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1.
Int J Mol Sci ; 22(17)2021 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-34502139

RESUMO

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causative agent of the COVID19 pandemic. The SARS-CoV-2 genome encodes for a small accessory protein termed Orf9b, which targets the mitochondrial outer membrane protein TOM70 in infected cells. TOM70 is involved in a signaling cascade that ultimately leads to the induction of type I interferons (IFN-I). This cascade depends on the recruitment of Hsp90-bound proteins to the N-terminal domain of TOM70. Binding of Orf9b to TOM70 decreases the expression of IFN-I; however, the underlying mechanism remains elusive. We show that the binding of Orf9b to TOM70 inhibits the recruitment of Hsp90 and chaperone-associated proteins. We characterized the binding site of Orf9b within the C-terminal domain of TOM70 and found that a serine in position 53 of Orf9b and a glutamate in position 477 of TOM70 are crucial for the association of both proteins. A phosphomimetic variant Orf9bS53E showed drastically reduced binding to TOM70 and did not inhibit Hsp90 recruitment, suggesting that Orf9b-TOM70 complex formation is regulated by phosphorylation. Eventually, we identified the N-terminal TPR domain of TOM70 as a second binding site for Orf9b, which indicates a so far unobserved contribution of chaperones in the mitochondrial targeting of the viral protein.


Assuntos
COVID-19/transmissão , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , SARS-CoV-2/patogenicidade , Animais , Sítios de Ligação/genética , COVID-19/imunologia , COVID-19/virologia , Chlorocebus aethiops , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/isolamento & purificação , Humanos , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/isolamento & purificação , Mutação , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Fosfoproteínas/isolamento & purificação , Fosfoproteínas/metabolismo , Fosforilação , Ligação Proteica/genética , Ligação Proteica/imunologia , Domínios Proteicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Células Vero
2.
Front Immunol ; 12: 706186, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34484202

RESUMO

Background: Sargramostim [recombinant human granulocyte-macrophage colony-stimulating factor (rhu GM-CSF)] was approved by US FDA in 1991 to accelerate bone marrow recovery in diverse settings of bone marrow failure and is designated on the list of FDA Essential Medicines, Medical Countermeasures, and Critical Inputs. Other important biological activities including accelerating tissue repair and modulating host immunity to infection and cancer via the innate and adaptive immune systems are reported in pre-clinical models but incompletely studied in humans. Objective: Assess safety and efficacy of sargramostim in cancer and other diverse experimental and clinical settings. Methods and Results: We systematically reviewed PubMed, Cochrane and TRIP databases for clinical data on sargramostim in cancer. In a variety of settings, sargramostim after exposure to bone marrow-suppressing agents accelerated hematologic recovery resulting in fewer infections, less therapy-related toxicity and sometimes improved survival. As an immune modulator, sargramostim also enhanced anti-cancer responses in solid cancers when combined with conventional therapies, for example with immune checkpoint inhibitors and monoclonal antibodies. Conclusions: Sargramostim accelerates hematologic recovery in diverse clinical settings and enhances anti-cancer responses with a favorable safety profile. Uses other than in hematologic recovery are less-well studied; more data are needed on immune-enhancing benefits. We envision significantly expanded use of sargramostim in varied immune settings. Sargramostim has the potential to reverse the immune suppression associated with sepsis, trauma, acute respiratory distress syndrome (ARDS) and COVID-19. Further, sargramostim therapy has been promising in the adjuvant setting with vaccines and for anti-microbial-resistant infections and treating autoimmune pulmonary alveolar proteinosis and gastrointestinal, peripheral arterial and neuro-inflammatory diseases. It also may be useful as an adjuvant in anti-cancer immunotherapy.


Assuntos
COVID-19/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/uso terapêutico , Fatores Imunológicos/uso terapêutico , Imunoterapia , Neoplasias/tratamento farmacológico , COVID-19/tratamento farmacológico , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/uso terapêutico , SARS-CoV-2/efeitos dos fármacos
3.
PLoS One ; 16(8): e0255796, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34375345

