RESUMO
Introduction: The early transcription unit 3 (E3) of human adenoviruses (HAdVs) encodes several immunoevasins, including the E3/49K protein, which is unique for species D of HAdVs. It is expressed as surface transmembrane protein and shed. E3/49K of HAdV-D64 binds to the protein tyrosine phosphatase surface receptor CD45, thereby modulating activation of T and NK cells. Methods: Considering that E3/49K represents the most polymorphic viral protein among species D HAdVs, we demonstrate here that all tested E3/49K orthologs bind to the immunologically important regulator CD45. Thus, this feature is conserved regardless of the pathological associations of the respective HAdV types. Results: It appeared that modulation of CD45 is a unique property restricted to HAdVs of species D. Moreover, E3/49K treatment inhibited B cell receptor (BCR) signaling and impaired BCR signal phenotypes. The latter were highly comparable to B cells having defects in the expression of CD45, suggesting E3/49K as a potential tool to investigate CD45 specific functions. Conclusion: We identified B cells as new direct target of E3/49K-mediated immune modulation, representing a novel viral immunosubversive mechanism.
Assuntos
Proteínas E3 de Adenovirus , Adenovírus Humanos , Linfócitos B , Antígenos Comuns de Leucócito , Receptores de Antígenos de Linfócitos B , Transdução de Sinais , Humanos , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais/imunologia , Antígenos Comuns de Leucócito/metabolismo , Antígenos Comuns de Leucócito/imunologia , Adenovírus Humanos/imunologia , Proteínas E3 de Adenovirus/imunologia , Proteínas E3 de Adenovirus/metabolismo , Proteínas E3 de Adenovirus/genética , Linfócitos B/imunologia , Linfócitos B/metabolismo , Infecções por Adenovirus Humanos/imunologia , Infecções por Adenovirus Humanos/virologia , Infecções por Adenovirus Humanos/metabolismo , Células HEK293RESUMO
Human adenoviruses (HAdVs) are small DNA viruses that generally cause mild disease. Certain strains, particularly those belonging to species B HAdVs, can cause severe pneumonia and have a relatively high mortality rate. Little is known about the molecular aspects of how these highly pathogenic species affect the infected cell and how they suppress innate immunity. The present study provides molecular insights into how species B adenoviruses suppress the interferon signaling pathway. Our study shows that these viruses, unlike HAdV-C2, are resistant to type I interferon. This resistance likely arises due to the highly efficient suppression of interferon-stimulated gene expression. Unlike in HAdV-C2, HAdV-B7 and B14 sequester STAT2 and RNA polymerase II from interferon-stimulated gene promoters in infected cells. This results in suppressed interferon- stimulated gene activation. In addition, we show that RuvBL1 and RuvBL2, cofactors important for RNA polymerase II recruitment to promoters and interferon-stimulated gene activation, are redirected to the cytoplasm forming high molecular weight complexes that, likely, are unable to associate with chromatin. Proteomic analysis also identified key differences in the way these viruses affect the host cell, providing insights into species B-associated high pathogenicity. Curiously, we observed that at the level of protein expression changes to the infected cell, HAdV-C2 and B7 were more similar than those of the same species, B7 and B14. Collectively, our study represents the first such study of innate immune suppression by the highly pathogenic HAdV-B7 and B14, laying an important foundation for future investigations.IMPORTANCEHuman adenoviruses form a large family of double-stranded DNA viruses known for a variety of usually mild diseases. Certain strains of human adenovirus cause severe pneumonia leading to much higher mortality and morbidity than most other strains. The reasons for this enhanced pathogenicity are unknown. Our study provides a molecular investigation of how these highly pathogenic strains might inactivate the interferon signaling pathway, highlighting the lack of sensitivity of these viruses to type I interferon in general while providing a global picture of how viral changes in cellular proteins drive worse disease outcomes.
