigen tests have a shorter turnaround time than laboratory-based nucleic acid amplification tests, which allows for more rapid identification of infected individuals. Antigen test sensitivity limitations are important to consider when developing a testing program.
Assuntos
COVID-19 , SARS-CoV-2 , Antígenos Virais , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e EspecificidadeRESUMO
We used the BinaxNOW COVID-19 Ag Card to screen 1,540 asymptomatic college students for severe acute respiratory syndrome coronavirus 2 in a low-prevalence setting. Compared with reverse transcription PCR, BinaxNOW showed 20% overall sensitivity; among participants with culturable virus, sensitivity was 60%. BinaxNOW provides point-of-care screening but misses many infections.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Sistemas Automatizados de Assistência Junto ao Leito , Sensibilidade e Especificidade , EstudantesRESUMO
We evaluated the performance of self-collected anterior nasal swab (ANS) and saliva samples compared with healthcare worker-collected nasopharyngeal swab specimens used to test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We used the same PCR diagnostic panel to test all self-collected and healthcare worker-collected samples from participants at a public hospital in Atlanta, Georgia, USA. Among 1,076 participants, 51.9% were men, 57.1% were >50 years of age, 81.2% were Black (non-Hispanic), and 74.9% reported >1 chronic medical condition. In total, 8.0% tested positive for SARS-CoV-2. Compared with nasopharyngeal swab samples, ANS samples had a sensitivity of 59% and saliva samples a sensitivity of 68%. Among participants tested 3-7 days after symptom onset, ANS samples had a sensitivity of 80% and saliva samples a sensitivity of 85%. Sensitivity varied by specimen type and patient characteristics. These findings can help physicians interpret PCR results for SARS-CoV-2.
Assuntos
COVID-19 , SARS-CoV-2 , Idoso de 80 Anos ou mais , Teste para COVID-19 , Georgia , Humanos , Masculino , Nasofaringe , Saliva , Manejo de EspécimesRESUMO
Rapid antigen tests, such as the Abbott BinaxNOW COVID-19 Ag Card (BinaxNOW), offer results more rapidly (approximately 15-30 minutes) and at a lower cost than do highly sensitive nucleic acid amplification tests (NAATs) (1). Rapid antigen tests have received Food and Drug Administration (FDA) Emergency Use Authorization (EUA) for use in symptomatic persons (2), but data are lacking on test performance in asymptomatic persons to inform expanded screening testing to rapidly identify and isolate infected persons (3). To evaluate the performance of the BinaxNOW rapid antigen test, it was used along with real-time reverse transcription-polymerase chain reaction (RT-PCR) testing to analyze 3,419 paired specimens collected from persons aged ≥10 years at two community testing sites in Pima County, Arizona, during November 3-17, 2020. Viral culture was performed on 274 of 303 residual real-time RT-PCR specimens with positive results by either test (29 were not available for culture). Compared with real-time RT-PCR testing, the BinaxNOW antigen test had a sensitivity of 64.2% for specimens from symptomatic persons and 35.8% for specimens from asymptomatic persons, with near 100% specificity in specimens from both groups. Virus was cultured from 96 of 274 (35.0%) specimens, including 85 (57.8%) of 147 with concordant antigen and real-time RT-PCR positive results, 11 (8.9%) of 124 with false-negative antigen test results, and none of three with false-positive antigen test results. Among specimens positive for viral culture, sensitivity was 92.6% for symptomatic and 78.6% for asymptomatic individuals. When the pretest probability for receiving positive test results for SARS-CoV-2 is elevated (e.g., in symptomatic persons or in persons with a known COVID-19 exposure), a negative antigen test result should be confirmed by NAAT (1). Despite a lower sensitivity to detect infection, rapid antigen tests can be an important tool for screening because of their quick turnaround time, lower costs and resource needs, high specificity, and high positive predictive value (PPV) in settings of high pretest probability. The faster turnaround time of the antigen test can help limit transmission by more rapidly identifying infectious persons for isolation, particularly when used as a component of serial testing strategies.
Assuntos
Teste Sorológico para COVID-19 , COVID-19/diagnóstico , Serviços de Saúde Comunitária , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Arizona/epidemiologia , COVID-19/epidemiologia , COVID-19/prevenção & controle , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Fatores de Tempo , Adulto JovemRESUMO
Somatic hypermutation generates a myriad of Ab mutants in Ag-specific B cells, from which high-affinity mutants are selected. Chickens, sheep, and rabbits use nontemplated point mutations and templated mutations via gene conversion to diversify their expressed Ig loci, whereas mice and humans rely solely on untemplated somatic point mutations. In this study, we demonstrate that, in addition to untemplated point mutations, templated mutagenesis readily occurs at the murine and human Ig loci. We provide two distinct lines of evidence that are not explained by the Neuberger model of somatic hypermutation: 1) across multiple data sets there is significant linkage disequilibrium between individual mutations, especially among close mutations, and 2) among those mutations, those <8 bp apart are significantly more likely to match microhomologous regions in the IgHV repertoire than predicted by the mutation profiles of somatic hypermutation. Together, this supports the role of templated mutagenesis during somatic diversification of Ag-activated B cells.