RESUMO

Serological assays to detect antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) might contribute to confirming the suspected coronavirus disease 2019 (COVID-19) in patients not detected with molecular assays. Human antibodies that target the host angiotensin-converting enzyme 2-binding domain of the viral spike protein are a target for serodiagnosis and therapeutics. This study aimed to characterize the classes and subclasses of antibody responses to a recombinant receptor-binding protein (RBD) of SARS-CoV-2 in COVID-19 patients and investigated the reactivity of these antibodies in patients with other tropical infections and healthy individuals in Thailand. ELISAs for IgM, IgA, IgG and IgG subclasses based on RBD antigen were developed and tested with time series of 27 serum samples from 15 patients with COVID-19 and 60 samples from pre-COVID-19 outbreaks including acute dengue fever, murine typhus, influenza, leptospirosis and healthy individuals. Both RBD-specific IgA and IgG were detected in only 21% of the COVID-19 patients in the acute phase. The median IgA and IgG levels were significantly higher in the convalescent serum sample compared to the acute serum sample (P < 0.05). We observed the highest correlation between levels of IgG and IgA (rho = 0. 92). IgG1 and IgG3 were the major IgG subclasses detected in SARS-CoV-2 infection. Only acute IgG3 level was negatively associated with viral detection based on RT-PCR of ORF1ab gene (rho = -0.57). The median IgA and IgG levels in convalescence sera of COVID-19 patients were significantly higher than healthy individuals and convalescent sera of other febrile infectious patients. The analyses of antibody classes and subclasses provide insights into human immune responses against SARS-CoV-2 during natural infection and interpretation of antibody assays.


Assuntos
Anticorpos Antivirais/sangue , Formação de Anticorpos , COVID-19/patologia , Isotipos de Imunoglobulinas/sangue , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , COVID-19/sangue , COVID-19/virologia , Feminino , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Domínios Proteicos/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Tailândia , Adulto Jovem
4.
J Gerontol A Biol Sci Med Sci ; 76(10): 1775-1783, 2021 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-34396395

RESUMO

Aging and comorbidities make individuals at greatest risk of COVID-19 serious illness and mortality due to senescence-related events and deleterious inflammation. Long-living individuals (LLIs) are less susceptible to inflammation and develop more resiliency to COVID-19. As demonstrated, LLIs are characterized by high circulating levels of BPIFB4, a protein involved in homeostatic response to inflammatory stimuli. Also, LLIs show enrichment of homozygous genotype for the minor alleles of a 4 missense single-nucleotide polymorphism haplotype (longevity-associated variant [LAV]) in BPIFB4, able to counteract progression of diseases in animal models. Thus, the present study was designed to assess the presence and significance of BPIFB4 level in COVID-19 patients and the potential therapeutic use of LAV-BPIFB4 in fighting COVID-19. BPIFB4 plasma concentration was found significantly higher in LLIs compared to old healthy controls while it significantly decreased in 64 COVID-19 patients. Further, the drop in BPIFB4 values correlated with disease severity. Accordingly to the LAV-BPIFB4 immunomodulatory role, while lysates of SARS-CoV-2-infected cells induced an inflammatory response in healthy peripheral blood mononuclear cells in vitro, the co-treatment with recombinant protein (rh) LAV-BPIFB4 resulted in a protective and self-limiting reaction, culminating in the downregulation of CD69 activating-marker for T cells (both TCD4+ and TCD8+) and in MCP-1 reduction. On the contrary, rhLAV-BPIFB4 induced a rapid increase in IL-18 and IL-1b levels, shown largely protective during the early stages of the virus infection. This evidence, along with the ability of rhLAV-BPIFB4 to counteract the cytotoxicity induced by SARS-CoV-2 lysate in selected target cell lines, corroborates BPIFB4 prognostic value and open new therapeutic possibilities in more vulnerable people.


Assuntos
COVID-19 , Peptídeos e Proteínas de Sinalização Intercelular , Longevidade/imunologia , Idoso de 80 Anos ou mais , Biomarcadores/sangue , COVID-19/diagnóstico , COVID-19/epidemiologia , COVID-19/imunologia , Linhagem Celular , Citocinas/sangue , Citotoxicidade Imunológica/efeitos dos fármacos , Feminino , Humanos , Fatores Imunológicos/imunologia , Fatores Imunológicos/farmacologia , Inflamação/sangue , Inflamação/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Itália/epidemiologia , Masculino , Prognóstico , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , SARS-CoV-2/imunologia , Índice de Gravidade de Doença
5.
Arch Virol ; 166(10): 2733-2741, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34322722