Assuntos
Adenovírus Humanos , Interferon Tipo I , Humanos , Adenovírus Humanos/genética , Adenovírus Humanos/patogenicidade , Adenovírus Humanos/fisiologia , Adenovírus Humanos/imunologia , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Interferon Tipo I/genética , Fator de Transcrição STAT2/metabolismo , Fator de Transcrição STAT2/genética , Imunidade Inata , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Transdução de Sinais , Infecções por Adenovirus Humanos/virologia , Infecções por Adenovirus Humanos/imunologia , Virulência , Interações Hospedeiro-Patógeno/imunologia , Animais , Regiões Promotoras Genéticas , Evasão da Resposta Imune , Células A549RESUMO
The exacerbation of pneumonia in children with human adenovirus type 3 (HAdV-3E) is secondary to a Staphylococcus aureus (S. aureus) infection. The influence of host-pathogen interactions on disease progression remains unclear. It is important to note that S. aureus infections following an HAdV-3E infection are frequently observed in clinical settings, yet the underlying susceptibility mechanisms are not fully understood. This study utilized an A549 cell model to investigate secondary infection with S. aureus following an HAdV-3E infection. The findings suggest that HAdV-3E exacerbates the S. aureus infection by intensifying lung epithelial cell damage. The results highlight the role of HAdV-3E in enhancing the interferon signaling pathway through RIG-I (DDX58), resulting in the increased expression of interferon-stimulating factors like MX1, RSAD2, and USP18. The increase in interferon-stimulating factors inhibits the NF-κB and MAPK/P38 pro-inflammatory signaling pathways. These findings reveal new mechanisms of action for HAdV-3E and S. aureus in secondary infections, enhancing our comprehension of pathogenesis.
Assuntos
Infecções por Adenovirus Humanos , Adenovírus Humanos , Proteína DEAD-box 58 , Transdução de Sinais , Infecções Estafilocócicas , Staphylococcus aureus , Humanos , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Infecções por Adenovirus Humanos/metabolismo , Infecções por Adenovirus Humanos/imunologia , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/fisiologia , Adenovírus Humanos/imunologia , Coinfecção/microbiologia , Proteína DEAD-box 58/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Inflamação/metabolismo , NF-kappa B/metabolismo , Receptores Imunológicos/metabolismo , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/patogenicidade , Ubiquitina TiolesteraseRESUMO
Human adenovirus type 7 (HAdV-7) is a significant viral pathogen that causes respiratory infections in children. Currently, there are no specific antiviral drugs or vaccines for children targeting HAdV-7, and the mechanisms of its pathogenesis remain unclear. The NLRP3 inflammasome-driven inflammatory cascade plays a crucial role in the host's antiviral immunity. Our previous study demonstrated that HAdV-7 infection activates the NLRP3 inflammasome. Building upon this finding, our current study has identified the L4 100 kDa protein encoded by HAdV-7 as the primary viral component responsible for NLRP3 inflammasome activation. By utilizing techniques such as co-immunoprecipitation, we have confirmed that the 100 kDa protein interacts with the NLRP3 protein and facilitates the assembly of the NLRP3 inflammasome by binding specifically to the NACHT and LRR domains of NLRP3. These insights offer a deeper understanding of HAdV-7 pathogenesis and contribute to the development of novel antiviral therapies.
Assuntos
Infecções por Adenovirus Humanos , Adenovírus Humanos , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteínas não Estruturais Virais , Humanos , Infecções por Adenovirus Humanos/imunologia , Infecções por Adenovirus Humanos/metabolismo , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/imunologia , Adenovírus Humanos/fisiologia , Células HEK293 , Inflamassomos/metabolismo , Inflamassomos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Ligação Proteica , Proteínas Virais/metabolismo , Proteínas Virais/imunologia , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismoRESUMO
BACKGROUND: Human adenoviruses (HAdVs) are extensively used as vectors for vaccines development and cancer therapy. People who already have antibodies against HAdVs, on the other hand, would have an impact on the preventative or therapeutic effect. This review focuses primarily on the prevalence of pre-existing antibodies against HAdVs in distinct geographical populations. SUMMARY: After screening, 64 studies from 31 countries between 1962 and 2021 were selected, totaling 39,427 samples. The total prevalence of preexisting antibodies to HAdVs varied by country or location, ranging from 2.00 to 95.70%. Southeast Asia had the highest prevalence (54.57%) while Europe had the lowest (18.17%). The prevalence in practically all developing nations was higher than in developed nations. Adults have a greater frequency than children and newborns in most nations. The primary HAdV antibody types varied by country. Adults in China, the USA, the United Kingdom, and Belgium had the lowest prevalence of preexisting antibodies against HAdV55, HAdV37, HAdV8, and HAdV36, respectively. Children in the USA, China, the United Kingdom, and Japan had the lowest rates of HAdV48, HAdV11, HAdV8, and HAdV40. The frequency of antibodies differed significantly between military and civilian groups. KEY MESSAGES: Preexisting antibodies against various types of HAdVs differed greatly throughout worldwide populations. Future development of HAdV-vector vaccines and medicines should focus on preexisting antibodies in target groups rather than a "one-size-fits-all" strategy. It might be advantageous in selecting HAdV vectors for studying the prevalence of preexisting antibodies against HAdVs in different locations and people throughout the world.
