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1.
mBio ; 12(4): e0150321, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34311582

RESUMO

Severe coronavirus disease 2019 (COVID-19) has been associated with T cell lymphopenia, but no causal effect of T cell deficiency on disease severity has been established. To investigate the specific role of T cells in recovery from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, we studied rhesus macaques that were depleted of either CD4+, CD8+, or both T cell subsets prior to infection. Peak virus loads were similar in all groups, but the resolution of virus in the T cell-depleted animals was slightly delayed compared to that in controls. The T cell-depleted groups developed virus-neutralizing antibody responses and class switched to IgG. When reinfected 6 weeks later, the T cell-depleted animals showed anamnestic immune responses characterized by rapid induction of high-titer virus-neutralizing antibodies, faster control of virus loads, and reduced clinical signs. These results indicate that while T cells play a role in the recovery of rhesus macaques from acute SARS-CoV-2 infections, their depletion does not induce severe disease, and T cells do not account for the natural resistance of rhesus macaques to severe COVID-19. Neither primed CD4+ nor CD8+ T cells appeared critical for immunoglobulin class switching, the development of immunological memory, or protection from a second infection. IMPORTANCE Patients with severe COVID-19 often have decreased numbers of T cells, a cell type important in fighting most viral infections. However, it is not known whether the loss of T cells contributes to severe COVID-19 or is a consequence of it. We studied rhesus macaques, which develop only mild COVID-19, similar to most humans. Experimental depletion of T cells slightly prolonged their clearance of virus, but there was no increase in disease severity. Furthermore, they were able to develop protection from a second infection and produced antibodies capable of neutralizing the virus. They also developed immunological memory, which allows a much stronger and more rapid response upon a second infection. These results suggest that T cells are not critical for recovery from acute SARS-CoV-2 infections in this model and point toward B cell responses and antibodies as the essential mediators of protection from re-exposure.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/patologia , Memória Imunológica/imunologia , Linfopenia/imunologia , SARS-CoV-2/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , COVID-19/imunologia , Feminino , Depleção Linfocítica/métodos , Macaca mulatta/imunologia , Masculino
2.
bioRxiv ; 2021 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-33821272

RESUMO

Severe COVID-19 has been associated with T cell lymphopenia 1,2, but no causal effect of T cell deficiency on disease severity has been established. To investigate the specific role of T cells in recovery from SARS-CoV-2 infections we studied rhesus macaques that were depleted of either CD4+, CD8+ or both T cell subsets prior to infection. Peak virus loads were similar in all groups, but the resolution of virus in the T cell-depleted animals was slightly delayed compared to controls. The T cell-depleted groups developed virus-neutralizing antibody responses and also class-switched to IgG. When re-infected six weeks later, the T cell-depleted animals showed anamnestic immune responses characterized by rapid induction of high-titer virus-neutralizing antibodies, faster control of virus loads and reduced clinical signs. These results indicate that while T cells play a role in the recovery of rhesus macaques from acute SARS-CoV-2 infections, their depletion does not induce severe disease, and T cells do not account for the natural resistance of rhesus macaques to severe COVID-19. Neither primed CD4+ or CD8+ T cells appeared critical for immunoglobulin class switching, the development of immunological memory or protection from a second infection.

3.
Am J Trop Med Hyg ; 104(6): 2195-2198, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33878029

RESUMO

The burden on diagnostic and research laboratories to provide reliable inactivation for biological specimens to allow for safe downstream processing is high during the coronavirus disease 2019 (COVID-19) pandemic. We provide safety data regarding commonly used chemical and physical inactivation procedures that verify their effectiveness against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).