Assuntos
Ligação Genética , Loci Gênicos , Cadeias Pesadas de Imunoglobulinas/genética , Mutagênese , Hipermutação Somática de Imunoglobulina , Animais , DNA Helicases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Centro Germinativo/imunologia , Humanos , Região Variável de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação , Plasmócitos/imunologiaRESUMO
Hepatitis C virus (HCV) is a significant public health concern with approximately 160 million people infected worldwide. HCV infection often results in chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. No vaccine is available and current therapies are effective against some, but not all, genotypes. HCV is an enveloped virus with two surface glycoproteins (E1 and E2). E2 binds to the host cell through interactions with scavenger receptor class B type I (SR-BI) and CD81, and serves as a target for neutralizing antibodies. Little is known about the molecular mechanism that mediates cell entry and membrane fusion, although E2 is predicted to be a class II viral fusion protein. Here we describe the structure of the E2 core domain in complex with an antigen-binding fragment (Fab) at 2.4 Å resolution. The E2 core has a compact, globular domain structure, consisting mostly of ß-strands and random coil with two small α-helices. The strands are arranged in two, perpendicular sheets (A and B), which are held together by an extensive hydrophobic core and disulphide bonds. Sheet A has an IgG-like fold that is commonly found in viral and cellular proteins, whereas sheet B represents a novel fold. Solution-based studies demonstrate that the full-length E2 ectodomain has a similar globular architecture and does not undergo significant conformational or oligomeric rearrangements on exposure to low pH. Thus, the IgG-like fold is the only feature that E2 shares with class II membrane fusion proteins. These results provide unprecedented insights into HCV entry and will assist in developing an HCV vaccine and new inhibitors.
Assuntos
Hepacivirus/química , Proteínas do Envelope Viral/química , Cristalografia por Raios X , Dissulfetos/química , Hepacivirus/fisiologia , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/química , Modelos Moleculares , Dobramento de Proteína , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Propriedades de Superfície , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão , Vacinas contra Hepatite Viral , Internalização do VírusRESUMO
AIMS: PD-1 inhibitors facilitate immune response against certain tumour types, including melanoma. These drugs have led to prolonged survival but can also result in autoimmune-type side effects, including gastrointestinal inflammation. The histopathological effects of this medication class have not been well studied. METHODS AND RESULTS: We identified 37 gastrointestinal tract biopsies from 20 patients taking a PD-1 or PD-L1 inhibitor and evaluated clinicopathological findings. Diarrhoea was the most common symptom, and endoscopic findings ranged from mild erythema to erosion/ulceration. Common histological findings included lamina propria expansion, villous blunting (if applicable), intra-epithelial neutrophils and increased crypt/gland apoptosis, although intra-epithelial lymphocytes were rarely prominent. A few cases showed crypt rupture with resultant histiocytic/granulomatous response. Most patients responded to drug cessation and/or steroids, but follow-up endoscopies were not performed. CONCLUSIONS: PD-1/PD-L1 inhibitors can cause gastritis, enteritis and colitis, similar to other immunomodulatory antibodies (such as CTLA-4 inhibitors and PI3Kδ inhibitors), but the histological findings vary somewhat among drug classes. Clinical history, lack of prominent intra-epithelial lymphocytes and crypt rupture may help to distinguish PD-1 inhibitor gastroenterocolitis from mimics, which include other medication effect, inflammatory bowel disease, graft-versus-host disease, cytomegalovirus infection and autoimmune enteropathy.