Assuntos
Infecções por Adenovirus Humanos , Adenovírus Humanos , Anticorpos Antivirais , Humanos , Adenovírus Humanos/imunologia , Anticorpos Antivirais/sangue , Infecções por Adenovirus Humanos/epidemiologia , Infecções por Adenovirus Humanos/imunologia , Prevalência , Saúde Global , Criança , Adulto , Estudos SoroepidemiológicosRESUMO
Human Adenoviruses (HAdV) are nearly ubiquitous pathogens comprising numerous sub-types that infect various tissues and organs. Among many encoded proteins that facilitate viral replication and subversion of host cellular processes, the viral E4orf1 protein has emerged as an intriguing yet under-investigated player in the complex interplay between the virus and its host. E4orf1 has gained attention as a metabolism activator and oncogenic agent, while recent research is showing that E4orf1 may play a more important role in modulating cellular pathways such as PI3K-Akt-mTOR, Ras, the immune response and further HAdV replication stages than previously anticipated. In this review, we aim to explore the structure, molecular mechanisms, and biological functions of E4orf1, shedding light on its potentially multifaceted roles during HAdV infection, including metabolic diseases and oncogenesis. Furthermore, we discuss the role of functional E4orf1 in biotechnological applications such as Adenovirus (AdV) vaccine vectors and oncolytic AdV. By dissecting the intricate relationships between HAdV types and E4orf1 proteins, this review provides valuable insights into viral pathogenesis and points to promising areas of future research.
Assuntos
Proteínas E4 de Adenovirus , Adenovírus Humanos , Carcinogênese , Replicação Viral , Humanos , Adenovírus Humanos/genética , Adenovírus Humanos/imunologia , Carcinogênese/imunologia , Carcinogênese/genética , Proteínas E4 de Adenovirus/metabolismo , Proteínas E4 de Adenovirus/genética , Infecções por Adenovirus Humanos/imunologia , Infecções por Adenovirus Humanos/virologia , Obesidade/imunologia , Obesidade/metabolismo , Obesidade/virologia , Animais , Vetores Genéticos/imunologia , Vetores Genéticos/genética , Terapia Viral Oncolítica/métodosRESUMO
Detection of pathogenic viruses for point-of-care applications has attracted great attention since the COVID-19 pandemic. Current virus diagnostic tools are laborious and expensive, while requiring medically trained staff. Although user-friendly and cost-effective biosensors are utilized for virus detection, many of them rely on recognition elements that suffer major drawbacks. Herein, computationally designed epitope-imprinted polymers (eIPs) are conjugated with a portable piezoelectric sensing platform to establish a sensitive and robust biosensor for the human pathogenic adenovirus (HAdV). The template epitope is selected from the knob part of the HAdV capsid, ensuring surface accessibility. Computational simulations are performed to evaluate the conformational stability of the selected epitope. Further, molecular dynamics simulations are executed to investigate the interactions between the epitope and the different functional monomers for the smart design of eIPs. The HAdV epitope is imprinted via the solid-phase synthesis method to produce eIPs using in silico-selected ingredients. The synthetic receptors show a remarkable detection sensitivity (LOD: 102 pfu mL-1) and affinity (dissociation constant (Kd): 6.48 × 10-12 M) for HAdV. Moreover, the computational eIPs lead to around twofold improved binding behavior than the eIPs synthesized with a well-established conventional recipe. The proposed computational strategy holds enormous potential for the intelligent design of ultrasensitive imprinted polymer binders.
Assuntos
Adenovírus Humanos , Epitopos , Humanos , Adenovírus Humanos/imunologia , Adenovírus Humanos/química , Epitopos/imunologia , Epitopos/química , Técnicas Biossensoriais/métodos , Polímeros/química , Simulação de Dinâmica Molecular , Polímeros Molecularmente Impressos/química , Impressão Molecular/métodos , Limite de Detecção , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/químicaRESUMO
Intramuscular delivery of human adenovirus (HAdV)-based vaccines leads to rapid recruitment of neutrophils, which then release antimicrobial peptides/proteins (AMPs). How these AMPs influence vaccine efficacy over the subsequent 24 h is poorly understood. In this study, we asked if human neutrophil protein 1 (HNP-1), an α-defensin that influences direct and indirect innate immune responses to a range of pathogens, impacts the response of human phagocytes to three HAdV species/types (HAdV-C5, -D26, -B35). We show that HNP-1 binds to the capsids and redirects HAdV-C5, -D26, and -B35 to Toll-like receptor 4 (TLR4), which leads to internalization, an NLRP3-mediated inflammasome response, and interleukin 1 beta (IL-1ß) release. Surprisingly, IL-1ß release was not associated with notable disruption of plasma membrane integrity. These data further our understanding of HAdV vaccine immunogenicity and may provide pathways to extend the efficacy. IMPORTANCE This study examines the interactions between danger-associated molecular patterns and human adenoviruses, and their impact on vaccines. HAdVs and HNP-1 can interact, and these interactions will modify the response of antigen-presenting cells, which will influence vaccine efficacy.