Assuntos
Detergentes/farmacologia , Desinfetantes/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/efeitos da radiação , Inativação de Vírus , Humanos , Laboratórios , RNA Viral/fisiologia , Manejo de Espécimes/métodos
4.
Am J Trop Med Hyg ; 100(5): 1275-1277, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30860018

RESUMO

Gamma irradiation using a cobalt-60 source is a commonly used method for the inactivation of infectious specimens to be handled safely in subsequent laboratory procedures. Here, we determined irradiation doses to safely inactivate liquid proteinaceous specimens harboring different emerging/reemerging viral pathogens known to cause neglected tropical and other diseases of regional or global public health importance. By using a representative arenavirus, bunyavirus, coronavirus, filovirus, flavivirus, orthomyxovirus, and paramyxovirus, we found that these enveloped viruses differed in their susceptibility to irradiation treatment with adsorbed doses for inactivation of a target dose of 1 × 106 50% tissue culture infectious dose (TCID50)/mL ranging from 1 to 5 MRads. This finding seemed generally inversely correlated with genome size. Our data may help to guide other facilities in testing and verifying safe inactivation procedures.


Assuntos
Raios gama , Inativação de Vírus/efeitos da radiação , Vírus/patogenicidade , Vírus/efeitos da radiação , Flavivirus/efeitos da radiação , Genoma Viral/efeitos da radiação , Orthobunyavirus/efeitos da radiação , Orthomyxoviridae/efeitos da radiação , Virologia/métodos
5.
J Infect Dis ; 218(suppl_5): S297-S300, 2018 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-29982557

RESUMO

Diagnostics and research analyses involving samples containing maximum-containment viruses present unique challenges, and inactivation protocols compatible with downstream testing are needed. Our aim was to identify a validated viral inactivation protocol compatible with bacterial identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We assessed a panel of bacteria with 6 validated maximum-containment virus-inactivation protocols and report that inactivation with TRIzol or γ-irradiation is compatible with MALDI-TOF MS. The availability, simplicity, and rapidity of TRIzol inactivation make this method the more suitable choice.


Assuntos
Bactérias/efeitos da radiação , Coinfecção/virologia , Inativação de Vírus/efeitos da radiação , Vírus/efeitos da radiação , Humanos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
6.
Science ; 348(6233): 439-42, 2015 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-25814063

RESUMO

Zaire ebolavirus is the causative agent of the current outbreak of hemorrhagic fever disease in West Africa. Previously, we showed that a whole Ebola virus (EBOV) vaccine based on a replication-defective EBOV (EBOVΔVP30) protects immunized mice and guinea pigs against lethal challenge with rodent-adapted EBOV. Here, we demonstrate that EBOVΔVP30 protects nonhuman primates against lethal infection with EBOV. Although EBOVΔVP30 is replication-incompetent, we additionally inactivated the vaccine with hydrogen peroxide; the chemically inactivated vaccine remained antigenic and protective in nonhuman primates. EBOVΔVP30 thus represents a safe, efficacious, whole-EBOV vaccine candidate that differs from other EBOV vaccine platforms in that it presents all viral proteins and the viral RNA to the host immune system, which might contribute to protective immune responses.


Assuntos
Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Animais , Vacinas contra Ebola/administração & dosagem , Vacinas contra Ebola/efeitos adversos , Ebolavirus/efeitos dos fármacos , Ebolavirus/efeitos da radiação , Raios gama , Cobaias , Peróxido de Hidrogênio/farmacologia , Macaca fascicularis , Camundongos , RNA Viral/imunologia , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/efeitos adversos , Vacinas Atenuadas/imunologia , Proteínas Virais/imunologia , Replicação Viral
7.
Proc Natl Acad Sci U S A ; 110(5): 1893-8, 2013 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-23319647

RESUMO

Ebola viruses cause hemorrhagic disease in humans and nonhuman primates with high fatality rates. These viruses pose a significant health concern worldwide due to the lack of approved therapeutics and vaccines as well as their potential misuse as bioterrorism agents. Although not licensed for human use, recombinant vesicular stomatitis virus (rVSV) expressing the filovirus glycoprotein (GP) has been shown to protect macaques from Ebola virus and Marburg virus infections, both prophylactically and postexposure in a homologous challenge setting. However, the immune mechanisms of protection conferred by this vaccine platform remain poorly understood. In this study, we set out to investigate the role of humoral versus cellular immunity in rVSV vaccine-mediated protection against lethal Zaire ebolavirus (ZEBOV) challenge. Groups of cynomolgus macaques were depleted of CD4+ T, CD8+ T, or CD20+ B cells before and during vaccination with rVSV/ZEBOV-GP. Unfortunately, CD20-depleted animals generated a robust IgG response. Therefore, an additional group of vaccinated animals were depleted of CD4+ T cells during challenge. All animals were subsequently challenged with a lethal dose of ZEBOV. Animals depleted of CD8+ T cells survived, suggesting a minimal role for CD8+ T cells in vaccine-mediated protection. Depletion of CD4+ T cells during vaccination caused a complete loss of glycoprotein-specific antibodies and abrogated vaccine protection. In contrast, depletion of CD4+ T cells during challenge resulted in survival of the animals, indicating a minimal role for CD4+ T-cell immunity in rVSV-mediated protection. Our results suggest that antibodies play a critical role in rVSV-mediated protection against ZEBOV.


