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1.
Virus Res ; 295: 198305, 2021 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-33482242

RESUMO

In this study, we showed that a codon optimized version of the spike (S) protein of SARS-CoV-2 can migrate to the cell membrane. However, efficient production of Moloney murine leukemia (MLV) infectious viral particles was only achieved with stable expression of a shorter S version in C-terminal (ΔS) in MLV Gag-pol expressing cells. As compared to transient transfections, this platform generated viruses with a 1000-fold higher titer. ΔS was 15-times more efficiently incorporated into VLPs as compared to S, and that was not due to steric interference between the cytoplasmic tail and the MLV capsid, as similar differences were also observed with extracellular vesicles. The amount of ΔS incorporated into VLPs released from producer cells was high and estimated at 1.25 µg/mL S2 equivalent (S is comprised of S1 and S2). The resulting VLPs could potentially be used alone or as a boost of other immunization strategies for COVID-19.


Assuntos
/imunologia , Glicoproteína da Espícula de Coronavírus/biossíntese , Vírion/genética , Linhagem Celular , Humanos , Vírus da Leucemia Murina de Moloney/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Sintéticas/imunologia , Vírion/imunologia
2.
Biochem Biophys Res Commun ; 543: 45-49, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33515911

RESUMO

In order to control the COVID-19 pandemic caused by SARS-CoV-2 infection, serious progress has been made to identify infected patients and to detect patients with a positive immune response against the virus. Currently, attempts to generate a vaccine against the coronavirus are ongoing. To understand SARS-CoV-2 immunoreactivity, we compared the IgG antibody response against SARS-CoV-2 in infected versus control patients by dot blot using recombinant viral particle proteins: N (Nucleocapsid), M (Membrane) and S (Spike). In addition, we used different protein fragments of the N and S protein to map immune epitopes. Most of the COVID-19 patients presented a specific immune response against the full length and fragments of the N protein and, to lesser extent, against a fragment containing amino acids 300-685 of the S protein. In contrast, immunoreactivity against other S protein fragments or the M protein was low. This response is specific for COVID-19 patients as very few of the control patients displayed immunoreactivity, likely reflecting an immune response against other coronaviruses. Altogether, our results may help develop method(s) for measuring COVID-19 antibody response, selectivity of methods detecting such SARS-CoV-2 antibodies and vaccine development.


Assuntos
/imunologia , /imunologia , /genética , /genética , Humanos , Soros Imunes/imunologia , Imunidade Humoral , Immunoblotting , Imunoglobulina G/sangue , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vírion/imunologia
3.
Virol J ; 18(1): 1, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397387

RESUMO

BACKGROUND: Virus neutralization by antibodies is an important prognostic factor in many viral diseases. To easily and rapidly measure titers of neutralizing antibodies in serum or plasma, we developed pseudovirion particles composed of the spike glycoprotein of SARS-CoV-2 incorporated onto murine leukemia virus capsids and a modified minimal murine leukemia virus genome encoding firefly luciferase. This assay design is intended for use in laboratories with biocontainment level 2 and therefore circumvents the need for the biocontainment level 3 that would be required for replication-competent SARS-CoV-2 virus. To validate the pseudovirion assay, we set up comparisons with other available antibody tests including those from Abbott, Euroimmun and Siemens, using archived, known samples. RESULTS: 11 out of 12 SARS-CoV-2-infected patient serum samples showed neutralizing activity against SARS-CoV-2-spike pseudotyped MLV viruses, with neutralizing titers-50 (NT50) that ranged from 1:25 to 1:1,417. Five historical samples from patients hospitalized for severe influenza infection in 2016 tested negative in the neutralization assay (NT50 < 25). Three serum samples with high neutralizing activity against SARS-CoV-2/MLV pseudoviruses showed no detectable neutralizing activity (NT50 < 25) against SARS-CoV-1/MLV pseudovirions. We also compared the semiquantitative Siemens SARS-CoV-2 IgG test, which measures binding of IgG to recombinantly expressed receptor binding domain of SARS-CoV-2 spike glycoprotein with the neutralization titers obtained in the pseudovirion assay and the results show high concordance between the two tests (R2 = 0.9344). CONCLUSIONS: SARS-CoV-2 spike/MLV pseudovirions provide a practical means of assessing neutralizing activity of antibodies in serum or plasma from infected patients under laboratory conditions consistent with biocontainment level 2. This assay offers promise also in evaluating immunogenicity of spike glycoprotein-based candidate vaccines in the near future.