Assuntos
Infecções por Adenoviridae , Vacinas contra Adenovirus , Adenovírus Humanos , Fagócitos , Receptor 4 Toll-Like , alfa-Defensinas , Infecções por Adenoviridae/imunologia , Vacinas contra Adenovirus/imunologia , Adenovírus Humanos/imunologia , Humanos , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fagócitos/citologia , Fagócitos/metabolismo , Receptor 4 Toll-Like/metabolismo , alfa-Defensinas/imunologiaRESUMO
Adenovirus (Ad) is a major causal agent of acute respiratory infections. However, they are a powerful delivery system for gene therapy and vaccines. Some Ad serotypes antagonize the immune system leading to meningitis, conjunctivitis, gastroenteritis, and/or acute hemorrhagic cystitis. Studies have shown that the release of small, membrane-derived extracellular vesicles (EVs) may offer a mechanism by which viruses can enter cells via receptor-independent entry and how they influence disease pathogenesis and/or host protection considering their existence in almost all bodily fluids. We proposed that Ad3 could alter EV biogenesis, composition, and trafficking and may stimulate various immune responses in vitro. In the present study, we evaluated the impact of in vitro infection with Ad3 vector on EV biogenesis and composition in the human adenocarcinoma lung epithelial cell line A549. Cells were infected in an exosome-free media at different multiplicity of infections (MOIs) and time points. The cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and fluorometric calcein-AM. EVs were isolated via ultracentrifugation. Isolated EV proteins were quantified and evaluated via nanoparticle tracking, transmission electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting assays. The cell viability significantly decreased with an increase in MOI and incubation time. A significant increase in particle mean sizes, concentrations, and total EV protein content was detected at higher MOIs when compared to uninfected cells (control group). A549 cell-derived EVs revealed the presence of TSG101, tetraspanins CD9 and CD63, and heat shock proteins 70 and 100 with significantly elevated levels of Rab5, 7, and 35 at higher MOIs (300, 750, and 1500) when compared to the controls. Our findings suggested Ad3 could modulate EV biogenesis, composition, and trafficking which could impact infection pathogenesis and disease progression. This study might suggest EVs could be diagnostic and therapeutic advancement to Ad infections and other related viral infections. However, further investigation is warranted to explore the underlying mechanism(s).
Assuntos
Infecções por Adenovirus Humanos/imunologia , Adenovírus Humanos/imunologia , Vesículas Extracelulares/imunologia , Pulmão/patologia , Pneumonia Viral/imunologia , Células A549 , Infecções por Adenovirus Humanos/patologia , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/genética , Sobrevivência Celular/imunologia , Vesículas Extracelulares/metabolismo , Humanos , Pulmão/citologia , Pulmão/imunologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , SorogrupoRESUMO
We have developed a dual-antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike protein (S-Fusion) and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to increase the potential for MHC class II responses. The vaccine antigens are delivered by a human adenovirus serotype 5 platform, hAd5 [E1-, E2b-, E3-], previously demonstrated to be effective in the presence of Ad immunity. Vaccination of rhesus macaques with the hAd5 S-Fusion + N-ETSD vaccine by subcutaneous prime injection followed by two oral boosts elicited neutralizing anti-S IgG and T helper cell 1-biased T-cell responses to both S and N that protected the upper and lower respiratory tracts from high titer (1 x 106 TCID50) SARS-CoV-2 challenge. Notably, viral replication was inhibited within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge.
Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adenovírus Humanos/genética , Adenovírus Humanos/imunologia , Adenovírus Humanos/metabolismo , Administração Oral , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vacinas contra COVID-19/administração & dosagem , Citocinas/sangue , Imunização Secundária/métodos , Imunoglobulina G/sangue , Pulmão/virologia , Macaca mulatta , Nariz/virologia , Fosfoproteínas/imunologia , Domínios Proteicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinação , Replicação Viral/imunologiaRESUMO
Human adenovirus types 4 (HAdV4) and 7 (HAdV7) often lead to severe respiratory diseases and occur epidemically in children, adults, immune deficiency patients, and other groups, leading to mild or severe symptoms and even death. However, no licensed adenovirus vaccine has been approved in the market for general use. E3 genes of adenovirus are generally considered nonessential for virulence and replication; however, a few studies have demonstrated that the products of these genes are also functional. In this study, most of the E3 genes were deleted, and two E3-deleted recombinant adenoviruses (ΔE3-rAdVs) were constructed as components of the vaccine. After E3 deletion, the replication efficiencies and cytopathogenicity of ΔE3-rAdVs were reduced, indicating that ΔE3-rAdVs were attenuated after E3 genes deletion. Furthermore, single immunization with live-attenuated bivalent vaccine candidate protects mice against challenge with wild-type human adenovirus types 4 and 7, respectively. Vaccinated mice demonstrated remarkably decreased viral loads in the lungs and less lung pathology compared to the control animals. Taken together, our study confirms the possibility of the two live-attenuated viruses as a vaccine for clinic use and illustrates a novel strategy for the construction of an adenovirus vaccine.
Assuntos
Proteínas E3 de Adenovirus/genética , Infecções por Adenovirus Humanos/prevenção & controle , Vacinas contra Adenovirus/imunologia , Adenovírus Humanos/imunologia , Vacinas Atenuadas/imunologia , Células A549 , Infecções por Adenovirus Humanos/imunologia , Adenovírus Humanos/classificação , Animais , Linhagem Celular , Feminino , Deleção de Genes , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Carga ViralRESUMO
Human adenoviruses (HAdVs) are a major cause for disease in children, in particular after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Currently, effective therapies for HAdV infections in immunocompromised hosts are lacking. To decipher immune recognition of HAdV infection and determine new targets for immune-mediated control, we used an HAdV infection 3D organoid system, based on primary human intestinal epithelial cells. HLA-F, the functional ligand for the activating NK cell receptor KIR3DS1, was strongly up-regulated and enabled enhanced killing of HAdV5-infected cells in organoids by KIR3DS1+ NK cells. In contrast, HLA-A and HLA-B were significantly down-regulated in HAdV5-infected organoids in response to adenoviral E3/glycoprotein19K, consistent with evasion from CD8+ T cells. Immunogenetic analyses in a pediatric allo-HSCT cohort showed a reduced risk to develop severe HAdV disease and faster clearance of HAdV viremia in children receiving KIR3DS1+/HLA-Bw4+ donor cells compared with children receiving nonKIR3DS1+/HLA-Bw4+ cells. These findings identify the KIR3DS1/HLA-F axis as a new target for immunotherapeutic strategies against severe HAdV disease.
Assuntos
Infecções por Adenovirus Humanos/imunologia , Células Matadoras Naturais/imunologia , Receptores KIR3DS1/imunologia , Células A549 , Adenovírus Humanos/imunologia , Células HEK293 , HumanosRESUMO
Following repeat exposure to many human adenoviruses (HAdVs), most adults harbour long-lived B- and T-cell responses. Combined, this response typically protects us for years from re-infection by the same HAdV type. In spite of these immune responses, some HAdV types are associated with persistent infections that constitute a life-threatening risk when an individual's T-cell response is compromised. By contrast, patients with B-cell deficiencies do not appear to be at a greater risk of HAdV disease. This dichotomy begs the question of the secondary role of anti-HAdV antibodies during host defence. In this study, we explored IgG-complexed (IC)-HAdV5 and primary human plasmacytoid dendritic cell (pDC) interactions. We found that IC-HAdV5 are efficiently internalized in pDCs, stimulate their activation through TLR9 signalling, and cause apoptosis. These data may help reconcile the enigma of robust immune response to HAdVs, while concurrently allowing persistence.
