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1.
Virol J ; 18(1): 28, 2021 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-33499880

RESUMO

BACKGROUND: Diverse vaccination outcomes and protection levels among different populations pose a serious challenge to the development of an effective malaria vaccine. Co-infections are among many factors associated with immune dysfunction and sub-optimal vaccination outcomes. Chronic, asymptomatic viral infections can contribute to the modulation of vaccine efficacy through various mechanisms. Human Pegivirus-1 (HPgV-1) persists in immune cells thereby potentially modulating immune responses. We investigated whether Pegivirus infection influences vaccine-induced responses and protection in African volunteers undergoing whole P. falciparum sporozoites-based malaria vaccination and controlled human malaria infections (CHMI). METHODS: HPgV-1 prevalence was quantified by RT-qPCR in plasma samples of 96 individuals before, post vaccination with PfSPZ Vaccine and after CHMI in cohorts from Tanzania and Equatorial Guinea. The impact of HPgV-1 infection was evaluated on (1) systemic cytokine and chemokine levels measured by Luminex, (2) PfCSP-specific antibody titers quantified by ELISA, (3) asexual blood-stage parasitemia pre-patent periods and parasite multiplication rates, (4) HPgV-1 RNA levels upon asexual blood-stage parasitemia induced by CHMI. RESULTS: The prevalence of HPgV-1 was 29.2% (28/96) and sequence analysis of the 5' UTR and E2 regions revealed the predominance of genotypes 1, 2 and 5. HPgV-1 infection was associated with elevated systemic levels of IL-2 and IL-17A. Comparable vaccine-induced anti-PfCSP antibody titers, asexual blood-stage multiplication rates and pre-patent periods were observed in HPgV-1 positive and negative individuals. However, a tendency for higher protection levels was detected in the HPgV-1 positive group (62.5%) compared to the negative one (51.6%) following CHMI. HPgV-1 viremia levels were not significantly altered after CHMI. CONCLUSIONS: HPgV-1 infection did not alter PfSPZ Vaccine elicited levels of PfCSP-specific antibody responses and parasite multiplication rates. Ongoing HPgV-1 infection appears to improve to some degree protection against CHMI in PfSPZ-vaccinated individuals. This is likely through modulation of immune system activation and systemic cytokines as higher levels of IL-2 and IL17A were observed in HPgV-1 infected individuals. CHMI is safe and well tolerated in HPgV-1 infected individuals. Identification of cell types and mechanisms of both silent and productive infection in individuals will help to unravel the biology of this widely present but largely under-researched virus.

2.
Clin Infect Dis ; 2020 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-33206173
3.
Viruses ; 12(10)2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33019624

RESUMO

Although extracellular vesicle (EV) surface electrostatic properties (measured as zeta potential, ζ-potential) have been reported by many investigators, the biophysical implications of charge and EV origin remains uncertain. Here, we compared the ζ-potential of human blood EVs (BEVs) and semen EVs (SEVs) from 26 donors that were HIV-infected (HIV+, n = 13) or HIV uninfected (HIV-, n = 13). We found that, compared to BEVs that bear neutral surface charge, SEVs were significantly more negatively charged, even when BEVs and SEVs were from the same individual. Comparison of BEVs and SEVs from HIV- and HIV+ groups revealed subtle HIV-induced alteration in the ζ-potential of EVs, with the effect being more significant in SEVs (∆ζ-potential = -8.82 mV, p-value = 0.0062) than BEVs (∆ζ-potential = -1.4 mV, p-value = 0.0462). These observations were validated by differences in the isoelectric point (IEP) of EVs, which was in the order of HIV + SEV ≤ HIV-SEV ≪ HIV + BEV ≤ HIV-BEV. Functionally, the rate and efficiency of SEV internalization by the human cervical epithelial cell line, primary peripheral blood lymphocytes, and primary blood-derived monocytes were significantly higher than those of BEVs. Mechanistically, removal of sialic acids from the surface of EVs using neuraminidase treatment significantly decreased SEV's surface charge, concomitant with a substantial reduction in SEV's internalization. The neuraminidase effect was independent of HIV infection and insignificant for BEVs. Finally, these results were corroborated by enrichment of glycoproteins in SEVs versus BEVs. Taken together, these findings uncover fundamental tissue-specific differences in surface electrostatic properties of EVs and highlight the critical role of surface charge in EV/target cell interactions.