Assuntos
/imunologia , Leucemia/imunologia , Testes de Neutralização/métodos , Glicoproteína da Espícula de Coronavírus/imunologia , Vírion/imunologia , /imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Células HEK293 , Humanos , Imunoglobulina G/sangue , Camundongos
4.
Signal Transduct Target Ther ; 5(1): 212, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963228

RESUMO

The outbreaks of severe acute respiratory syndrome (SARS) and Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV and SARS-CoV-2, respectively, have posed severe threats to global public health and the economy. Treatment and prevention of these viral diseases call for the research and development of human neutralizing monoclonal antibodies (NMAbs). Scientists have screened neutralizing antibodies using the virus receptor-binding domain (RBD) as an antigen, indicating that RBD contains multiple conformational neutralizing epitopes, which are the main structural domains for inducing neutralizing antibodies and T-cell immune responses. This review summarizes the structure and function of RBD and RBD-specific NMAbs against SARS-CoV and SARS-CoV-2 currently under development.


Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Síndrome Respiratória Aguda Grave/prevenção & controle , Glicoproteína da Espícula de Coronavírus/química , Anticorpos Monoclonais/biossíntese , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/imunologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Reações Cruzadas , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Humanos , Modelos Moleculares , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/imunologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Ligação Proteica , Estrutura Secundária de Proteína , Receptores Virais/química , Receptores Virais/imunologia , Receptores Virais/metabolismo , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/imunologia , Vírus da SARS/patogenicidade , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Vírion/imunologia , Vírion/ultraestrutura
5.
Anal Bioanal Chem ; 412(28): 7685-7699, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32870351

RESUMO

Pathogen-host cell interactions play an important role in many human infectious and inflammatory diseases. Several pathogens, including Escherichia coli (E. coli), Mycobacterium tuberculosis (M. tb), and even the recent 2019 novel coronavirus (2019-nCoV), can cause serious breathing and brain disorders, tissue injury and inflammation, leading to high rates of mortality and resulting in great loss to human physical and mental health as well as the global economy. These infectious diseases exploit the microbial and host factors to induce serious inflammatory and immunological symptoms. Thus the development of anti-inflammatory drugs targeting bacterial/viral infection is an urgent need. In previous studies, YojI-IFNAR2, YojI-IL10RA, YojI-NRP1,YojI-SIGLEC7, and YojI-MC4R membrane-protein interactions were found to mediate E. coli invasion of the blood-brain barrier (BBB), which activated the downstream anti-inflammatory proteins NACHT, LRR and PYD domains-containing protein 2(NLRP2), using a proteomic chip conjugated with cell immunofluorescence labeling. However, the studies of pathogen (bacteria/virus)-host cell interactions mediated by membrane protein interactions did not extend their principles to broad biomedical applications such as 2019-nCoV infectious disease therapy. The first part of this feature article presents in-depth analysis of the cross-talk of cellular anti-inflammatory transduction signaling among interferon membrane protein receptor II (IFNAR2), interleukin-10 receptor subunit alpha (IL-10RA), NLRP2 and [Ca2+]-dependent phospholipase A2 (PLA2G5), based on experimental results and important published studies, which lays a theoretical foundation for the high-throughput construction of the cytokine and virion solution chip. The paper then moves on to the construction of the novel GPCR recombinant herpes virion chip and virion nano-oscillators for profiling membrane protein functions, which drove the idea of constructing the new recombinant virion and cytokine liquid chips for HTS of leading drugs. Due to the different structural properties of GPCR, IFNAR2, ACE2 and Spike of 2019-nCoV, their ligands will either bind the extracellular domain of IFNAR2/ACE2/Spike or the specific loops of the GPCR on the envelope of the recombinant herpes virions to induce dynamic charge distribution changes that lead to the variable electron transition for detection. Taken together, the combined overview of two of the most innovative and exciting developments in the immunoinflammatory field provides new insight into high-throughput construction of ultrasensitive cytokine and virion liquid chips for HTS of anti-inflammatory drugs or clinical diagnosis and treatment of inflammatory diseases including infectious diseases, acute or chronic inflammation (acute gouty arthritis or rheumatoid arthritis), cardiovascular disease, atheromatosis, diabetes, obesity, tissue injury and tumors. It has significant value in the prevention and treatment of these serious and painful diseases. Graphical abstract.