RESUMO

Congenital tremor (CT) type A-II in piglets is a worldwide disease caused by an emerging atypical porcine pestivirus (APPV). Preparation and evaluation of vaccines in laboratory animals is an important preliminary step toward prevention and control of the disease. Here, virus-like particles (VLPs) of APPV were prepared and VLPs vaccine was evaluated in BALB/c mice. Purified Erns and E2 proteins expressed in E. coli were allowed to self-assemble into VLPs, which had the appearance of hollow spherical particles with a diameter of about 100 nm by transmission electron microscopy (TEM). The VLPs induced strong antibody responses and reduced the viral load in tissues of BALB/c mice. The data from animal challenge experiments, RT-PCR, and immunohistochemical analysis demonstrated that BALB/c mice are an appropriate laboratory model for APPV. These results suggest the feasibility of using VLPs as a vaccine for the prevention and control of APPV and provide useful information for further study of APPV in laboratory animals.


Assuntos
Infecções por Pestivirus/prevenção & controle , Pestivirus/imunologia , Vacinação/veterinária , Replicação Viral/efeitos dos fármacos , Animais , Anticorpos Antivirais/sangue , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Pestivirus/virologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Suínos , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/virologia , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Carga Viral , Vacinas Virais/genética , Vacinas Virais/imunologia
6.
Front Immunol ; 12: 634738, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34248932

RESUMO

P48/45 is a conserved gametocyte antigen involved in Plasmodium parasite fertilization. A recombinant Plasmodium vivax P48/45 (Pvs48/45) protein expressed in Escherichia coli (E. coli) was highly antigenic and immunogenic in experimental animals and elicited specific transmission-blocking (TB) antibodies in a previous pilot study. Here, a similar Pvs48/45 gene was expressed in Chinese Hamster Ovary (CHO) cells and we compared its immunoreactivity with the E. coli product. Specific antibody titers were determined using plasma from Colombian individuals (n=227) living in endemic areas where both P. vivax and P. falciparum are prevalent and from Guatemala (n=54) where P. vivax is highly prevalent. In Colombia, plasma seroprevalence to CHO-rPvs48/45 protein was 46.3%, while for E. coli-rPvs48/45 protein was 36.1% (p<0.001). In Guatemala, the sero prevalence was 24.1% and 14.8% (p<0.001), respectively. Reactivity index (RI) against both proteins showed an age-dependent increase. IgG2 was the predominant subclass and the antibody avidity index evaluated by ELISA ranged between 4-6 mol/L. Ex vivo P. vivax mosquito direct membrane feeding assays (DMFA) performed in presence of study plasmas, displayed significant parasite transmission-blocking (TB), however, there was no direct correlation between antibody titers and oocysts transmission reduction activity (%TRA). Nevertheless, DMFA with CHO rPvs48/45 affinity purified IgG showed a dose response; 90.2% TRA at 100 µg/mL and 71.8% inhibition at 10 µg/mL. In conclusion, the CHO-rPvs48/45 protein was more immunoreactive in most of the malaria endemic places studied, and CHO-rPvs48/45 specific IgG showed functional activity, supporting further testing of the protein vaccine potential.


Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Doenças Endêmicas , Escherichia coli/metabolismo , Imunoglobulina G/sangue , Malária Vivax/diagnóstico , Plasmodium vivax/imunologia , Testes Sorológicos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Especificidade de Anticorpos , Antígenos de Protozoários/genética , Antígenos de Protozoários/metabolismo , Células CHO , Criança , Colômbia/epidemiologia , Cricetulus , Escherichia coli/genética , Feminino , Guatemala/epidemiologia , Humanos , Malária Vivax/sangue , Malária Vivax/epidemiologia , Malária Vivax/imunologia , Masculino , Pessoa de Meia-Idade , Plasmodium vivax/patogenicidade , Valor Preditivo dos Testes , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Estudos Soroepidemiológicos , Adulto Jovem
7.
Genes (Basel) ; 12(6)2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34205689