4.
Infect Control Hosp Epidemiol ; : 1-8, 2020 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-32783787

RESUMO

BACKGROUND: Personal protective equipment (PPE) is a critical need during the coronavirus disease 2019 (COVID-19) pandemic. Alternative sources of surgical masks, including 3-dimensionally (3D) printed approaches that may be reused, are urgently needed to prevent PPE shortages. Few data exist identifying decontamination strategies to inactivate viral pathogens and retain 3D-printing material integrity. OBJECTIVE: To test viral disinfection methods on 3D-printing materials. METHODS: The viricidal activity of common disinfectants (10% bleach, quaternary ammonium sanitizer, 3% hydrogen peroxide, or 70% isopropanol and exposure to heat (50°C, and 70°C) were tested on four 3D-printed materials used in the healthcare setting, including a surgical mask design developed by the Veterans' Health Administration. Inactivation was assessed for several clinically relevant RNA and DNA pathogenic viruses, including severe acute respiratory coronavirus virus 2 (SARS-CoV-2) and human immunodeficiency virus 1 (HIV-1). RESULTS: SARS-CoV-2 and all viruses tested were completely inactivated by a single application of bleach, ammonium quaternary compounds, or hydrogen peroxide. Similarly, exposure to dry heat (70°C) for 30 minutes completely inactivated all viruses tested. In contrast, 70% isopropanol reduced viral titers significantly less well following a single application. Inactivation did not interfere with material integrity of the 3D-printed materials. CONCLUSIONS: Several standard decontamination approaches effectively disinfected 3D-printed materials. These approaches were effective in the inactivation SARS-CoV-2, its surrogates, and other clinically relevant viral pathogens. The decontamination of 3D-printed surgical mask materials may be useful during crisis situations in which surgical mask supplies are limited.

5.
mBio ; 11(3)2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32371599

RESUMO

The vaginal microbiota influences sexual transmission of human immunodeficiency virus type 1 (HIV-1). Colonization of the vaginal tract is normally dominated by Lactobacillus species. Both Lactobacillus and Enterococcus faecalis may secrete reutericyclin, which inhibits the growth of a variety of pathogenic bacteria. Increasing evidence suggests a potential therapeutic role for an analogue of reutericyclin, glycerol monolaurate (GML), against microbial pathogens. Previous studies using a macaque vaginal simian immunodeficiency virus (SIV) transmission model demonstrated that GML reduces transmission and alters immune responses to infection in vitro Previous studies showed that structural analogues of GML negatively impact other enveloped viruses. We sought to expand understanding of how GML inhibits HIV-1 and other enveloped viruses and show that GML restricts HIV-1 entry post-CD4 engagement at the step of coreceptor binding. Further, HIV-1 and yellow fever virus (YFV) particles were more sensitive to GML interference than particles "matured" by proteolytic processing. We show that high-pressure-liquid-chromatography (HPLC)-purified reutericyclin and reutericyclin secreted by Lactobacillus inhibit HIV-1. These data emphasize the importance and protective nature of the normal vaginal flora during viral infections and provide insights into the antiviral mechanism of GML during HIV-1 infection and, more broadly, to other enveloped viruses.IMPORTANCE A total of 340 million sexually transmitted infections (STIs) are acquired each year. Antimicrobial agents that target multiple infectious pathogens are ideal candidates to reduce the number of newly acquired STIs. The antimicrobial and immunoregulatory properties of GML make it an excellent candidate to fit this critical need. Previous studies established the safety profile and antibacterial activity of GML against both Gram-positive and Gram-negative bacteria. GML protected against high-dose SIV infection and reduced inflammation, which can exacerbate disease, during infection. We found that GML inhibits HIV-1 and other human-pathogenic viruses (yellow fever virus, mumps virus, and Zika virus), broadening its antimicrobial range. Because GML targets diverse infectious pathogens, GML may be an effective agent against the broad range of sexually transmitted pathogens. Further, our data show that reutericyclin, a GML analog expressed by some lactobacillus species, also inhibits HIV-1 replication and thus may contribute to the protective effect of Lactobacillus in HIV-1 transmission.


Assuntos
Antivirais/farmacologia , Lactobacillus/metabolismo , Lauratos/farmacologia , Monoglicerídeos/farmacologia , Proteínas do Envelope Viral/metabolismo , Vírus/efeitos dos fármacos , Animais , Antivirais/metabolismo , Feminino , HIV-1/efeitos dos fármacos , HIV-1/metabolismo , HIV-1/fisiologia , Humanos , Lauratos/metabolismo , Monoglicerídeos/metabolismo , Receptores Virais/metabolismo , Ácido Tenuazônico/análogos & derivados , Ácido Tenuazônico/metabolismo , Ácido Tenuazônico/farmacologia , Vagina/microbiologia , Ligação Viral , Internalização do Vírus , Vírus/metabolismo
6.
Vaccine ; 38(23): 3934-3941, 2020 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-32295718