Assuntos
Anti-Inflamatórios/farmacologia , Antivirais/farmacologia , Ensaios de Triagem em Larga Escala/instrumentação , Dispositivos Lab-On-A-Chip , Testes de Sensibilidade Microbiana/instrumentação , Animais , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/imunologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/imunologia , Citocinas/imunologia , Descoberta de Drogas/instrumentação , Descoberta de Drogas/métodos , Desenho de Equipamento , Ensaios de Triagem em Larga Escala/métodos , Humanos , Testes de Sensibilidade Microbiana/métodos , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/imunologia , Bibliotecas de Moléculas Pequenas/farmacologia , Vírion/efeitos dos fármacos , Vírion/imunologia , Viroses/tratamento farmacológico , Viroses/imunologia
6.
J Virol ; 94(21)2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-32788194

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS-COV-2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here, we characterized the use of a codon-optimized SARS-COV-2 Spike glycoprotein for the generation of pseudotyped HIV-1, murine leukemia virus (MLV), and vesicular stomatitis virus (VSV) particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail. Infection of 293FT target cells was possible only if the cells were engineered to stably express the human angiotensin-converting enzyme 2 (ACE2) receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike-activating protease TMPRSS2 further enhanced susceptibility to infection by 5- to 10-fold. Replacement of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modifications D614G and R682Q further enhanced infectious particle production. With all enhancing elements combined, the titer of pseudotyped HIV-1 particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV-2 Spike were successfully used to detect neutralizing antibodies in plasma from coronavirus disease 2019 (COVID-19) patients, but not in plasma from uninfected individuals.IMPORTANCE In work with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus, leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here, we describe a series of modifications that result in the production of relatively high-titer SARS-COV-2 pseudoparticles that are suitable for the detection of neutralizing antibodies from COVID-19 patients.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Glicoproteína da Espícula de Coronavírus/fisiologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Betacoronavirus/genética , Betacoronavirus/imunologia , Betacoronavirus/metabolismo , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/metabolismo , Células HEK293 , HIV-1/genética , HIV-1/metabolismo , Humanos , Vírus da Leucemia Murina , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/imunologia , Pneumonia Viral/metabolismo , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/metabolismo , Vírion/genética , Vírion/imunologia , Vírion/metabolismo , Internalização do Vírus
7.
Emerg Microbes Infect ; 9(1): 1712-1721, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32619390

RESUMO

Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) has resulted in a pandemic and is continuing to spread rapidly around the globe. No effective vaccine is currently available to prevent COVID-19, and intense efforts are being invested worldwide into vaccine development. In this context, all technology platforms must overcome several challenges resulting from the use of an incompletely characterized new virus. These include finding the right conditions for virus amplification for the development of vaccines based on inactivated or attenuated whole viral particles. Here, we describe a shotgun tandem mass spectrometry workflow, the data produced can be used to guide optimization of the conditions for viral amplification. In parallel, we analysed the changes occurring in the host cell proteome following SARS-CoV-2 infection to glean information on the biological processes modulated by the virus that could be further explored as potential drug targets to deal with the pandemic.


Assuntos
Antígenos Virais/biossíntese , Betacoronavirus/imunologia , Proteômica/métodos , Vacinas Virais/imunologia , Vírion/imunologia , Animais , Antígenos Virais/imunologia , Chlorocebus aethiops , Espectrometria de Massas em Tandem , Células Vero
8.
J Med Microbiol ; 69(7): 960-970, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32510304

RESUMO

Introduction. Persistent human papillomavirus (HPV) type 16 infection is the main causal agent of cervical cancer. Most HPV infections clear spontaneously within 1-2 years. Although not all infected women develop detectable HPV antibodies, about 60-70 % seroconvert and retain their antibodies at low levels.Aim. We investigated if cervical HPV16 DNA positivity was associated with HPV16 seroreactivity measured with two different antigen formulations. We assessed if associations were influenced by co-infection with other HPV types and HPV16 viral load.Methodology. We used baseline data for women participating in the Ludwig-McGill cohort, a longitudinal investigation of the natural history of HPV infection and cervical neoplasia. The study enrolled 2462 Brazilian women from 1993 to 1997 (pre-vaccination). ELISA assays were based on L1-only or L1+L2 virus-like particles (VLPs). Seroreactivity was expressed as normalized absorbance ratios. HPV genotyping and viral load were evaluated by PCR protocols. Pearson's r was used to measure correlations between interval-scaled variables. Serological accuracy in HPV16 DNA detection was assessed using receiver operating characteristic (ROC) curves. We analysed the association between HPV DNA positivity and HPV16 seroreactivity by linear regression.Results. Correlations between L1+L2 and L1-only VLPs for detection of HPV16 were poor (r=0.43 and 0.44 for dilutions 1 : 10 and 1 : 50, respectively). The protocol with the best accuracy was L1+L2 VLPs at serum dilution 1 : 10 (ROC area=0.73, 95 % CI: 0.65-0.85). HPV16 DNA positivity was correlated with HPV16 seroreactivity and was not influenced by co-infection or viral load. To a lesser degree, HPV16 seroreactivity was correlated with infection by other Alpha-9 papillomavirus species.Conclusion. HPV16 DNA positivity and HPV16 seroreactivity are strongly correlated. L1+L2 VLPs perform better than L1-only VLPs for detecting IgG antibodies to HPV16 in women infected with HPV16 or other Alpha-9 HPV species. This study advances our understanding of humoral immune responses against HPV16 by providing insights about the influence of VLP antigen composition to measure humoral immune response against naturally acquired HPV infection.


