RESUMO
CONTEXT: In contrast with other respiratory viruses, children infected with SARS-CoV-2 are largely spared from severe COVID-19. OBJECTIVES: To critically assess age-related differences in three host proteins involved in SARS-CoV-2 cellular entry: angiotensin-converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2) and furin. METHODS: We systematically searched Medline, Embase, and PubMed databases for relevant publications. Studies were eligible if they evaluated ACE2, TMPRSS2 or furin expression, methylation, or protein level in children. RESULTS: Sixteen papers were included. Age-dependent differences in membrane-bound and soluble ACE2 were shown in several studies, with ACE2 expression increasing with age. TMPRSS2 and furin are key proteases involved in SARS-CoV-2 spike protein cleavage. TMPRSS2 expression is increased by circulating androgens and is thus low in pre-pubertal children. Furin has not currently been well researched. LIMITATIONS: High levels of study heterogeneity. CONCLUSIONS: Low expression of key host proteins may partially explain the reduced incidence of severe COVID-19 among children, although further research is needed.
Assuntos
COVID-19 , SARS-CoV-2 , Criança , Humanos , SARS-CoV-2/metabolismo , Peptidil Dipeptidase A/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
BACKGROUND: Different classes of latency reversing agents (LRAs) are being evaluated to measure their effects in reactivating HIV replication from latently infected cells. A limited number of studies have demonstrated additive effects of LRAs with the viral protein Tat in initiating transcription, but less is known about how LRAs interact with Tat, particularly through basic residues that may be post-translationally modified to alter the behaviour of Tat for processive transcription and co-transcriptional RNA processing. RESULTS: Here we show that various lysine and arginine mutations reduce the capacity of Tat to induce both transcription and mRNA splicing. The lysine 28 and lysine 50 residues of Tat, or the acetylation and methylation modifications of these basic amino acids, were essential for Tat transcriptional control, and also for the proviral expression effects elicited by histone deacetylase inhibitors (HDACi) or the bromodomain inhibitor JQ1. We also found that JQ1 was the only LRA tested that could induce HIV mRNA splicing in the absence of Tat, or rescue splicing for Tat lysine mutants in a BRD4-dependent manner. CONCLUSIONS: Our data provide evidence that Tat activities in both co-transcriptional RNA processing together with transcriptional initiation and processivity are crucial during reactivation of latent HIV infection. The HDACi and JQ1 LRAs act with Tat to increase transcription, but JQ1 also enables post-transcriptional mRNA splicing. Tat residues K28 and K50, or their modifications through acetylation or methylation, are critical for LRAs that function in conjunction with Tat.
Assuntos
Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Latência Viral/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Substituição de Aminoácidos , Fármacos Anti-HIV/farmacologia , Azepinas/farmacologia , Proteínas de Ciclo Celular , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Humanos , Mutação , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Splicing de RNA , Fatores de Transcrição/metabolismo , Transcrição Gênica/efeitos dos fármacos , Triazóis/farmacologia , Ativação Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Despite the success of combination antiretroviral therapy (cART), HIV persists in long lived latently infected cells in the blood and tissue, and treatment is required lifelong. Recent clinical studies have trialed latency-reversing agents (LRA) as a method to eliminate latently infected cells; however, the effects of LRA on the central nervous system (CNS), a well-known site of virus persistence on cART, are unknown. In this study, we evaluated the toxicity and potency of a panel of commonly used and well-known LRA (panobinostat, romidepsin, vorinostat, chaetocin, disulfiram, hexamethylene bisacetamide [HMBA], and JQ-1) in primary fetal astrocytes (PFA) as well as monocyte-derived macrophages as a cellular model for brain perivascular macrophages. We show that most LRA are non-toxic in these cells at therapeutic concentrations. Additionally, romidepsin, JQ-1, and panobinostat were the most potent at inducing viral transcription, with greater magnitude observed in PFA. In contrast, vorinostat, chaetocin, disulfiram, and HMBA all demonstrated little or no induction of viral transcription. Together, these data suggest that some LRA could potentially activate transcription in latently infected cells in the CNS. We recommend that future trials of LRA also examine the effects of these agents on the CNS via examination of cerebrospinal fluid.