RESUMO

INTRODUCTION: Subjects with rheumatoid arthritis (RA) receiving tumor necrosis factor-inhibiting (TNFi) therapies are at risk for severe influenza, and may respond less well to influenza vaccine. We examined the safety and immunogenicity of high dose influenza vaccine (HD) compared to standard dose vaccine (SD) in participants with RA receiving stable TNFi. METHODS: A randomized, double-blinded, Phase II study was conducted in adults with RA receiving TNFi, and healthy, gender and age-matched control subjects. Participants were immunized with HD (Sanofi Pasteur Fluzone High Dose [60 mcg × 3 strains]) or SD (Sanofi Pasteur Fluzone® [15 mcg × 3 strains]) intramuscularly (IM). A self-administered memory aid recorded temperature and systemic and local adverse events (AEs) for 8 days, and safety was evaluated and serum obtained to measure HAI activity on days 7, 21 and 180 days following vaccination. RESULTS: A greater proportion of RA subjects who received HD seroconverted at day 21 compared to SD, although this was not statistically significant. GMT antibody responses in RA subjects who received HD compared to SD were greater for all strains on day 21, and this was significant for H1N1. Seroconversion rates and GMT values were not different between RA subjects and control subjects. There were no safety concerns for HD or SD in RA subjects, and RA-related symptoms did not differ between SD and HD recipients by a RA-symptom questionnaire (RAPID 3). CONCLUSIONS: TNF-inhibitor therapy in people with RA did not appear to influence the immunogenicity of either SD or HD. Influenza seroconversion and GMT values were higher among RA subjects receiving HD compared to SD; however, differences were small and a larger study is needed to validate these findings. Given the apparent risk of increased influenza-related morbidity and mortality among immune compromised subjects, the higher GMT values generated by HD may be beneficial.

7.
Clin Infect Dis ; 2020 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-32198521

RESUMO

BACKGROUND: The increasing global prevalence of pulmonary nontuberculous mycobacteria (NTM) disease has called attention to challenges in NTM diagnosis and management. This study is conducted to understand management and outcomes of patients with pulmonary NTM disease at diverse centers across the US. METHODS: We conducted a 10-year (2005-2015) retrospective study at seven Vaccine and Treatment Evaluation Units to evaluate pulmonary NTM treatment outcomes in human immunodeficiency virus-negative adults. Demographic and clinical information were abstracted through medical record review. Microbiologic and clinical cure were evaluated using previously defined criteria. RESULTS: Of 297 patients diagnosed with pulmonary NTM, the most frequent NTM species were Mycobacterium avium-intracellulare complex (83.2%), M. kansasii (7.7%), and M. abscessus (3.4%). Two hundred forty-five (82.5%) patients received treatment, while 45 (15.2%) were followed without treatment. Eighty-six patients had available drug susceptibility results; of these, >40% exhibited resistance to rifampin, ethambutol, or amikacin. Of the 138 patients with adequate outcome data, 78 (56.5%) experienced clinical and/or microbiologic cure. Adherence to the American Thoracic Society/Infectious Diseases Society of America (ATS/IDSA) treatment guidelines was significantly more common in patients who were cured (odds ratio [OR] 4.5, 95% confidence interval [CI] 2.0-10.4, P < 0.001). Overall mortality was 15.7%. CONCLUSIONS: Despite ATS/IDSA Guidelines, management of pulmonary NTM disease was heterogeneous and cure rates were relatively low. Further work is required to understand which patients are suitable for monitoring without treatment and the impact of antimicrobial therapy on pulmonary NTM morbidity and mortality.

8.
Vaccine ; 38(11): 2600-2607, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32057574

RESUMO

Traditional replicating smallpox vaccines are associated with serious safety concerns in the general population and are contraindicated in immunocompromised individuals. However, this very population remains at greatest risk for severe complications following viral infections, making vaccine prevention particularly relevant. MVA-BN was developed as a non-replicating smallpox vaccine that is potentially safer for people who are immunocompromised. In this phase II trial, 3 MVA-BN dosing regimens were evaluated for safety, tolerability, and immunogenicity in persons with HIV (PWH) who had a history of AIDS. Following randomization, 87 participants who were predominately male and African American received either 2 standard doses on weeks 0 and 4 in the standard dose (SD) group (N = 27), 2 double-standard doses on the same schedule in the double dose (DD) group (N = 29), or 3 standard doses on weeks 0, 4 and 12 in the booster dose (BD) group (N = 31). No safety concerns were identified, and injection site pain was the most commonly reported solicited adverse event (AE) in all groups (66.7%), with no meaningful differences between groups. The incidence of severe (Grade 3) AEs was low across groups and no serious AEs or AEs of special interest considered related to study vaccine were reported. Doubling the standard MVA-BN dose had no significant effect on induction of neutralizing antibodies, with 100% seroconversion and comparable GMTs at week 6 in the SD and DD groups (78.9 and 100.3, respectively). A booster dose significantly increased peak neutralizing titers in the BD group (GMT: 281.1), which remained elevated at 12 months (GMT: 45.3) compared to the SD (GMT: 6.2) and DD (GMT: 10.6) groups. However, based on the immune response previously reported for healthy participants, a third dose (booster) does not appear necessary, even for immunocompromised participants. Clinical Trial Registry Number: NCT02038881.