Assuntos
Papillomavirus Humano 16/genética , Papillomavirus Humano 16/imunologia , Infecções por Papillomavirus/diagnóstico , Adulto , Anticorpos Antivirais/sangue , Brasil , Capsídeo/imunologia , Proteínas do Capsídeo/genética , Colo do Útero/virologia , Estudos de Coortes , Feminino , Papillomavirus Humano 16/patogenicidade , Humanos , Complexo Antígeno L1 Leucocitário/imunologia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Papillomaviridae/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/imunologia , Neoplasias do Colo do Útero/etiologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia , Carga Viral/métodos , Vírion/imunologia
9.
Virology ; 544: 42-54, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32174513

RESUMO

Only a small subset of the hundreds of proteins encoded by the poxvirus genome have been shown to be effective as vaccine and/or therapeutic targets. One of these proteins is A33. Here we assess and dissect the ability of an anti-A33 humanized monoclonal antibody, c6C, to affect vaccinia virus infection in vitro. Enveloped virions (EV) released from infected cells can be sensitive or resistant to neutralization by c6C indicating there are different types of EV particles, extracellular enveloped virions (EEV) and released cellular-associated virions (rCEV), that are biologically distinct. Through a combination of plaque phenotype, confocal imaging, and neutralization assays, we found that c6C differentially affects EV from two different virus strains, IHD-J and WR. Evidence for an anti-A33 resistant EV particle, and strain differences in this phenotype, provides a logical answer as to why certain functional assays in the literature have been unable to detect anti-viral effects of anti-A33 antibodies.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Glicoproteínas de Membrana/imunologia , Vírus Vaccinia/imunologia , Proteínas do Envelope Viral/imunologia , Vírion/imunologia , Linhagem Celular , Humanos , Testes de Neutralização , Ensaio de Placa Viral
10.
Int J Mol Sci ; 21(4)2020 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-32098424

RESUMO

Influenza (flu) is a contagious viral disease, which targets the human respiratory tract and spreads throughout the world each year. Every year, influenza infects around 10% of the world population and between 290,000 and 650,000 people die from it according to the World Health Organization (WHO). Influenza viruses belong to the Orthomyxoviridae family and have a negative sense eight-segment single-stranded RNA genome that encodes 11 different proteins. The only control over influenza seasonal epidemic outbreaks around the world are vaccines, annually updated according to viral strains in circulation, but, because of high rates of mutation and recurrent genetic assortment, new viral strains of influenza are constantly emerging, increasing the likelihood of pandemics. Vaccination effectiveness is limited, calling for new preventive and therapeutic approaches and a better understanding of the virus-host interactions. In particular, grasping the role of influenza non-structural protein 1 (NS1) and related known interactions in the host cell is pivotal to better understand the mechanisms of virus infection and replication, and thus propose more effective antiviral approaches. In this review, we assess the structure of NS1, its dynamics, and multiple functions and interactions, to highlight the central role of this protein in viral biology and its potential use as an effective therapeutic target to tackle seasonal and pandemic influenza.


Assuntos
Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Infecções por Orthomyxoviridae/imunologia , Proteínas não Estruturais Virais/imunologia , Vírion/imunologia , Animais , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/metabolismo , Vacinas contra Influenza/administração & dosagem , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Mutação , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Conformação Proteica , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Vírion/genética , Vírion/metabolismo
11.
Sci Rep ; 10(1): 3229, 2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-32094377