Assuntos
HIV-1/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Neurônios/efeitos dos fármacos , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Acetamidas/farmacologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/virologia , Azepinas/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Depsipeptídeos/farmacologia , Dissulfiram/farmacologia , Feto , HIV-1/genética , HIV-1/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/virologia , Neurônios/metabolismo , Neurônios/virologia , Panobinostat , Piperazinas/farmacologia , Cultura Primária de Células , Transcrição Gênica/efeitos dos fármacos , Triazóis/farmacologia , Ativação Viral/genética , Latência Viral/genética , Replicação Viral/genética , VorinostatRESUMO
Pleural empyema is a serious complication of pneumonia in children. Negative bacterial cultures commonly impede optimal antibiotic therapy. To improve bacterial identification, we developed a molecular assay and evaluated its performance compared with bacterial culture. Our multiplex-quantitative PCR to detect Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus and Haemophilus influenzae was assessed using bacterial genomic DNA and laboratory-prepared samples (n = 267). To evaluate clinical performance, we conducted the Molecular Assessment of Thoracic Empyema (MATE) observational study, enrolling children hospitalised with empyema. Pleural fluids were tested by bacterial culture and multiplex-qPCR, and performance determined using a study gold standard. We determined clinical sensitivity and time-to-organism-identification to assess the potential of the multiplex-qPCR to reduce the duration of empiric untargeted antibiotic therapy. Using spiked samples, the multiplex-qPCR demonstrated 213/215 (99.1%) sensitivity and 52/52 (100%) specificity for all organisms. During May 2019-March 2023, 100 children were enrolled in the MATE study; median age was 3.9 years (IQR 2-5.6). A bacterial pathogen was identified in 90/100 (90%) specimens by multiplex-qPCR, and 24/100 (24%) by bacterial culture (P <0.001). Multiplex-qPCR identified a bacterial cause in 68/76 (90%) culture-negative specimens. S. pneumoniae was the most common pathogen, identified in 67/100 (67%) specimens. We estimate our multiplex-qPCR would have reduced the duration of untargeted antibiotic therapy in 61% of cases by a median 20 days (IQR 17.5-23, range 1-55). Multiplex-qPCR significantly increased pathogen detection compared with culture and may allow for reducing the duration of untargeted antibiotic therapy.
Assuntos
Empiema Pleural , Reação em Cadeia da Polimerase Multiplex , Humanos , Pré-Escolar , Empiema Pleural/microbiologia , Empiema Pleural/tratamento farmacológico , Empiema Pleural/diagnóstico , Masculino , Feminino , Reação em Cadeia da Polimerase Multiplex/métodos , Criança , Haemophilus influenzae/genética , Haemophilus influenzae/isolamento & purificação , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificação , Streptococcus pyogenes/genética , Streptococcus pyogenes/isolamento & purificação , Lactente , Hospitalização , Antibacterianos/uso terapêutico , Sensibilidade e Especificidade , DNA Bacteriano/genéticaRESUMO
Bovine colostrum (first milk) contains very high concentrations of IgG, and on average 1 kg (500 g/liter) of IgG can be harvested from each immunized cow immediately after calving. We used a modified vaccination strategy together with established production systems from the dairy food industry for the large-scale manufacture of broadly neutralizing HIV-1 IgG. This approach provides a low-cost mucosal HIV preventive agent potentially suitable for a topical microbicide. Four cows were vaccinated pre- and/or postconception with recombinant HIV-1 gp140 envelope (Env) oligomers of clade B or A, B, and C. Colostrum and purified colostrum IgG were assessed for cross-clade binding and neutralization against a panel of 27 Env-pseudotyped reporter viruses. Vaccination elicited high anti-gp140 IgG titers in serum and colostrum with reciprocal endpoint titers of up to 1 × 10(5). While nonimmune colostrum showed some intrinsic neutralizing activity, colostrum from 2 cows receiving a longer-duration vaccination regimen demonstrated broad HIV-1-neutralizing activity. Colostrum-purified polyclonal IgG retained gp140 reactivity and neutralization activity and blocked the binding of the b12 monoclonal antibody to gp140, showing specificity for the CD4 binding site. Colostrum-derived anti-HIV antibodies offer a cost-effective option for preparing the substantial quantities of broadly neutralizing antibodies that would be needed in a low-cost topical combination HIV-1 microbicide.