Assuntos
Anticorpos Antivirais/imunologia , Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/imunologia , Glicoproteínas de Membrana/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Antivirais/sangue , Citocinas/sangue , Citocinas/imunologia , Vacinas contra Ebola/administração & dosagem , Ebolavirus/genética , Ensaio de Imunoadsorção Enzimática , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Fígado/imunologia , Fígado/parasitologia , Fígado/patologia , Linfócitos/imunologia , Macaca fascicularis , Masculino , Marburgvirus/genética , Marburgvirus/imunologia , Glicoproteínas de Membrana/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/imunologia , Baço/parasitologia , Baço/patologia , Fatores de Tempo , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia , Proteínas do Envelope Viral/genética , Carga Viral/genética
8.
J Virol ; 85(3): 1214-23, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21084481

RESUMO

The first influenza pandemic of the new millennium was caused by a newly emerged swine-origin influenza virus (SOIV) (H1N1). This new virus is characterized by a previously unknown constellation of gene segments derived from North American and Eurasian swine lineages and the absence of common markers predictive of human adaptation. Overall, human infections appeared to be mild, but an alarming number of young individuals presented with symptoms atypical for seasonal influenza. The new SOIV also showed a sustained human-to-human transmissibility and higher reproduction ratio than common seasonal viruses, altogether indicating a higher pathogenic potential for this newly emerged virus. To study the virulence of the SOIV, we used a recently established cynomolgus macaque model and compared parameters of clinical disease, virology, host responses, and pathology/histopathology with a current seasonal H1N1 virus. We here show that infection of macaques with two genetically similar but clinically distinct SOIV isolates from the early stage of the pandemic (A/Mexico/4108/2009 and A/Mexico/InDRE4487/2009) resulted in upper and lower respiratory tract infections and clinical disease ranging from mild to severe pneumonia that was clearly advanced over the mild infection caused by A/Kawasaki/UTK-4/2009, a current seasonal strain. Unexpectedly, we observed heterogeneity among the two SOIV isolates in virus replication, host transcriptional and cytokine responses, and disease progression, demonstrating a higher pathogenic potential for A/Mexico/InDRE4487/2009. Differences in virulence may explain more severe disease, as was seen with certain individuals infected with the emerged pandemic influenza virus. Thus, the nonhuman primate model closely mimics influenza in humans.


Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H1N1/patogenicidade , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Doenças dos Primatas/patologia , Doenças dos Primatas/virologia , Animais , Pré-Escolar , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Perfilação da Expressão Gênica , Variação Genética , Humanos , Influenza Humana/virologia , Macaca , Masculino , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Infecções Respiratórias/patologia , Infecções Respiratórias/virologia , Índice de Gravidade de Doença , Virulência
9.
J Virol ; 84(7): 3503-15, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20106931