Assuntos
Adenovírus Humanos/imunologia , Apoptose/imunologia , Células Dendríticas/patologia , Células Dendríticas/virologia , Imunidade Inata , Imunoglobulina G/imunologia , Apresentação de Antígeno , Células Cultivadas , Células Dendríticas/imunologia , Humanos , Transdução de SinaisRESUMO
Preexisting immune responses toward adenoviral vectors limit the use of a vector based on particular serotypes and its clinical applicability for gene therapy and/or vaccination. Therefore, there is a significant interest in vectorizing novel adenoviral types that have low seroprevalence in the human population. Here, we describe the discovery and vectorization of a chimeric human adenovirus, which we call HAdV-20-42-42. Full-genome sequencing revealed that this virus is closely related to human serotype 42, except for the penton base, which is derived from serotype 20. The HAdV-20-42-42 vector could be propagated stably to high titers on existing E1-complementing packaging cell lines. Receptor-binding studies revealed that the vector utilized both CAR and CD46 as receptors for cell entry. Furthermore, the HAdV-20-42-42 vector was potent in transducing human and murine cardiovascular cells and tissues, irrespective of the presence of blood coagulation factor X. In vivo characterizations demonstrate that when delivered intravenously (i.v.) in mice, HAdV-20-42-42 mainly targeted the lungs, liver, and spleen and triggered robust inflammatory immune responses. Finally, we demonstrate that potent T-cell responses against vector-delivered antigens could be induced upon intramuscular vaccination in mice. In summary, from the data obtained we conclude that HAdV-20-42-42 provides a valuable addition to the portfolio of adenoviral vectors available to develop efficacious products in the fields of gene therapy and vaccination. IMPORTANCE Adenoviral vectors are under investigation for a broad range of therapeutic indications in diverse fields, such as oncology and gene therapy, as well as for vaccination both for human and veterinary use. A wealth of data shows that preexisting immune responses may limit the use of a vector. Particularly in the current climate of global pandemic, there is a need to expand the toolbox with novel adenoviral vectors for vaccine development. Our data demonstrate that we have successfully vectorized a novel adenovirus type candidate with low seroprevalence. The cell transduction data and antigen-specific immune responses induced in vivo demonstrate that this vector is highly promising for the development of gene therapy and vaccine products.
Assuntos
Adenovírus Humanos , Terapia Genética/métodos , Vetores Genéticos , Desenvolvimento de Vacinas/métodos , Células A549 , Adenovírus Humanos/genética , Adenovírus Humanos/imunologia , Animais , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Células HEK293 , Humanos , Masculino , Camundongos , Estudos SoroepidemiológicosRESUMO
Reactivation of Human Cytomegalovirus (HCMV) and Human Adenovirus (HAdV) in immunocompromised patients following stem cell transplantation (SCT) or solid organ transplantation (SOT) is associated with high morbidity and mortality. The adoptive transfer of virus-specific CD8+ and CD4+ T cells has been shown to re-establish the antiviral T-cell response and improve clinical outcome. The viral load in immunocompromised patients can efficiently be reduced solely by the infusion of virus-specific CD4+ T cells. The identification of CD4+ T-cell epitopes has mainly focused on a limited number of viral proteins that were characterized as immunodominant. Here, we used in silico prediction to determine promiscuous CD4+ T-cell epitopes from the entire proteomes of HCMV and HAdV. Immunogenicity testing with enzyme-linked immuno spot (ELISpot) assays and intracellular cytokine staining (ICS) revealed numerous novel CD4+ T-cell epitopes derived from a broad spectrum of viral antigens. We identified 17 novel HCMV-derived and seven novel HAdV-derived CD4+ T-cell epitopes that were recognized by > 50% of the assessed peripheral blood mononuclear cell (PBMC) samples. The newly identified epitopes were pooled with previously published, retested epitopes to stimulate virus-specific memory T cells in PBMCs from numerous randomly selected blood donors. Our peptide pools induced strong IFNγ secretion in 46 out of 48 (HCMV) and 31 out of 31 (HAdV) PBMC cultures. In conclusion, we applied an efficient method to screen large viral proteomes for promiscuous CD4+ T-cell epitopes to improve the detection and isolation of virus-specific T cells in a clinical setting.
Assuntos
Adenovírus Humanos/imunologia , Linfócitos T CD4-Positivos/imunologia , Citomegalovirus/imunologia , Mapeamento de Epitopos/métodos , Epitopos de Linfócito T/imunologia , Antígenos Virais/imunologia , Humanos , Células T de Memória/imunologiaRESUMO
Adenovirus vector-based genetic vaccines have emerged as a powerful strategy against the SARS-CoV-2 health crisis. This success is not unexpected because adenoviruses combine many desirable features of a genetic vaccine. They are highly immunogenic and have a low and well characterized pathogenic profile paired with technological approachability. Ongoing efforts to improve adenovirus-vaccine vectors include the use of rare serotypes and non-human adenoviruses. In this review, we focus on the viral capsid and how the choice of genotypes influences the uptake and subsequent subcellular sorting. We describe how understanding capsid properties, such as stability during the entry process, can change the fate of the entering particles and how this translates into differences in immunity outcomes. We discuss in detail how mutating the membrane lytic capsid protein VI affects species C viruses' post-entry sorting and briefly discuss if such approaches could have a wider implication in vaccine and/or vector development.