Assuntos
Anticorpos Neutralizantes/imunologia , Colostro/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS , Animais , Anticorpos Monoclonais/imunologia , Bovinos , Produtos do Gene env/imunologia , Anticorpos Anti-HIV/sangue , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Imunoglobulina G/imunologia , Testes de Neutralização , VacinaçãoRESUMO
Streptococcus pneumoniae (the pneumococcus) is a leading cause of pneumonia in children under 5 years of age. Coinfection by pneumococci and respiratory viruses enhances disease severity. Little is known about pneumococcal coinfections with respiratory syncytial virus (RSV). Here, we developed a novel infant mouse model of coinfection using pneumonia virus of mice (PVM), a murine analogue of RSV, to examine the dynamics of coinfection in the upper respiratory tract, an anatomical niche that is essential for host-to-host transmission and progression to disease. Coinfection increased damage to the nasal tissue and increased production of the chemokine CCL3. Nasopharyngeal pneumococcal density and shedding in nasal secretions were increased by coinfection. In contrast, coinfection reduced PVM loads in the nasopharynx, an effect that was independent of pneumococcal strain and the order of infection. We showed that this "antagonistic" effect was absent using either ethanol-killed pneumococci or a pneumococcal mutant deficient in capsule production and incapable of nasopharyngeal carriage. Colonization with a pneumococcal strain naturally unable to produce capsule also reduced viral loads. The pneumococcus-mediated reduction in PVM loads was caused by accelerated viral clearance from the nasopharynx. Although these synergistic and antagonistic effects occurred with both wild-type pneumococcal strains used in this study, the magnitude of the effects was strain dependent. Lastly, we showed that pneumococci can also antagonize influenza virus. Taken together, our study has uncovered multiple novel facets of bacterial-viral coinfection. Our findings have important public health implications, including for bacterial and viral vaccination strategies in young children. IMPORTANCE Respiratory bacterial-viral coinfections (such as pneumococci and influenza virus) are often synergistic, resulting in enhanced disease severity. Although colonization of the nasopharynx is the precursor to disease and transmission, little is known about bacterial-viral interactions that occur within this niche. In this study, we developed a novel mouse model to examine pneumococcal-viral interactions in the nasopharynx with pneumonia virus of mice (PVM) and influenza. We found that PVM infection benefits pneumococci by increasing their numbers in the nasopharynx and shedding of these bacteria in respiratory secretions. In contrast, we discovered that pneumococci decrease PVM numbers by accelerating viral clearance. We also report a similar effect of pneumococci on influenza. By showing that coinfections lead to both synergistic and antagonistic outcomes, our findings challenge the existing dogma in the field. Our work has important applications and implications for bacterial and viral vaccines that target these microbes.
Assuntos
Antibiose , Coinfecção/microbiologia , Coinfecção/virologia , Infecções Pneumocócicas/virologia , Infecções por Pneumovirus/virologia , Sistema Respiratório/virologia , Fatores Etários , Animais , Coinfecção/imunologia , Citocinas/análise , Citocinas/imunologia , Modelos Animais de Doenças , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Vírus da Pneumonia Murina/genética , Vírus da Pneumonia Murina/imunologia , Nasofaringe/virologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/virologia , Infecções por Pneumovirus/imunologia , Sistema Respiratório/imunologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/imunologia , Carga ViralRESUMO
A persistent latent reservoir of virus in CD4+ T cells is a major barrier to cure HIV. Activating viral transcription in latently infected cells using small molecules is one strategy being explored to eliminate latency. We previously described the use of a FlpIn.FM HEK293 cellular assay to identify and then optimize the 2-acylaminothiazole class to exhibit modest activation of HIV gene expression. Here, we implement two strategies to further improve the activation of viral gene expression and physicochemical properties of this class. Firstly, we explored rigidification of the central oxy-carbon linker with a variety of saturated heterocycles, and secondly, investigated bioisosteric replacement of the 2-acylaminothiazole moiety. The optimization process afforded lead compounds (74 and 91) from the 2-piperazinyl thiazolyl urea and the imidazopyridine class. The lead compounds from each class demonstrate potent activation of HIV gene expression in the FlpIn.FM HEK293 cellular assay (both with LTR EC50s of 80 nM) and in the Jurkat Latency 10.6 cell model (LTR EC50 220 and 320 nM respectively), but consequently activate gene expression non-specifically in the FlpIn.FM HEK293 cellular assay (CMV EC50 70 and 270 nM respectively) manifesting in cellular cytotoxicity. The lead compounds have potential for further development as novel latency reversing agents.