RESUMO

Flaviviruses transmitted by arthropods represent a tremendous disease burden for humans, causing millions of infections annually. All vector-borne flaviviruses studied to date suppress host innate responses to infection by inhibiting alpha/beta interferon (IFN-alpha/beta)-mediated JAK-STAT signal transduction. The viral nonstructural protein NS5 of some flaviviruses functions as the major IFN antagonist, associated with inhibition of IFN-dependent STAT1 phosphorylation (pY-STAT1) or with STAT2 degradation. West Nile virus (WNV) infection prevents pY-STAT1 although a role for WNV NS5 in IFN antagonism has not been fully explored. Here, we report that NS5 from the virulent NY99 strain of WNV prevented pY-STAT1 accumulation, suppressed IFN-dependent gene expression, and rescued the growth of a highly IFN-sensitive virus (Newcastle disease virus) in the presence of IFN, suggesting that this protein can function as an efficient IFN antagonist. In contrast, NS5 from Kunjin virus (KUN), a naturally attenuated subtype of WNV, was a poor suppressor of pY-STAT1. Mutation of a single residue in KUN NS5 to the analogous residue in WNV-NY99 NS5 (S653F) rendered KUN NS5 an efficient inhibitor of pY-STAT1. Incorporation of this mutation into recombinant KUN resulted in 30-fold greater inhibition of JAK-STAT signaling than with the wild-type virus and enhanced KUN replication in the presence of IFN. Thus, a naturally occurring mutation is associated with the function of NS5 in IFN antagonism and may influence virulence of WNV field isolates.


Assuntos
Interferon Tipo I/antagonistas & inibidores , Janus Quinases/antagonistas & inibidores , Fator de Transcrição STAT1/antagonistas & inibidores , Transdução de Sinais/fisiologia , Proteínas não Estruturais Virais/fisiologia , Animais , Chlorocebus aethiops , Humanos , Células Vero , Proteínas não Estruturais Virais/química , Vírus do Nilo Ocidental/fisiologia
10.
Nat Genet ; 39(6): 733-40, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17496894

RESUMO

Allotypes of the natural killer (NK) cell receptor KIR3DL1 vary in both NK cell expression patterns and inhibitory capacity upon binding to their ligands, HLA-B Bw4 molecules, present on target cells. Using a sample size of over 1,500 human immunodeficiency virus (HIV)+ individuals, we show that various distinct allelic combinations of the KIR3DL1 and HLA-B loci significantly and strongly influence both AIDS progression and plasma HIV RNA abundance in a consistent manner. These genetic data correlate very well with previously defined functional differences that distinguish KIR3DL1 allotypes. The various epistatic effects observed here for common, distinct KIR3DL1 and HLA-B Bw4 combinations are unprecedented with regard to any pair of genetic loci in human disease, and indicate that NK cells may have a critical role in the natural history of HIV infection.


Assuntos
Infecções por HIV/imunologia , HIV-1/genética , Antígenos HLA-B/metabolismo , Receptores Imunológicos/metabolismo , Alelos , Estudos de Coortes , Progressão da Doença , Infecções por HIV/genética , Infecções por HIV/metabolismo , Antígenos HLA-B/genética , Antígenos HLA-B/imunologia , Humanos , Imunidade Inata , Células Matadoras Naturais/imunologia , RNA Viral/sangue , RNA Viral/genética , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores KIR , Receptores KIR3DL1 , Carga Viral
11.
J Virol ; 77(20): 10900-9, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-14512540

RESUMO

Virus-specific CD4(+) T-cell function is thought to play a central role in induction and maintenance of effective CD8(+) T-cell responses in experimental animals or humans. However, the reasons that diminished proliferation of human immunodeficiency virus (HIV)-specific CD4(+) T cells is observed in the majority of infected patients and the role of these diminished responses in the loss of control of replication during the chronic phase of HIV infection remain incompletely understood. In a cohort of 15 patients that were selected for particularly strong HIV-specific CD4(+) T-cell responses, the effects of viremia on these responses were explored. Restriction of HIV replication was not observed during one to eight interruptions of antiretroviral therapy in the majority of patients (12 of 15). In each case, proliferative responses to HIV antigens were rapidly inhibited during viremia. The frequencies of cells that produce IFN-gamma in response to Gag, Pol, and Nef peptide pools were maintained during an interruption of therapy. In a subset of patients with elevated frequencies of interleukin-2 (IL-2)-producing cells, IL-2 production in response to HIV antigens was diminished during viremia. Addition of exogenous IL-2 was sufficient to rescue in vitro proliferation of DR0101 class II Gag or Pol tetramer(+) or total-Gag-specific CD4(+) T cells. These observations suggest that, during viremia, diminished in vitro proliferation of HIV-specific CD4(+) T cells is likely related to diminished IL-2 production. These results also suggest that relatively high frequencies of HIV-specific CD4(+) T cells persist in the peripheral blood during viremia, are not replicatively senescent, and proliferate when IL-2 is provided exogenously.