RESUMO

Diabetes mellitus (DM) patients are at an increased risk of complications following influenza-virus infection, seasonal vaccination (SV) is recommended. However, SV with trivalent influenza vaccine (TIV) can induce antibody and type-I interferon (IFN) responses, and the effect of anti-DM treatment on these responses is incompletely understood. We evaluated the antibody response and IFN-α expression in individuals with and without type 2 DM (T2DM) following SV, and examined the effects on anti-DM treatment. TIV elicited sero-protection in all groups, but antibody persistency was <8 months, except for the antibody response to B-antigens in non-DM. T2DM impaired the IgG avidity index, and T2DM showed a significantly decreased response against H1N1 and H3N2, in addition to delaying and reducing haemagglutination-inhibition persistency against influenza B-antigens in DM groups treated with metformin (Met-DM) or glibenclamide (GB-DM). Following TIV, the Met-DM and GB-DM groups exhibited reduced IFN-α expression upon stimulation with whole- and split-virion influenza vaccines. Suppression of IFN-α expression in the Met-DM group was associated with a reduction in the mechanistic target of rapamycin complex-1 pathway and impaired IgG avidity index. Thus, single-dose TIV each year might not be suitable for T2DM. Our data could aid the development of an efficacious influenza vaccine for T2DM.


Assuntos
Formação de Anticorpos/efeitos dos fármacos , Diabetes Mellitus Tipo 2/imunologia , Interferon-alfa/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metformina/farmacologia , Estações do Ano , Transdução de Sinais , Vacinação , Idoso , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos/imunologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Feminino , Glibureto/farmacologia , Glibureto/uso terapêutico , Testes de Inibição da Hemaglutinação , Humanos , Imunoglobulina G/metabolismo , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza B/efeitos dos fármacos , Vírus da Influenza B/imunologia , Vacinas contra Influenza/imunologia , Masculino , Metformina/uso terapêutico , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vírion/efeitos dos fármacos , Vírion/imunologia
12.
Virology ; 543: 20-26, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32056843

RESUMO

Human adenovirus serotype 7 (HAdV-7), belonging to species B, has caused severe lower respiratory tract diseases and even deaths recently. However, no adenovirus vaccine or therapeutic is available thus far. In this study, a HAdV-7-specific human monoclonal antibody (HMAb), 3-3E, isolated from single plasma cells obtained from the peripheral blood mononuclear cells of HAdV-7-infected patients showed potent HAdV-7 neutralization activity. The results showed HMAb 3-3E only binds to the hexon protein of intact HAdV-7 or the recombinant hexon protein and it does not bind to other intact virion particles. This could mean the antibody recognizes a conformational epitope of the hexon protein. Further, HMAb 3-3E potently neutralized HAdV-7 in vitro at low concentrations. In vivo studies showed HMAb 3-3E protected from HAdV-7 infection in a murine model. Therefore, HMAb 3-3E is promising as a safe and effective prophylactic and therapeutic treatment for HAdV-7 infection.


Assuntos
Infecções por Adenovirus Humanos/imunologia , Adenovírus Humanos/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Vírion/imunologia , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , Animais , Linhagem Celular , Mapeamento de Epitopos , Epitopos/imunologia , Expressão Gênica/genética , Humanos , Leucócitos Mononucleares/imunologia , Camundongos , Camundongos SCID , Proteínas Recombinantes/genética , Sorogrupo , Vírion/genética , Vírion/metabolismo
13.
Cell Host Microbe ; 27(2): 249-261.e5, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32027857

RESUMO

Hand, foot, and mouth disease is a common childhood illness primarily caused by coxsackievirus A16 (CVA16), for which there are no current vaccines or treatments. We identify three CVA16-specific neutralizing monoclonal antibodies (nAbs) with therapeutic potential: 18A7, 14B10, and NA9D7. We present atomic structures of these nAbs bound to all three viral particle forms-the mature virion, A-particle, and empty particle-and show that each Fab can simultaneously occupy the mature virion. Additionally, 14B10 or NA9D7 provide 100% protection against lethal CVA16 infection in a neonatal mouse model. 18A7 binds to a non-conserved epitope present in all three particles, whereas 14B10 and NA9D7 recognize broad protective epitopes but only bind the mature virion. NA9D7 targets an immunodominant site, which may overlap the receptor-binding site. These findings indicate that CVA16 vaccines should be based on mature virions and that these antibodies could be used to discriminate optimal virion-based immunogens.