Assuntos
Antineoplásicos/efeitos adversos , Enterocolite/induzido quimicamente , Enterocolite/patologia , Gastrite/induzido quimicamente , Gastrite/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Humanizados/efeitos adversos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/tratamento farmacológico , Nivolumabe , Receptor de Morte Celular Programada 1/antagonistas & inibidoresRESUMO
BACKGROUND: Understanding the immune response kinetics to SARS-CoV-2 infection and COVID-19 vaccination is important in nursing home (NH) residents, a high-risk population. METHODS: An observational longitudinal evaluation of 37 consenting vaccinated NH residents with/without SARS-CoV-2 infection from October 2020 to July 2022 was conducted to characterize the immune response to spike protein due to infection and/or mRNA COVID-19 vaccine. Antibodies (IgG) to SARS-CoV-2 full-length spike, nucleocapsid, and receptor binding domain protein antigens were measured, and surrogate virus neutralization capacity was assessed using Meso Scale Discovery immunoassays. The participant's spike exposure status varied depending on the acquisition of infection or receipt of a vaccine dose. Longitudinal linear mixed effects modeling was used to describe trajectories based on the participant's last infection or vaccination; the primary series mRNA COVID-19 vaccine was considered two spike exposures. Mean antibody titer values from participants who developed an infection post receipt of mRNA COVID-19 vaccine were compared with those who did not. In a subset of participants (n = 15), memory B cell (MBC) S-specific IgG (%S IgG) responses were assessed using an ELISPOT assay. RESULTS: The median age of the 37 participants at enrollment was 70.5 years; 30 (81%) had prior SARS-CoV-2 infection, and 76% received Pfizer-BioNTech and 24% Moderna homologous vaccines. After an observed augmented effect with each spike exposure, a decline in the immune response, including %S IgG MBCs, was observed over time; the percent decline decreased with increasing spike exposures. Participants who developed an infection at least two weeks post-receipt of a vaccine were observed to have lower humoral antibody levels than those who did not develop an infection post-receipt. CONCLUSIONS: These findings suggest that understanding the durability of immune responses in this vulnerable NH population can help inform public health policy regarding the timing of booster vaccinations as new variants display immune escape.
Assuntos
COVID-19 , Humanos , Idoso , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Georgia , SARS-CoV-2 , Vacinação , Imunidade , Casas de Saúde , RNA Mensageiro , Imunoglobulina G , Anticorpos AntiviraisRESUMO
Influenza infections cause several million cases of severe respiratory illness, hospitalizations, and hundreds of thousands of deaths globally. Secondary infections are a leading cause of influenza's high morbidity and mortality, and significantly factored into the severity of the 1918, 1968, and 2009 pandemics. Furthermore, there is an increased incidence of other respiratory infections even in vaccinated individuals during influenza season. Putative mechanisms responsible for vaccine failures against influenza as well as other respiratory infections during influenza season are investigated. Peripheral blood mononuclear cells (PBMCs) are used from influenza vaccinated individuals to assess antigen-specific responses to influenza, measles, and varicella. The observations made in humans to a mouse model to unravel the mechanism is confirmed and extended. Infection with influenza virus suppresses an ongoing adaptive response to vaccination against influenza as well as other respiratory pathogens, i.e., Adenovirus and Streptococcus pneumoniae by preferentially infecting and killing activated lymphocytes which express elevated levels of sialic acid receptors. These findings propose a new mechanism for the high incidence of secondary respiratory infections due to bacteria and other viruses as well as vaccine failures to influenza and other respiratory pathogens even in immune individuals due to influenza viral infections.
Assuntos
Imunidade Adaptativa/imunologia , Influenza Humana/imunologia , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Malaria control and interventions including long-lasting insecticide-treated nets, indoor residual spraying, and intermittent preventative treatment in pregnancy have resulted in a significant reduction in the number of Plasmodium falciparum cases. Considerable efforts have been devoted to P. falciparum vaccines development with much less to P. vivax. Transmission-blocking vaccines, which can elicit antibodies targeting Plasmodium antigens expressed during sexual stage development and interrupt transmission, offer an alternative strategy to achieve malaria control. The post-fertilization antigen P25 mediates several functions essential to ookinete survival but is poorly immunogenic in humans. Previous clinical trials targeting this antigen have suggested that conjugation to a carrier protein could improve the immunogenicity of P25. Here we report the production, and characterization of a vaccine candidate composed of a chimeric P. vivax Merozoite Surface Protein 1 (cPvMSP1) genetically fused to P. vivax P25 (Pvs25) designed to enhance CD4+ T cell responses and its assessment in a murine model. We demonstrate that antibodies elicited by immunization with this chimeric protein recognize both the erythrocytic and sexual stages and are able to block the transmission of P. vivax field isolates in direct membrane-feeding assays. These findings provide support for the continued development of multi-stage transmission blocking vaccines targeting the life-cycle stage responsible for clinical disease and the sexual-stage development accountable for disease transmission simultaneously.