Assuntos
Linfócitos T CD4-Positivos/imunologia , HIV/imunologia , Interleucina-2/biossíntese , Ativação Linfocitária , Replicação Viral , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Interleucina-2/farmacologia , Viremia/imunologia , Viremia/virologia
12.
J Virol ; 77(12): 6889-98, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12768008

RESUMO

Although the HLA B(*)5701 class I allele is highly overrepresented among human immunodeficiency virus (HIV)-infected long-term nonprogressors (LTNPs), it is also present at the expected frequency (11%) in patients with progressive HIV infection. Whether B57(+) progressors lack restriction of viral replication because of escape from recognition of highly immunodominant B57-restricted gag epitopes by CD8(+) T cells remains unknown. In this report, we investigate the association between restriction of virus replication and recognition of autologous virus sequences in 27 B(*)57(+) patients (10 LTNPs and 17 progressors). Amplification and direct sequencing of single molecules of viral cDNA or proviral DNA revealed low frequencies of genetic variations in these regions of gag. Furthermore, CD8(+) T-cell recognition of autologous viral variants was preserved in most cases. In two patients, responses to autologous viral variants were not demonstrable at one epitope. By using a novel technique to isolate primary CD4(+) T cells expressing autologous viral gene products, it was found that 1 to 13% of CD8(+) T cells were able to respond to these cells by gamma interferon production. In conclusion, escape-conferring mutations occur infrequently within immunodominant B57-restricted gag epitopes and are not the primary mechanism of virus evasion from immune control in B(*)5701(+) HIV-infected patients. Qualitative features of the virus-specific CD8(+) T-cell response not measured by current assays remain the most likely determinants of the differential abilities of HLA B(*)5701(+) LTNPs and progressors to restrict virus replication.


Assuntos
Sequência de Aminoácidos , Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/imunologia , Produtos do Gene gag/química , Infecções por HIV/imunologia , Sobreviventes de Longo Prazo ao HIV , Antígenos HLA-B/metabolismo , Replicação Viral , Apresentação de Antígeno , Linfócitos T CD4-Positivos/metabolismo , Citometria de Fluxo , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/imunologia , Antígenos HLA-B/genética , Humanos , Epitopos Imunodominantes/imunologia , Dados de Sequência Molecular , Análise de Sequência de DNA , Proteínas Virais/genética , Proteínas Virais/imunologia
13.
Nat Immunol ; 3(11): 1061-8, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12368910

RESUMO

It is unclear why immunological control of HIV replication is incomplete in most infected individuals. We examined here the CD8+ T cell response to HIV-infected CD4+ T cells in rare patients with immunological control of HIV. Although high frequencies of HIV-specific CD8+ T cells were present in nonprogressors and progressors, only those of nonprogressors maintained a high proliferative capacity. This proliferation was coupled to increases in perforin expression. These results indicated that nonprogressors were differentiated by increased proliferative capacity of HIV-specific CD8+ T cells linked to enhanced effector function. In addition, the relative absence of these functions in progressors may represent a mechanism by which HIV avoids immunological control.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , Sobreviventes de Longo Prazo ao HIV , HIV-1/imunologia , HIV-2/imunologia , Ativação Linfocitária , Glicoproteínas de Membrana/fisiologia , Subpopulações de Linfócitos T/imunologia , Adulto , Idoso , Fármacos Anti-HIV/uso terapêutico , Antígenos CD/análise , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Estudos de Coortes , Progressão da Doença , Exocitose , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/fisiologia , HIV-2/fisiologia , Humanos , Integrina beta1/imunologia , Masculino , Pessoa de Meia-Idade , Perforina , Fenótipo , Proteínas Citotóxicas Formadoras de Poros , Receptores de Antígenos de Linfócitos T/imunologia , Carga Viral , Replicação Viral
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