Assuntos
Anticorpos Neutralizantes , Enterovirus Humano A/imunologia , Doença de Mão, Pé e Boca/virologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/ultraestrutura , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/ultraestrutura , Proteínas do Capsídeo/imunologia , Linhagem Celular , Microscopia Crioeletrônica , Enterovirus/imunologia , Enterovirus/ultraestrutura , Enterovirus Humano A/ultraestrutura , Doença de Mão, Pé e Boca/imunologia , Doença de Mão, Pé e Boca/prevenção & controle , Humanos , Camundongos , Vacinas Virais/imunologia , Vírion/imunologia
14.
Nat Microbiol ; 5(4): 584-598, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32015498

RESUMO

Internal N6-methyladenosine (m6A) modification is one of the most common and abundant modifications of RNA. However, the biological roles of viral RNA m6A remain elusive. Here, using human metapneumovirus (HMPV) as a model, we demonstrate that m6A serves as a molecular marker for innate immune discrimination of self from non-self RNAs. We show that HMPV RNAs are m6A methylated and that viral m6A methylation promotes HMPV replication and gene expression. Inactivating m6A addition sites with synonymous mutations or demethylase resulted in m6A-deficient recombinant HMPVs and virion RNAs that induced increased expression of type I interferon, which was dependent on the cytoplasmic RNA sensor RIG-I, and not on melanoma differentiation-associated protein 5 (MDA5). Mechanistically, m6A-deficient virion RNA induces higher expression of RIG-I, binds more efficiently to RIG-I and facilitates the conformational change of RIG-I, leading to enhanced interferon expression. Furthermore, m6A-deficient recombinant HMPVs triggered increased interferon in vivo and were attenuated in cotton rats but retained high immunogenicity. Collectively, our results highlight that (1) viruses acquire m6A in their RNA as a means of mimicking cellular RNA to avoid detection by innate immunity and (2) viral RNA m6A can serve as a target to attenuate HMPV for vaccine purposes.


Assuntos
Adenosina/análogos & derivados , Proteína DEAD-box 58/genética , Evasão da Resposta Imune/genética , Interferon beta/genética , Metapneumovirus/imunologia , RNA Viral/genética , Células A549 , Adenosina/imunologia , Adenosina/metabolismo , Animais , Chlorocebus aethiops , Proteína DEAD-box 58/imunologia , Regulação da Expressão Gênica , Genoma Viral/imunologia , Células HeLa , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/imunologia , Interferon beta/imunologia , Metapneumovirus/genética , Metapneumovirus/crescimento & desenvolvimento , NF-kappa B/genética , NF-kappa B/imunologia , Infecções por Paramyxoviridae/genética , Infecções por Paramyxoviridae/imunologia , Infecções por Paramyxoviridae/virologia , RNA Viral/imunologia , Sigmodontinae , Transdução de Sinais , Células THP-1 , Células Vero , Vírion/genética , Vírion/crescimento & desenvolvimento , Vírion/imunologia
15.
J Gen Virol ; 101(4): 420-425, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31985394

RESUMO

The γ-herpesviruses have proved hard to vaccination against, with no convincing protection against long-term latent infection by recombinant viral subunits. In experimental settings, whole-virus vaccines have proved more effective, even when the vaccine virus itself establishes latent infection poorly. The main alternative is replication-deficient virus particles. Here high-dose, replication-deficient murid herpesvirus-4 only protected mice partially against wild-type infection. By contrast, latency-deficient but replication-competent vaccine protected mice strongly, even when delivered non-invasively to the olfactory epithelium. Thus, this approach seems to provide the best chance of a safe and effective γ-herpesvirus vaccine.


Assuntos
Infecções por Herpesviridae/prevenção & controle , Rhadinovirus/imunologia , Vacinas Virais , Animais , Anticorpos Antivirais/sangue , Gammaherpesvirinae/imunologia , Infecções por Herpesviridae/imunologia , Proteínas Imediatamente Precoces/genética , Camundongos , Camundongos Endogâmicos C57BL , Testes de Neutralização , Transativadores/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologia , Vírion/imunologia , Latência Viral/imunologia , Replicação Viral/genética
16.
Virol Sin ; 35(1): 1-13, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31916022

RESUMO

Antibodies play critical roles in neutralizing viral infections and are increasingly used as therapeutic drugs and diagnostic tools. Structural studies on virus-antibody immune complexes are important for better understanding the molecular mechanisms of antibody-mediated neutralization and also provide valuable information for structure-based vaccine design. Cryo-electron microscopy (cryo-EM) has recently matured as a powerful structural technique for studying bio-macromolecular complexes. When combined with X-ray crystallography, cryo-EM provides a routine approach for structurally characterizing the immune complexes formed between icosahedral viruses and their antibodies. In this review, recent advances in the structural understanding of virus-antibody interactions are outlined for whole virions with icosahedral T = pseudo 3 (picornaviruses) and T = 3 (flaviviruses) architectures, focusing on the dynamic nature of viral shells in different functional states. Glycoprotein complexes from pleomorphic enveloped viruses are also discussed as immune complex antigens. Improving our understanding of viral epitope structures using virus-based platforms would provide a fundamental road map for future vaccine development.