RESUMO

Accumulation of α-Synuclein (αSyn) in nigral dopaminergic neurons is commonly seen in patients with Parkinson's disease (PD). We recently reported that transduction of intracellular single-chain intrabody targeting the 53-87 amino acid residues of human αSyn by recombinant adeno associated viral vector (AAV-NAC32) downregulated αSyn protein in SH-SY5Y cells and rat brain. This study characterizes the behavioral phenotype and dopaminergic protection in animals receiving AAV-NAC32. Our results show that adult DAT-Cre rats selectively overexpress αSyn in nigra dopaminergic neurons after local administration of AAV-DIO-αSyn. These animals develop PD-like phenotype, including bradykinesia and loss of tyrosine hydroxylase (TH) immunoreactivity in substantia nigra pars compacta dorsal tier (SNcd). An injection of AAV-NAC32 to nigra produces a selective antibody against αSyn and normalizes the behavior. AAV-NAC32 significantly increases TH, while reduces αSyn immunoreactivity in SNcd. Altogether, our data suggest that an AAV-mediated gene transfer of NAC32 antibody effectively antagonizes αSyn-mediated dopaminergic degeneration in nigra, which may be a promising therapeutic candidate for synucleinopathy or PD.


Assuntos
Anticorpos/uso terapêutico , Imunoterapia/métodos , Locomoção , Doença de Parkinson/terapia , alfa-Sinucleína/imunologia , Animais , Anticorpos/imunologia , Células CHO , Cricetinae , Cricetulus , Dependovirus/genética , Neurônios Dopaminérgicos/metabolismo , Vetores Genéticos/genética , Masculino , Doença de Parkinson/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Ratos , Ratos Long-Evans , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , alfa-Sinucleína/química , alfa-Sinucleína/genética
8.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209932

RESUMO

Enzymatic transamidation of gliadins by microbial transglutaminase (mTG) inhibits interferon-γ (IFN-γ) secretion by intestinal T cell lines in patients with celiac disease (CD). To gain insight into the cellular mechanisms underlying the down-regulatory effects of transamidation, we tested a single recombinant α-gliadin (r-gliadin) harbouring two immunodominant peptides, p13 (aa. 120-139) and p23 (aa. 220-239), in HLA-DQ8 transgenic mice, a model of gluten sensitivity. Mice were intranasally immunised with r-gliadin or r-gliadin transamidated by mTG (K-r-gliadin) along with cholera toxin, and the response of mesenteric lymph node cells was analysed by cytokine multiplex assay. An in vitro challenge with r-gliadin was characterised by secretion of specific cytokines featuring both innate immunity and the Th1/Th2/Th17 pattern of the adaptive response. Notably, transamidation specifically down-regulated the Th1 response. Structural studies performed on K-r-gliadin confirmed that specific glutamine residues in p13 and p23, previously found to be deamidated by tissue transglutaminase, were also transamidated by mTG. In silico analysis, simulating p13 and p23 peptide binding to HLA-DQ8 showed that these glutamines, in the form of glutamate, could interact by means of salt bridges with peculiar amino acids of the alpha chain of HLA-DQ8, suggesting that their transamidation may influence the HLA-restricted recognition of these peptides. Thus, the structural findings provided a rationale to explain the down-regulation of the r-gliadin-specific Th1 response following transamidation.


Assuntos
Doença Celíaca/tratamento farmacológico , Toxina da Cólera/administração & dosagem , Citocinas/metabolismo , Gliadina/administração & dosagem , Antígenos HLA-DQ/genética , Transglutaminases/metabolismo , Administração Intranasal , Animais , Doença Celíaca/genética , Doença Celíaca/imunologia , Toxina da Cólera/imunologia , Citocinas/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo , Regulação da Expressão Gênica , Gliadina/química , Gliadina/genética , Gliadina/imunologia , Antígenos HLA-DQ/metabolismo , Imunização , Epitopos Imunodominantes/imunologia , Camundongos , Camundongos Transgênicos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Células Th1/imunologia , Células Th17/imunologia , Células Th2/imunologia
9.
Epidemiol Infect ; 149: e140, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34099081

RESUMO

The novel coronavirus, severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), is the causative agent of the 2020 worldwide coronavirus pandemic. Antibody testing is useful for diagnosing historic infections of a disease in a population. These tests are also a helpful epidemiological tool for predicting how the virus spreads in a community, relating antibody levels to immunity and for assessing herd immunity. In the present study, SARS-CoV-2 viral proteins were recombinantly produced and used to analyse serum from individuals previously exposed, or not, to SARS-CoV-2. The nucleocapsid (Npro) and spike subunit 2 (S2Frag) proteins were identified as highly immunogenic, although responses to the former were generally greater. These two proteins were used to develop two quantitative enzyme-linked immunosorbent assays (ELISAs) that when used in combination resulted in a highly reliable diagnostic test. Npro and S2Frag-ELISAs could detect at least 10% more true positive coronavirus disease-2019 (COVID-19) cases than the commercially available ARCHITECT test (Abbott). Moreover, our quantitative ELISAs also show that specific antibodies to SARS-CoV-2 proteins tend to wane rapidly even in patients who had developed severe disease. As antibody tests complement COVID-19 diagnosis and determine population-level surveillance during this pandemic, the alternative diagnostic we present in this study could play a role in controlling the spread of the virus.