9.
Mol Cell Proteomics ; 19(1): 78-100, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676584

RESUMO

Blood and semen are important body-fluids that carry exosomes for bioinformation transmission. Therefore, characterization of their proteomes is necessary for understanding body-fluid-specific physiologic and pathophysiologic functions. Using systematic multifactorial proteomic profiling, we characterized the proteomes of exosomes and exosome-free fractions from autologous blood and semen from three HIV-uninfected and three HIV-infected participants (total of 24 samples). We identified exosome-based protein signatures specific to blood and semen along with HIV-induced tissue-dependent proteomic perturbations. We validated our findings with samples from 16 additional donors and showed that unlike blood exosomes (BE), semen exosomes (SE) are enriched in clusterin. SE but not BE promote Protein·Nucleic acid binding and increase cell adhesion irrespective of HIV infection. This is the first comparative study of the proteome of autologous BE and SE. The proteins identified may be developed as biomarkers applicable to different fields of medicine, including reproduction and infectious diseases.


Assuntos
Sangue/metabolismo , Exossomos/metabolismo , Infecções por HIV/metabolismo , HIV-1/genética , Proteoma , Proteômica/métodos , Sêmen/metabolismo , Adulto , Biomarcadores/metabolismo , Infecções por HIV/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Mapas de Interação de Proteínas , RNA Viral/genética , Adulto Jovem
10.
FEBS Lett ; 594(4): 695-709, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31665815

RESUMO

Semen exosomes (SE) inhibit HIV infection. However, the effect of SE on cell activation and inflammation remains unknown. We characterized the response of peripheral blood mononuclear cells (PBMCs) from HIV-uninfected and antiretroviral therapy-suppressed HIV-infected (HIV+) subjects to SE. Quiescent PBMCs or T-cell receptor (TCR)-activated PBMCs from HIV- and HIV+ donors were stimulated with SE in the presence/absence of ex vivo HIV infection. In HIV-infected PBMCs, SE did not reactivate HIV, did not induce lymphoblast development, nor increase CD69+/CD25+ numbers. Furthermore, SE inhibited de novo HIV infection without altering cell activation. SE also asynchronously downregulated HIV-inducible IL1ß, IL8, and TNFα and upregulated CXCL10. These data suggest that SE inhibits HIV infection and production of HIV-induced proinflammatory cytokines while preserving lymphocyte activation.


Assuntos
Citocinas/biossíntese , Exossomos/metabolismo , HIV-1/fisiologia , Ativação Linfocitária , Linfócitos/imunologia , Linfócitos/virologia , Sêmen/citologia , Feminino , Células HEK293 , Humanos , Inflamação/metabolismo , Masculino , Replicação Viral
11.
Clin Infect Dis ; 71(5): 1221-1228, 2020 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31671178

RESUMO

BACKGROUND: Human pegivirus (HPgV) is a single-strand RNA virus belonging to the Flaviviridae. Although no definitive association between HPgV infection and disease has been identified, previous studies have suggested an association of HPgV viremia with risk of lymphomas. METHODS: We conducted a systematic review and meta-analysis, including 1 cohort study and 14 case-control studies, assessing the association of HPgV viremia with adult lymphomas. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using a random-effects model, overall and by geographic region and lymphoma subtype. RESULTS: The overall OR for lymphoma was 2.85 (95% CI, 1.98-4.11), with statistically significantly elevated ORs observed in 8 of 15 studies. There was a small amount of heterogeneity among studies (I2 = 28.9%; Q = 18.27, P = .16), and the funnel plot provided no evidence for publication bias. The strongest association with lymphoma risk was observed for studies from Southern Europe (OR, 5.68 [95% CI, 1.98-16.3]), whereas weaker ORs (with 95% CIs) were observed for studies from North America (2.24 [1.76-2.85]), Northern Europe (2.90 [.45-18.7), and the Middle East (2.51 [.87-7.27]), but all of similar magnitude. Participants with HPgV viremia had statistically significantly increased risks (OR [95% CI]) for developing diffuse large B-cell (3.29 [1.63-6.62]), follicular (3.01 [1.95-4.63]), marginal zone (1.90 [1.13-3.18]), and T-cell (2.11 [1.17-3.89]) lymphomas, while the risk for Hodgkin lymphoma (3.53 [.48-25.9]) and chronic lymphocytic leukemia (1.45 [.45-4.66]) were increased but did not achieve statistical significance. CONCLUSIONS: This meta-analysis supports a positive association of HPgV viremia with lymphoma risk, overall and for the major lymphoma subtypes.