Assuntos
Anticorpos Antivirais/ultraestrutura , Complexo Antígeno-Anticorpo/ultraestrutura , Microscopia Crioeletrônica , Vírion/ultraestrutura , Animais , Anticorpos Antivirais/imunologia , Epitopos/imunologia , Epitopos/ultraestrutura , Flavivirus/imunologia , Flavivirus/ultraestrutura , Humanos , Picornaviridae/imunologia , Picornaviridae/ultraestrutura , Ligação Proteica , Conformação Proteica , Vírion/imunologia
17.
J Immunol ; 204(2): 335-347, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31836655

RESUMO

Epitope density has a profound impact on B cell responses to particulate Ags, the molecular mechanisms of which remain to be explored. To dissect the role of epitope density in this process, we have synthesized a series of liposomal particles, similar to the size of viruses, that display a model self-antigen peptide at defined surface densities. Immunization of C57BL/6J mice using these particles elicited both IgM and class-switched IgG1, IgG2b, and IgG3 autoreactive Abs that depended on the epitope density. In C57BL/6 gene knockout mice lacking either functional TCRs or MHC class II molecules on B cells, the liposomal particles also elicited IgM, IgG1, IgG2b, and IgG3 responses that were comparable in magnitudes to wild-type mice, suggesting that this B cell response was independent of cognate T cell help. Notably, the titer of the IgG in wild-type animals could be increased by more than 200-fold upon replacement of liposomes with bacteriophage Qß virus-like particles that displayed the same self-antigen peptide at comparable epitope densities. This enhancement was lost almost completely in gene knockout mice lacking either TCRs or MHC class II molecules on B cells. In conclusion, epitope density above a threshold on particulate Ags can serve as a stand-alone signal to trigger secretion of autoreactive and class-switched IgG in vivo in the absence of cognate T cell help or any adjuvants. The extraordinary immunogenicity of Qß viral-like particles relies, in large part, on their ability to effectively recruit T cell help after B cell activation.


Assuntos
Autoanticorpos/sangue , Imunoglobulina G/sangue , Lipossomos/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Autoantígenos/imunologia , Células Cultivadas , Citocinas/metabolismo , Epitopos de Linfócito B/metabolismo , Switching de Imunoglobulina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nanopartículas/metabolismo , Peptídeos/imunologia , Fator de Necrose Tumoral alfa/imunologia , Vírion/imunologia
18.
Methods Mol Biol ; 2099: 117-126, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31883092

RESUMO

Pseudotyped viral particle production has been used extensively and broadly for many viruses to evaluate levels of neutralizing antibodies, viral entry inhibitors and vaccine immunogenicity. This assay is extremely safe and useful alternative to live virus-based assay without the need for high containment facilities. In this chapter, we describe the generation of MERS-CoV pseudotyped viral particles (MERSpp) expressing full-length spike protein using second-generation lentiviral packaging system. This platform is optimized to generate high titer of MERSpp and to test sera from different mammalian species.


Assuntos
Anticorpos Neutralizantes/sangue , Infecções por Coronavirus/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Vírion/imunologia , Animais , Células HEK293 , Humanos , Imunogenicidade da Vacina/imunologia , Mamíferos , Coronavírus da Síndrome Respiratória do Oriente Médio/isolamento & purificação , Testes de Neutralização , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Vírion/genética , Vírion/metabolismo , Internalização do Vírus
19.
PLoS Negl Trop Dis ; 13(11): e0007800, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31725816

RESUMO

B cell activating factor (BAFF) is a member of the tumor necrosis factor (TNF) superfamily of cytokines that links innate with adaptive immunity. BAFF signals through receptors on B cells, making it an attractive molecule to potentiate vaccine-induced B cell responses. We hypothesized that a rabies virus (RABV)-based vaccine displaying both antigen and BAFF on the surface of the same virus particle would target antigen-specific B cells for activation and improve RABV-specific antibody responses. To test this hypothesis, we constructed a recombinant RABV-based vector expressing virus membrane-anchored murine BAFF (RABV-ED51-mBAFF). BAFF was incorporated into the RABV particle and determined to be biologically functional, as demonstrated by increased B cell survival of primary murine B cells treated ex-vivo with RABV-ED51-mBAFF. B cell survival was inhibited by pre-treating RABV-ED51-mBAFF with an antibody that blocks BAFF functions. RABV-ED51-mBAFF also activated primary murine B cells ex-vivo more effectively than RABV as shown by significant upregulation of CD69, CD40, and MHCII on the surface of infected B cells. In-vivo, RABV-ED51-mBAFF induced significantly faster and higher virus neutralizing antibody (VNA) titers than RABV while not adversely affecting the longevity of the vaccine-induced antibody response. Since BAFF was incorporated into the virus particle and genome replication was not required for BAFF expression in-vivo, we hypothesized that RABV-ED51-mBAFF would be effective as an inactivated vaccine. Mice immunized with 250 ng/mouse of ß-propriolactone-inactivated RABV-ED51-mBAFF showed faster and higher anti-RABV VNA titers compared to mice immunized with inactivated RABV. Together, this model stands as a potential foundation for exploring other virus membrane-anchored molecular adjuvants to make safer, more effective inactivated RABV-based vaccines.