Assuntos
Adenovírus Humanos/imunologia , Adenovírus Humanos/fisiologia , Capsídeo/metabolismo , Vetores Genéticos , Vacinas Virais/imunologia , Internalização do Vírus , Imunidade Adaptativa , Adenovírus Humanos/genética , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/imunologia , Capsídeo/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Ensaios Clínicos como Assunto , Humanos , Imunidade Inata , Camundongos , SARS-CoV-2/imunologiaRESUMO
Despite decades of clinical and preclinical investigations, we still poorly grasp our innate immune response to human adenoviruses (HAdVs) and their vectors. In this study, we explored the impact of lactoferrin on three HAdV types that are being used as vectors for vaccines. Lactoferrin is a secreted globular glycoprotein that influences direct and indirect innate immune response against a range of pathogens following a breach in tissue homeostasis. The mechanism by which lactoferrin complexes increases HAdV uptake and induce maturation of human phagocytes is unknown. We show that lactoferrin redirects HAdV types from species B, C, and D to Toll-like receptor 4 (TLR4) cell surface complexes. TLR4-mediated internalization of the HAdV-lactoferrin complex induced an NLRP3-associated response that consisted of cytokine release and transient disruption of plasma membrane integrity, without causing cell death. These data impact our understanding of HAdV immunogenicity and may provide ways to increase the efficacy of HAdV-based vectors/vaccines.
Assuntos
Adenovírus Humanos/imunologia , Lactoferrina/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fagócitos/virologia , Receptor 4 Toll-Like/metabolismo , Infecções por Adenoviridae/imunologia , Infecções por Adenoviridae/patologia , Adenovírus Humanos/genética , Citocinas/metabolismo , Citometria de Fluxo , Humanos , Imunidade Inata , Lactoferrina/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Receptor 4 Toll-Like/genéticaRESUMO
Human adenoviruses (HAdVs) are a large family of DNA viruses that include more than 100 genotypes divided into seven species (A to G) and induce respiratory tract infections, gastroenteritis, and conjunctivitis. Genetically modified adenoviruses are also used as vaccines, gene therapies, and anticancer treatments. The APOBEC3s are a family of cytidine deaminases that restrict viruses by introducing mutations in their genomes. Viruses developed different strategies to cope with the APOBEC3 selection pressure, but nothing is known on the interplay between the APOBEC3s and the HAdVs. In this study, we focused on three HAdV strains: the B3 and C2 strains, as they are very frequent, and the A12 strain, which is less common but is oncogenic in animal models. We demonstrated that the three HAdV strains induce a similar APOBEC3B upregulation at the transcriptional level. At the protein level, however, APOBEC3B is abundantly expressed during HAdV-A12 and -C2 infection and shows a nuclear distribution. On the contrary, APOBEC3B is barely detectable in HAdV-B3-infected cells. APOBEC3B deaminase activity is detected in total protein extracts upon HAdV-A12 and -C2 infection. Bioinformatic analysis demonstrates that the HAdV-A12 genome bears a stronger APOBEC3 evolutionary footprint than that of the HAdV-C2 and HAdV-B3 genomes. Our results show that HAdV infection triggers the transcriptional upregulation of the antiviral innate effector APOBEC3B. The discrepancies between the APOBEC3B mRNA and protein levels might reflect the ability of some HAdV strains to antagonize the APOBEC3B protein. These findings point toward an involvement of APOBEC3B in HAdV restriction and evolution. IMPORTANCE The APOBEC3 family of cytosine deaminases has important roles in antiviral innate immunity and cancer. Notably, APOBEC3A and APOBEC3B are actively upregulated by several DNA tumor viruses and contribute to transformation by introducing mutations in the cellular genome. Human adenoviruses (HAdVs) are a large family of DNA viruses that cause generally asymptomatic infections in immunocompetent adults. HAdVs encode several oncogenes, and some HAdV strains, like HAdV-A12, induce tumors in hamsters and mice. Here, we show that HAdV infection specifically promotes the expression of the APOBEC3B gene. We report that infection with the A12 strain induces a strong expression of an enzymatically active APOBEC3B protein in bronchial epithelial cells. We provide bioinformatic evidence that HAdVs' genomes and notably the A12 genome are under APOBEC3 selection pressure. Thus, APOBEC3B might contribute to adenoviral restriction, diversification, and oncogenic potential of particular strains.