12.
J Acquir Immune Defic Syndr ; 83(1): 90-98, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31809364

RESUMO

BACKGROUND: Extracellular vesicles (EVs) are cell-derived vesicles with diverse functions in intercellular communication including disease and infection, and EVs seem to influence HIV-1 pathogenesis. EVs isolated from HIV-1-uninfected semen (SE), but not blood (BE), contain factors that interfere with HIV-1 infection and replication in target cells. The reason for this dichotomy is unknown. Furthermore, the effect of HIV-1 infection and antiretroviral (ARV) drugs on the anti-HIV-1 effects of SE and BE is unknown. Here, we characterize EVs and EV-free plasma isolated from HIV-infected donor semen and blood and their effects on HIV infection. METHODS: EVs and EV-free plasma were purified from autologous blood and semen of HIV-negative, HIV-infected antiretroviral therapy (ART)-naïve, and HIV-infected ART-treated participants. HIV infection was assessed in a TZM-bl cell reporter system. ARV concentrations were analyzed using liquid chromatography-mass spectrometry. RESULTS: SE isolated from both HIV-negative and HIV-infected, ART-naïve donors inhibited HIV-1 infection, but BE and semen and blood EV-free plasma did not. By contrast, BE, SE, and EV-free plasma from HIV-infected, ART-treated donors inhibited HIV-1. Importantly, exosomes isolated from ART-treated donors contained concentrations of ARV drugs (ART-EVs) at biologically relevant inhibitory levels. CONCLUSIONS: The HIV-1-inhibitory phenotype of SE is independent of donor HIV-1 or ART status, and ARV drugs and their metabolites are SE- and BE-associated in vivo.


Assuntos
Fármacos Anti-HIV/farmacologia , Vesículas Extracelulares/metabolismo , Infecções por HIV/metabolismo , Sêmen/metabolismo , Replicação Viral/efeitos dos fármacos , Infecções por HIV/virologia , HIV-1/isolamento & purificação , HIV-1/fisiologia , Humanos , Técnicas In Vitro
13.
Cells ; 8(9)2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31484431

RESUMO

Semen exosomes (SE) from HIV-uninfected (HIV-) individuals potently inhibit HIV infection in vitro. However, morphological changes in target cells in response to SE have not been characterized or have the effect of HIV infection or the use of illicit substances, specifically psychostimulants, on the function of SE been elucidated. The objective of this study was to evaluate the effect of HIV infection, psychostimulant use, and both together on SE-mediated regulation of monocyte function. SE were isolated from semen of HIV- and HIV-infected (HIV+) antiretroviral therapy (ART)-naive participants who reported either using or not using psychostimulants. The SE samples were thus designated as HIV-Drug-, HIV-Drug+, HIV+Drug-, and HIV+Drug+. U937 monocytes were treated with different SEs and analyzed for changes in transcriptome, morphometrics, actin reorganization, adhesion, and chemotaxis. HIV infection and/or use of psychostimulants had minimal effects on the physical characteristics of SE. However, different SEs had diverse effects on the messenger RNA signature of monocytes and rapidly induced monocyte adhesion and spreading. SE from HIV infected or psychostimulants users but not HIV-Drug- SE, stimulated actin reorganization, leading to the formation of filopodia-like structures and membrane ruffles containing F-actin and vinculin that in some cases were colocalized. All SE stimulated monocyte chemotaxis to HIV secretome and activated the secretion of matrix metalloproteinases, a phenotype exacerbated by HIV infection and psychostimulant use. SE-directed regulation of cellular morphometrics and chemotaxis depended on the donor clinical status because HIV infection and psychostimulant use altered SE function. Although our inclusion criteria specified the use of cocaine, humans are poly-drug and alcohol users and our study participants used psychostimulants, marijuana, opiates, and alcohol. Thus, it is possible that the effects observed in this study may be due to one of these other substances or due to an interaction between different substances.


Assuntos
Adesão Celular , Quimiotaxia , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Exossomos/metabolismo , Infecções por HIV/metabolismo , Monócitos/metabolismo , Sêmen/metabolismo , Actinas/metabolismo , Adulto , Transtornos Relacionados ao Uso de Cocaína/complicações , Infecções por HIV/complicações , Humanos , Masculino , Metaloproteinases da Matriz/metabolismo , Monócitos/fisiologia , Transcriptoma , Células U937
14.
Gastroenterol Clin North Am ; 48(2): 259-279, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31046974

RESUMO

Many microbes, toxins, autoimmune diseases, and neoplastic diseases may cause liver inflammation; however, 5 viruses whose main pathogenesis is liver disease are referred to as hepatitis A, B, C, D, and E viruses. These viruses cause a significant burden of global illness. With the exception of hepatitis A virus, all may cause chronic infection potentially leading to cirrhosis and hepatocellular carcinoma. Excellent serologic and nucleic acid detection methods are available for determining the precise cause and, in some cases, the duration of infection. Diagnostics are critical for identifying individuals needing treatment and for monitoring the treatment success.