Assuntos
Formação de Anticorpos/imunologia , Fator Ativador de Células B/imunologia , Linfócitos B/imunologia , Vacinas Antirrábicas/imunologia , Raiva/imunologia , Raiva/prevenção & controle , Vírion/imunologia , Adjuvantes Imunológicos , Animais , Anticorpos Antivirais/sangue , Citocinas/metabolismo , Feminino , Imunização , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Vacinas Antirrábicas/efeitos adversos , Vírus da Raiva/genética , Vírus da Raiva/crescimento & desenvolvimento , Vírus da Raiva/imunologia , Vacinação , Vacinas Atenuadas/imunologia , Vacinas de Produtos Inativados/efeitos adversos , Vacinas de Produtos Inativados/imunologia , Vacinas Sintéticas
20.
Vaccine ; 37(51): 7509-7518, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31585726

RESUMO

Enteric viruses cause diverse infections with substantial morbidity and mortality in children, rotavirus (RV) and norovirus (NoV) being the leading agents of severe pediatric gastroenteritis. Coxsackie B viruses (CVB) are common enteroviruses (EV), associated with increased incidence of severe neonatal CVB disease with potentially fatal consequences. To prevent majority of childhood gastroenteritis, we have developed a non-live NoV-RV combination vaccine consisting of NoV virus-like particles (VLPs) and RV oligomeric rVP6 protein that induced protective immune responses to NoV and RV in mice. Moreover, rVP6 acted as an adjuvant for NoV VLPs. Here, we investigated a possibility to include a third enteric virus-derived antigen in the candidate NoV-RV vaccine, by adding recombinant nanoparticles derived from EV CVB1. To examine immunogenicity of EV-NoV-RV vaccine, BALB/c mice were immunized intramuscularly twice with 10 µg CVB1 VLPs, GII.4 VLPs and rVP6 nanotubes, either separately or combined. To evaluate the adjuvant effect of rVP6 on EV responses, mice received 0.3 µg CVB1 VLPs with or without 10 µg rVP6. Comparable serum IgG antibodies were detected whether the antigens were administered separately or in combination. Each formulation generated IgG1 and IgG2a antibodies, indicating a mixed Th2/Th1-type response. CVB1 VLPs skewed the isotype distribution slightly towards IgG1 subtype, while EV-NoV-RV combination vaccine induced unbiased Th1/Th2 responses to CVB1. Each antigen also induced T cell mediated immunity measured by IFN-γ secretion to specific stimulants ex vivo. Antisera raised by single antigens and combined formulation also exhibited strong neutralizing ability against CVB1 and NoV GII.4. Further, rVP6 showed an adjuvant effect on CVB1 responses, sparing the VLP dose and homogenizing the responses. Finally, the results support inclusion of additional antigens in the candidate NoV-RV combination vaccine to combat severe childhood infections and confirm adjuvant effect of rVP6 nanostructures.


Assuntos
Infecções por Caliciviridae/prevenção & controle , Infecções por Enterovirus/prevenção & controle , Gastroenterite/prevenção & controle , Infecções por Rotavirus/prevenção & controle , Vacinação/métodos , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas Virais/administração & dosagem , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/química , Antígenos Virais/imunologia , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Criança , Enterovirus Humano B/química , Enterovirus Humano B/efeitos dos fármacos , Enterovirus Humano B/imunologia , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Feminino , Gastroenterite/imunologia , Gastroenterite/virologia , Humanos , Esquemas de Imunização , Imunogenicidade da Vacina , Imunoglobulina G/sangue , Imunoglobulina G/classificação , Camundongos , Camundongos Endogâmicos BALB C , Norovirus/química , Norovirus/efeitos dos fármacos , Norovirus/imunologia , Rotavirus/química , Rotavirus/efeitos dos fármacos , Rotavirus/imunologia , Infecções por Rotavirus/imunologia , Infecções por Rotavirus/virologia , Vacinas Sintéticas , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas Virais/biossíntese , Vírion/química , Vírion/imunologia
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