Assuntos
Teste Sorológico para COVID-19/métodos , COVID-19/diagnóstico , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Idoso , Anticorpos Antivirais/sangue , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/isolamento & purificação , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Cinética , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Fosfoproteínas/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/isolamento & purificação
10.
Int J Mol Sci ; 22(9)2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-34066920

RESUMO

Diagnostic evaluation of specific antibodies against the SARS-CoV-2 virus is mainly based on spike (S) and nucleocapsid (N) proteins. Despite the critical functions in virus infection and contribution to the pattern of immunodominance in COVID-19, exploitation of the most abundant membrane (M) protein in the SARS-CoV-2 serology tests is minimal. This study investigated the recombinant M protein's immunoreactivity with the sera from COVID-19 convalescents. In silico designed protein was created from the outer N-terminal part (19 aa) and internal C-terminal tail (101-222 aa) of the M protein (YP_009724393.1) and was recombinantly produced and purified. The designed M protein (16,498.74 Da, pI 8.79) revealed both IgM and IgG reactivity with serum samples from COVID-19 convalescents in Western blot. In ELISA, more than 93% (28/30) of COVID-19 sera were positive for IgM detection, and more than 96% (29/30) were positive for specific IgG detection to M protein. Based on the capacity to provoke an immune response and its strong antigenic properties, as shown here, and the fact that it is also involved in the virion entry into host cells, the M protein of the SARS-CoV-2 virus as a good antigen has the potential in diagnostic purposes and vaccine design.


Assuntos
COVID-19/sangue , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , SARS-CoV-2/imunologia , Proteínas da Matriz Viral/imunologia , COVID-19/imunologia , Humanos , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/isolamento & purificação
11.
Structure ; 29(7): 655-663.e4, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34111408

RESUMO

Emerging SARS-CoV-2 strains, B.1.1.7 and B.1.351, from the UK and South Africa, respectively, show decreased neutralization by monoclonal antibodies and convalescent or vaccinee sera raised against the original wild-type virus, and are thus of clinical concern. However, the neutralization potency of two antibodies, 1-57 and 2-7, which target the receptor-binding domain (RBD) of the spike, was unaffected by these emerging strains. Here, we report cryo-EM structures of 1-57 and 2-7 in complex with spike, revealing each of these antibodies to utilize a distinct mechanism to bypass or accommodate RBD mutations. Notably, each antibody represented an immune response with recognition distinct from those of frequent antibody classes. Moreover, many epitope residues recognized by 1-57 and 2-7 were outside hotspots of evolutionary pressure for ACE2 binding and neutralizing antibody escape. We suggest the therapeutic use of antibodies, such as 1-57 and 2-7, which target less prevalent epitopes, could ameliorate issues of monoclonal antibody escape.


Assuntos
Enzima de Conversão de Angiotensina 2/química , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Receptores Virais/química , SARS-CoV-2/química , Glicoproteína da Espícula de Coronavírus/química , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/genética , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/metabolismo , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Epitopos/metabolismo , Expressão Gênica , Células HEK293 , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Receptores Virais/genética , Receptores Virais/imunologia , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo
13.
Sci Rep ; 11(1): 10475, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006961

RESUMO

Infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes COVID-19 disease. Therapeutic antibodies are being developed that interact with the viral spike proteins to limit viral infection of epithelium. We have applied a method to dramatically improve the performance of anti-SARS-CoV-2 antibodies by enhancing avidity through multimerization using simple engineering to yield tetrameric antibodies. We have re-engineered six anti-SARS-CoV-2 antibodies using the human p53 tetramerization domain, including three clinical trials antibodies casirivimab, imdevimab and etesevimab. The method yields tetrameric antibodies, termed quads, that retain efficient binding to the SARS-CoV-2 spike protein, show up to two orders of magnitude enhancement in neutralization of pseudovirus infection and retain potent interaction with virus variant of concern spike proteins. The tetramerization method is simple, general and its application is a powerful methodological development for SARS-CoV-2 antibodies that are currently in pre-clinical and clinical investigation.