Assuntos
Técnicas de Laboratório Clínico , Hepatite B/diagnóstico , Hepatite C/diagnóstico , Hepatite D/diagnóstico , Técnicas de Laboratório Clínico/métodos , Humanos , Monitorização Fisiológica , Técnicas de Amplificação de Ácido Nucleico , Testes Sorológicos/métodos
15.
J Gen Virol ; 100(3): 350-366, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30702421

RESUMO

The terms extracellular vesicles, microvesicles, oncosomes, or exosomes are often used interchangeably as descriptors of particles that are released from cells and comprise a lipid membrane that encapsulates nucleic acids and proteins. Although these entities are defined based on a specific size range and/or mechanism of release, the terminology is often ambiguous. Nevertheless, these vesicles are increasingly recognized as important modulators of intercellular communication. The generic characterization of extracellular vesicles could also be used as a descriptor of enveloped viruses, highlighting the fact that extracellular vesicles and enveloped viruses are similar in both composition and function. Their high degree of similarity makes differentiating between vesicles and enveloped viruses in biological specimens particularly difficult. Because viral particles and extracellular vesicles are produced simultaneously in infected cells, it is necessary to separate these populations to understand their independent functions. We summarize current understanding of the similarities and differences of extracellular vesicles, which henceforth we will refer to as exosomes, and the enveloped retrovirus, HIV-1. Here, we focus on the presence of these particles in semen, as these are of particular importance during HIV-1 sexual transmission. While there is overlap in the terminology and physical qualities between HIV-1 virions and exosomes, these two types of intercellular vehicles may differ depending on the bio-fluid source. Recent data have demonstrated that exosomes from human semen serve as regulators of HIV-1 infection that may contribute to the remarkably low risk of infection per sexual exposure.


Assuntos
Exossomos/virologia , Vesículas Extracelulares/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Animais , Exossomos/metabolismo , Infecções por HIV/metabolismo , HIV-1/genética , Humanos
16.
J Virol ; 92(21)2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30111566

RESUMO

Exosomes play various roles in host responses to cancer and infective agents, and semen exosomes (SE) inhibit HIV-1 infection and transmission, although the mechanism(s) by which this occurs is unclear. Here, we show that SE block HIV-1 proviral transcription at multiple transcriptional checkpoints, including transcription factor recruitment to the long terminal repeat (LTR), transcription initiation, and elongation. Biochemical and functional studies show that SE inhibit HIV-1 LTR-driven viral gene expression and virus replication. Through partitioning of the HIV-1 RNA, we found that SE reduced the optimal expression of various viral RNA species. Chromatin immunoprecipitation-real-time quantitative PCR (ChIP-RT-qPCR) and electrophoretic mobility shift assay (EMSA) analysis of infected cells identified the human transcription factors NF-κB and Sp1, as well as RNA polymerase (Pol) II and the viral protein transcriptional activator (Tat), as targets of SE. Of interest, SE inhibited HIV-1 LTR activation mediated by HIV-1 or Tat, but not by the mitogen phorbol myristate acetate (PMA) or tumor necrosis factor alpha (TNF-α). SE inhibited the DNA binding activities of NF-κB and Sp1 and blocked the recruitment of these transcription factors and Pol II to the HIV-1 LTR promoter. Importantly, SE directly blocked NF-κB, Sp1, and Pol II binding to the LTR and inhibited the interactions of Tat/NF-κB and Tat/Sp1, suggesting that SE-mediated inhibition of the functional quadripartite complex NF-κB-Sp1-Pol II-Tat may be a novel mechanism of proviral transcription repression. These data provide a novel molecular basis for SE-mediated inhibition of HIV-1 and identify Tat as a potential target of SE.IMPORTANCE HIV is most commonly transmitted sexually, and semen is the primary vector. Despite progress in studies of HIV pathogenesis and the success of combination antiretroviral therapy in controlling viral replication, current therapy cannot completely control sexual transmission. Thus, there is a need to identify effective methods of controlling HIV replication and transmission. Recently, it was shown that human semen contains exosomes that protect against HIV infection in vitro In this study, we identified a mechanism by which semen exosomes inhibited HIV-1 RNA expression. We found that semen exosomes inhibit recruitment of transcription factors NF-κB and Sp1, as well as RNA Pol II, to the promoter region in the 5' long terminal repeat (LTR) of HIV-1. The HIV-1 early protein transcriptional activator (Tat) was a target of semen exosomes, and semen exosomes inhibited the binding and recruitment of Tat to the HIV-1 LTR.