Assuntos
Infecções por Adenovirus Humanos/patologia , Adenovírus Humanos/imunologia , Citidina Desaminase/metabolismo , Imunidade Inata/imunologia , Antígenos de Histocompatibilidade Menor/metabolismo , Mucosa Respiratória/virologia , Infecções por Adenovirus Humanos/imunologia , Brônquios/citologia , Brônquios/virologia , Linhagem Celular , Células Epiteliais/virologia , Genoma Viral/genética , Humanos , Mucosa Respiratória/citologia , Infecções Respiratórias/patologia , Infecções Respiratórias/virologia , Transcrição Gênica/genética , Regulação para Cima/genéticaRESUMO
BACKGROUNDTo understand the features of a replicating vaccine that might drive potent and durable immune responses to transgene-encoded antigens, we tested a replication-competent adenovirus type 4 encoding influenza virus H5 HA (Ad4-H5-Vtn) administered as an oral capsule or via tonsillar swab or nasal spray.METHODSViral shedding from the nose, mouth, and rectum was measured by PCR and culturing. H5-specific IgG and IgA antibodies were measured by bead array binding assays. Serum antibodies were measured by a pseudovirus entry inhibition, microneutralization, and HA inhibition assays.RESULTSAd4-H5-Vtn DNA was shed from most upper respiratory tract-immunized (URT-immunized) volunteers for 2 to 4 weeks, but cultured from only 60% of participants, with a median duration of 1 day. Ad4-H5-Vtn vaccination induced increases in H5-specific CD4+ and CD8+ T cells in the peripheral blood as well as increases in IgG and IgA in nasal, cervical, and rectal secretions. URT immunizations induced high levels of serum neutralizing antibodies (NAbs) against H5 that remained stable out to week 26. The duration of viral shedding correlated with the magnitude of the NAb response at week 26. Adverse events (AEs) were mild, and peak NAb titers were associated with overall AE frequency and duration. Serum NAb titers could be boosted to very high levels 2 to 5 years after Ad4-H5-Vtn vaccination with recombinant H5 or inactivated split H5N1 vaccine.CONCLUSIONReplicating Ad4 delivered to the URT caused prolonged exposure to antigen, drove durable systemic and mucosal immunity, and proved to be a promising platform for the induction of immunity against viral surface glycoprotein targets.TRIAL REGISTRATIONClinicalTrials.gov NCT01443936 and NCT01806909.FUNDINGIntramural and Extramural Research Programs of the NIAID, NIH (U19 AI109946) and the Centers of Excellence for Influenza Research and Surveillance (CEIRS), NIAID, NIH (contract HHSN272201400008C).
Assuntos
Adenovírus Humanos/genética , Vetores Genéticos , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Adenovírus Humanos/imunologia , Adenovírus Humanos/fisiologia , Administração Oral , Adolescente , Adulto , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Imunidade nas Mucosas , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/genética , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Masculino , Sprays Nasais , Tonsila Palatina , Replicação Viral , Eliminação de Partículas Virais , Adulto JovemRESUMO
Human adenoviruses (HAdV) cause a variety of infections in human hosts, from self-limited upper respiratory tract infections in otherwise healthy people to fulminant pneumonia and death in immunocompromised patients. Many HAdV enter polarized epithelial cells by using the primary receptor, the Coxsackievirus and adenovirus receptor (CAR). Recently published data demonstrate that a potent neutrophil (PMN) chemoattractant, interleukin-8 (IL-8), stimulates airway epithelial cells to increase expression of the apical isoform of CAR (CAREx8), which results in increased epithelial HAdV type 5 (HAdV5) infection. However, the mechanism for PMN-enhanced epithelial HAdV5 transduction remains unclear. In this manuscript, the molecular mechanisms behind PMN mediated enhancement of epithelial HAdV5 transduction are characterized using an MDCK cell line that stably expresses human CAREx8 under a doxycycline inducible promoter (MDCK-CAREx8 cells). Contrary to our hypothesis, PMN exposure does not enhance HAdV5 entry by increasing CAREx8 expression nor through activation of non-specific epithelial endocytic pathways. Instead, PMN serine proteases are responsible for PMN-mediated enhancement of HAdV5 transduction in MDCK-CAREx8 cells. This is evidenced by reduced transduction upon inhibition of PMN serine proteases and increased transduction upon exposure to exogenous human neutrophil elastase (HNE). Furthermore, HNE exposure activates epithelial autophagic flux, which, even when triggered through other mechanisms, results in a similar enhancement of epithelial HAdV5 transduction. Inhibition of F-actin with cytochalasin D partially attenuates PMN mediated enhancement of HAdV transduction. Taken together, these findings suggest that HAdV5 can leverage innate immune responses to establish infections.