Assuntos
HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Imidazóis/química , Imidazóis/farmacologia , Piridinas/química , Piridinas/farmacologia , Tiazóis/química , Ureia/química , Latência Viral/efeitos dos fármacos , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Desenho de Fármacos , Células HEK293 , HumanosRESUMO
The persistent reservoir of cells latently infected with human immunodeficiency virus (HIV)-integrated proviral DNA necessitates lifelong suppressive antiretroviral therapy (ART). Epigenetic targeted compounds have shown promise as potential latency-reversing agents; however, these drugs have undesirable toxicity and lack specificity for HIV. We utilized a novel HEK293-derived FlpIn dual-reporter cell line, which quantifies specific HIV provirus reactivation (LTR promoter) relative to nonspecific host cell gene expression (CMV promoter), to identify the 5-substituted 2-acylaminothiazole hit class. Here, we describe the optimization of the hit class, defining the functionality necessary for HIV gene activation and for improving in vitro metabolism and solubility. The optimized compounds displayed enhanced HIV gene expression in HEK293 and Jurkat 10.6 latency cellular models and increased unspliced HIV RNA in resting CD4+ T cells isolated from HIV-infected individuals on ART, demonstrating the potential of the 2-acylaminothiazole class as latency-reversing agents.
Assuntos
Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Tiazóis/síntese química , Tiazóis/farmacologia , Latência Viral/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/biossíntese , Produtos do Gene tat do Vírus da Imunodeficiência Humana/efeitos dos fármacos , Animais , Fármacos Anti-HIV/farmacocinética , Terapia Antirretroviral de Alta Atividade/métodos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Desenho de Fármacos , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Células Jurkat , Camundongos , Microssomos Hepáticos , RNA Viral/biossíntese , RNA Viral/efeitos dos fármacos , RNA Viral/genética , Relação Estrutura-Atividade , Tiazóis/farmacocinética , Transcrição GênicaRESUMO
OBJECTIVE: To determine whether latency can be established and reversed in both proliferating and nonproliferating CD4+ T cells in the same model in vitro. METHODS: Activated CD4+ T cells were infected with either a nonreplication competent, luciferase reporter virus or wild-type full-length enhanced green fluorescent protein (EGFP) reporter virus and cultured for 12 days. The cells were then sorted by flow cytometry to obtain two distinct T-cell populations that did not express the T-cell activation markers, CD69, CD25 and human leukocyte antigen (HLA)-DR: CD69CD25HLA-DR small cells (nonblasts) that had not proliferated in vitro following mitogen stimulation and CD69CD25HLA-DR large cells (which we here call transitional blasts) that had proliferated. The cells were then reactivated with latency-reversing agents and either luciferase or EGFP quantified. RESULTS: Inducible luciferase expression, consistent with latent infection, was observed in nonblasts and transitional blasts following stimulation with either phorbol-myristate-acetate/phytohemagglutinin (3.8â±â1 and 2.9â±â0.5 fold above dimethyl sulfoxide, respectively) or romidepsin (2.1â±â0.6 and 1.8â±â0.2 fold above dimethyl sulfoxide, respectively). Constitutive expression of luciferase was higher in transitional blasts compared with nonblasts. Using wild-type full-length EGFP reporter virus, inducible virus was observed in nonblasts but not in transitional blasts. No significant difference was observed in the response to latency-reversing agents in either nonblasts or transitional blasts. CONCLUSION: HIV latency can be established in vitro in resting T cells that have not proliferated (nonblasts) and blasts that have proliferated (transitional blasts). This model could potentially be used to assess new strategies to eliminate latency.