Assuntos
Exossomos/metabolismo , Infecções por HIV/genética , HIV-1/genética , NF-kappa B/metabolismo , Sêmen/metabolismo , Fator de Transcrição Sp1/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Sítios de Ligação , Exossomos/genética , Regulação Viral da Expressão Gênica , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Repetição Terminal Longa de HIV/genética , Humanos , Masculino , NF-kappa B/genética , Regiões Promotoras Genéticas , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Fator de Transcrição Sp1/genética , Transcrição Genética , Ativação Transcricional , Replicação Viral , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
17.
Br J Haematol ; 182(5): 644-653, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29808922

RESUMO

We evaluated the association of Human Pegivirus (HPgV) viraemia with risk of developing lymphoma, overall and by major subtypes. Because this virus has also been associated with better prognosis in the setting of co-infection with human immunodeficiency virus, we further assessed the association of HPgV with prognosis. We used risk factor data and banked plasma samples from 2094 lymphoma cases newly diagnosed between 2002 and 2009 and 1572 frequency-matched controls. Plasma samples were tested for HPgV RNA by reverse transcription polymerase chain reaction (RT-PCR), and those with RNA concentrations <5000 genome equivalents/ml were confirmed using nested RT-PCR methods. To assess the role of HPgV in lymphoma prognosis, we used 2948 cases from a cohort study of newly diagnosed lymphoma patients (included all cases from the case-control study). There was a positive association of HPgV viraemia with risk of lymphoma overall (Odds ratio = 2·14; 95% confidence interval [CI] 1·63-2·80; P < 0·0001), and for all major subtypes except Hodgkin lymphoma and chronic lymphocytic leukaemia/small lymphocytic lymphoma, and this was not confounded by other lymphoma risk factors. In contrast, there was no association of HPgV viraemia with event-free survival (Hazard ratio [HR] = 1·00; 95% CI 0·85-1·18) or overall survival (HR = 0·97; 95% CI 0·79-1·20) for lymphoma overall, or any of the subtypes. These data support the hypothesis for a role of HPgV in the aetiology of multiple lymphoma subtypes.


Assuntos
Infecções por Flaviviridae/complicações , Linfoma/etiologia , Idoso , Infecções por Flaviviridae/mortalidade , Humanos , Pessoa de Meia-Idade , Prognóstico , RNA Viral/sangue , Risco , Fatores de Risco , Análise de Sobrevida
18.
J Virol ; 92(2)2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29093095

RESUMO

HIV vaccine development is focused on designing immunogens and delivery methods that elicit protective immunity. We evaluated a combination of adenovirus (Ad) vectors expressing HIV 1086.C (clade C) envelope glycoprotein (Env), SIV Gag p55, and human pegivirus GBV-C E2 glycoprotein. We compared replicating simian (SAd7) with nonreplicating human (Ad4) adenovirus-vectored vaccines paired with recombinant proteins in a novel prime-boost regimen in rhesus macaques, with the goal of eliciting protective immunity against SHIV challenge. In both vaccine groups, plasma and buccal Env-specific IgG, tier 1 heterologous neutralizing antibodies, and antibody-dependent cell-mediated viral inhibition were readily generated. High Env-specific T cell responses elicited in all vaccinees were significantly greater than responses targeting Gag. After three intrarectal exposures to heterologous tier 1 clade C SHIV, all 10 sham-vaccinated controls were infected, whereas 4/10 SAd7- and 3/10 Ad4-vaccinated macaques remained uninfected or maintained tightly controlled plasma viremia. Time to infection was significantly delayed in SAd7-vaccinated macaques compared to the controls. Cell-associated and plasma virus levels were significantly lower in each group of vaccinated macaques compared to controls; the lowest plasma viral burden was found in animals vaccinated with the SAd7 vectors, suggesting superior immunity conferred by the replicating simian vectors. Furthermore, higher V1V2-specific binding antibody titers correlated with viral control in the SAd7 vaccine group. Thus, recombinant Ad plus protein vaccines generated humoral and cellular immunity that was effective in either protecting from SHIV acquisition or significantly reducing viremia in animals that became infected, consequently supporting additional development of replicating Ad vectors as HIV vaccines.IMPORTANCE There is a well-acknowledged need for an effective AIDS vaccine that protects against HIV infection and limits in vivo viral replication and associated pathogenesis. Although replicating virus vectors have been advanced as HIV vaccine platforms, there have not been any direct comparisons of the replicating to the nonreplicating format. The present study directly compared the replicating SAd7 to nonreplicating Ad4 vectors in macaques and demonstrated that in the SAd7 vaccine group, the time to infection was significantly delayed compared to the control group, and V1V2 Env-specific binding antibodies correlated with viral outcomes. Viral control was significantly enhanced in vaccinated macaques compared to controls, and in infected SAd7-vaccinated macaques compared to Ad4-vaccinated macaques, suggesting that this vector may have conferred more effective immunity. Because blocking infection is so difficult with current vaccines, development of a vaccine that can limit viremia if infection occurs would be valuable. These data support further development of replicating adenovirus vectors.