Assuntos
Anticorpos Antiprotozoários/sangue , Formação de Anticorpos , Antígenos de Protozoários/imunologia , Antígenos de Superfície/imunologia , Transmissão de Doença Infecciosa/prevenção & controle , Vacinas Antimaláricas/imunologia , Malária Vivax/prevenção & controle , Plasmodium vivax/imunologia , Animais , Homólogo 5 da Proteína Cromobox , Vacinas Antimaláricas/administração & dosagem , Malária Vivax/transmissão , Proteína 1 de Superfície de Merozoito/imunologia , Camundongos , Proteínas Recombinantes de Fusão/imunologia , Fatores de Tempo , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
B cells play a major role in the adaptive immune response by producing antigen-specific antibodies against pathogens and imparting immunological memory. Following infection or vaccination, antibody-secreting B cells and memory B cells are generated in specialized regions of lymph nodes and spleens, called germinal centers. Here, we report a fully synthetic ex-vivo system that recapitulates the generation of antigen-specific germinal-center (GC) like B cells using material-surface driven polyvalent signaling. This synthetic germinal center (sGC) reaction was effectively induced using biomaterial-based artificial "follicular T helper cells (TFH)" that provided both natural CD40-CD40L ligation as well as crosslinking of CD40 and by mimicking artificial "follicular dendritic cells (FDC)" to provide efficient, polyvalent antigen presentation. The artificial sGC reaction resulted in efficient B cell expansion, immunoglobulin (Ig) class switching, and expression of germinal center phenotypes. Antigen presentation during sGC reaction selectively enhanced the antigen-specific B cell population and induced somatic hyper-mutations for potential affinity maturation. The resulting B cell population consisted primarily of GC-like B cells (centrocytes) as well as some plasma-like B cells expressing CD138. With concurrent cell sorting, we successfully created highly enriched populations of antigen-specific B cells. Adoptive transfer of these GC-like B cells into non-irradiated isogeneic or non-lethally irradiated congenic recipient mice showed successful engraftment and survival of the donor cells for the 4 week test period. We show that this material-surface driven sGC reaction can be successfully applied to not only splenic B cells but also B cells isolated from more therapeutically relevant sources such as peripheral blood mononuclear cells (PBMCs), thus making our current work an exciting prospect in the new era of personalized medicine and custom-immunotherapy.
Assuntos
Centro Germinativo , Imunidade Humoral , Imunidade Adaptativa , Animais , Linfócitos B/imunologia , Antígenos CD40/imunologia , Centro Germinativo/imunologia , Humanos , Memória Imunológica , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Avian influenza virus infection is a serious public health threat and preventive vaccination is the most cost-effective public health intervention strategy. Unfortunately, currently available unadjuvanted avian influenza vaccines are poorly immunogenic and alternative vaccine formulations and delivery strategies are in urgent need to reduce the high risk of avian influenza pandemics. Cationic polymers have been widely used as vectors for gene delivery in vitro and in vivo. In this study, we formulated H5N1 influenza vaccines with GenJet™ or in vivo-jetPEI®, and showed that these formulations significantly enhanced the immunogenicity of H5N1 vaccines and conferred protective immunity in a mouse model. Detailed analyses of adaptive immune responses revealed that both formulations induced mixed TH1/TH2 antigen-specific CD4 T-cell responses, antigen-specific cytotoxic CD8 T-cell and memory B-cell responses. Our findings suggest that cationic polymers merit future development as potential adjuvants for mucosal delivery of poorly immunogenic vaccines.
Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Imunidade Celular/efeitos dos fármacos , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/administração & dosagem , Influenza Aviária/imunologia , Vacinas Sintéticas/administração & dosagem , Administração Intranasal , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/biossíntese , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Composição de Medicamentos , Feminino , Imunogenicidade da Vacina , Indicadores e Reagentes/química , Indicadores e Reagentes/metabolismo , Vacinas contra Influenza/genética , Vacinas contra Influenza/metabolismo , Vacinas contra Influenza/uso terapêutico , Influenza Aviária/metabolismo , Influenza Aviária/prevenção & controle , Influenza Aviária/virologia , Camundongos Endogâmicos BALB C , Aves Domésticas , Análise de Sobrevida , Vacinas Sintéticas/genética , Vacinas Sintéticas/metabolismo , Vacinas Sintéticas/uso terapêutico , Redução de Peso/efeitos dos fármacosRESUMO
Long-lived plasma cells are critical to humoral immunity as a lifelong source of protective antibodies. Antigen-activated B cells-with T-cell help-undergo affinity maturation within germinal centres and persist as long-lived IgG plasma cells in the bone marrow. Here we show that antigen-specific, induced IgM plasma cells also persist for a lifetime. Unlike long-lived IgG plasma cells, which develop in germinal centres and then home to the bone marrow, IgM plasma cells are primarily retained within the spleen and can develop even in the absence of germinal centres. Interestingly, their expressed IgV loci exhibit somatic mutations introduced by the activation-induced cytidine deaminase (AID). However, these IgM plasma cells are probably not antigen-selected, as replacement mutations are spread through the variable segment and not enriched within the CDRs. Finally, antibodies from long-lived IgM plasma cells provide protective host immunity against a lethal virus challenge.