Assuntos
Adenoviridae , Vetores Genéticos , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/imunologia , Vacinas Sintéticas , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos/imunologia , Contagem de Linfócito CD4 , Linhagem Celular , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Genótipo , HIV/imunologia , Humanos , Imunidade Humoral , Imunização/métodos , Estimativa de Kaplan-Meier , Macaca mulatta , Masculino , Ligação Proteica/imunologia , Vacinas contra a SAIDS/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Proteínas do Envelope Viral/imunologia , Carga Viral
19.
Front Immunol ; 8: 1339, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29104574

RESUMO

In autoimmune diseases, toll-like receptor (TLR)-stimulated pro-inflammatory IL-6-secreting B cells exert pathogenic roles. Similarly, B cell Fc receptor-like 4 (FcRL4) expression amplifies TLR stimulation, and in rheumatoid arthritis patients, FcRL4 expression identifies a pro-inflammatory B cell subset. B cells from HIV-infected subjects also express heightened levels of FcRL4 and secrete high levels of IL-6: a critical mediator of HIV disease progression. In this study, we sought to determine if FcRL4 identifies a pro-inflammatory B cell subset in HIV-infected subjects and further elucidate the mechanisms underlying FcRL4 amplification of TLR stimulation. We determine that tissue-like memory B cells express the highest endogenous levels of FcRL4 positively correlating with IL-6 expression (p = 0.0022, r = 0.8667), but activated memory B cells exhibit the highest frequency of FcRL4hiIL-6hi cells. FcRL4hi B cells exhibit an activated TLR-signaling pathway identified by elevated phosphorylation levels of: pERK (p = 0.0373), p38 (p = 0.0337), p65 (p = 0.1097), and cJUN (p = 0.0239), concomitant with significantly elevated expression of the TLR-signaling modulator hematopoietic cell kinase (HcK, p = 0.0414). Compared to FcRL4neg B cells from healthy controls, TLR9-stimulated FcRL4pos B cells express significantly higher levels of lL-6 (p = 0.0179). Further, TLR9-stimulated B cells also upregulate the expression of FcRL4 (p = 0.0415) and HcK (p = 0.0386). In B-cell lines, siRNA-mediated HcK knockdown downmodulates TLR9-induced FcRL4-mediated activation quantified by CD23 upregulation (p = 0.0553). We present data suggesting that, in viremic HIV-infected individuals, FcRL4 expression identifies unique IL-6 producing pro-inflammatory B-cell subsets. Further, TLR stimulation likely modulates FcRL4 expression and FcRL4 expression is associated with Hck, potentially enhancing the activation of TLR-signaling associated transcription factors. Pathogenic B-cells have been identified in other disease settings, and this study represents a novel report describing a pro-inflammatory B cell subset in HIV-infected patients.

20.
J Infect Dis ; 216(9): 1164-1175, 2017 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-28968905

RESUMO

The Flavivirus genus within the Flaviviridae family is comprised of many important human pathogens including yellow fever virus (YFV), dengue virus (DENV), and Zika virus (ZKV), all of which are global public health concerns. Although the related flaviviruses hepatitis C virus and human pegivirus (formerly named GBV-C) interfere with T-cell receptor (TCR) signaling by novel RNA and protein-based mechanisms, the effect of other flaviviruses on TCR signaling is unknown. Here, we studied the effect of YFV, DENV, and ZKV on TCR signaling. Both YFV and ZKV replicated in human T cells in vitro; however, only YFV inhibited TCR signaling. This effect was mediated at least in part by the YFV envelope (env) protein coding RNA. Deletion mutagenesis studies demonstrated that expression of a short, YFV env RNA motif (vsRNA) was required and sufficient to inhibit TCR signaling. Expression of this vsRNA and YFV infection of T cells reduced the expression of a Src-kinase regulatory phosphatase (PTPRE), while ZKV infection did not. YFV infection in mice resulted in impaired TCR signaling and PTPRE expression, with associated reduction in murine response to experimental ovalbumin vaccination. Together, these data suggest that viruses within the flavivirus genus inhibit TCR signaling in a species-dependent manner.


Assuntos
Vírus da Dengue/genética , RNA/genética , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/genética , Replicação Viral/genética , Vírus da Febre Amarela/genética , Zika virus/genética , Vírus da Dengue/patogenicidade , Humanos , Vírus da Febre Amarela/patogenicidade , Zika virus/patogenicidade
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