Assuntos
SARS-CoV-2/metabolismo , Anticorpos de Cadeia Única/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Reações Antígeno-Anticorpo , COVID-19/tratamento farmacológico , COVID-19/virologia , Ensaio de Imunoadsorção Enzimática , Células HEK293 , Humanos , Testes de Neutralização , Domínios Proteicos , Multimerização Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/uso terapêutico , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/uso terapêutico , Ressonância de Plasmônio de Superfície , Proteína Supressora de Tumor p53/química
14.
Sci Rep ; 11(1): 9825, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972631

RESUMO

In the current global emergency due to SARS-CoV-2 outbreak, passive immunotherapy emerges as a promising treatment for COVID-19. Among animal-derived products, equine formulations are still the cornerstone therapy for treating envenomations due to animal bites and stings. Therefore, drawing upon decades of experience in manufacturing snake antivenom, we developed and preclinically evaluated two anti-SARS-CoV-2 polyclonal equine formulations as potential alternative therapy for COVID-19. We immunized two groups of horses with either S1 (anti-S1) or a mixture of S1, N, and SEM mosaic (anti-Mix) viral recombinant proteins. Horses reached a maximum anti-viral antibody level at 7 weeks following priming, and showed no major adverse acute or chronic clinical alterations. Two whole-IgG formulations were prepared via hyperimmune plasma precipitation with caprylic acid and then formulated for parenteral use. Both preparations had similar physicochemical and microbiological quality and showed ELISA immunoreactivity towards S1 protein and the receptor binding domain (RBD). The anti-Mix formulation also presented immunoreactivity against N protein. Due to high anti-S1 and anti-RBD antibody content, final products exhibited high in vitro neutralizing capacity of SARS-CoV-2 infection, 80 times higher than a pool of human convalescent plasma. Pre-clinical quality profiles were similar among both products, but clinical efficacy and safety must be tested in clinical trials. The technological strategy we describe here can be adapted by other producers, particularly in low- and middle-income countries.


Assuntos
COVID-19/imunologia , COVID-19/terapia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Cavalos/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/genética , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Ensaio de Imunoadsorção Enzimática , Humanos , Imunização/métodos , Imunização Passiva/métodos , Imunoglobulina G/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo
15.
Biochem Biophys Res Commun ; 560: 126-131, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-33989903

RESUMO

Brucellosis has placed a heavy economic burden on numerous countries and has consumed considerable medical resources worldwide. To improve the specificity and sensitivity of serological methods for diagnosing brucellosis, it is important to develop new diagnostic antigens. Brucella outer membrane proteins(omps) possess good immunogenicity, but there is a scarcity of comparative studies of these proteins in the clinical diagnosis of brucellosis. In this study, six recombinant Brucella outer membrane proteins, omp10, omp16, omp19, omp25, omp31 and BP26, were expressed in prokaryotic cells and utilized as diagnostic antigens. The clinical sera of humans, bovines and goats with brucellosis were analyzed by indirect ELISA using these proteins, lipopolysaccharide(LPS) and Rose Bengale Ag, served as positive-control antigens. In diagnosing human and goat serum, BP26 exhibited the highest diagnostic accuracy of 96.45% and 95.00%, respectively, while omp31 exhibited the strongest ability to detect Brucella in bovine serum with an accuracy of 84.03%. Cross-reaction experiments also confirmed that the diagnostic specificities of omp31 and BP26 were higher than those of the LPS and Rose Bengale Ag antigens. The results of this study indicate that omp31 and BP26 are candidate antigens with high potential application value in the clinical diagnosis of brucellosis.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Brucella/imunologia , Brucelose Bovina/diagnóstico , Brucelose/diagnóstico , Brucelose/veterinária , Doenças das Cabras/diagnóstico por imagem , Animais , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Bovinos , Reações Cruzadas , Doenças das Cabras/diagnóstico , Cabras , Humanos , Proteínas Recombinantes/análise , Proteínas Recombinantes/imunologia
16.
J Immunol ; 206(10): 2312-2321, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33952617

RESUMO

IL-8 is a potent chemokine that recruits neutrophils and basophils to promote inflammation in many species. IL-8 is produced by many cell types, including monocytes. In this study, we report a novel role for IgE-binding monocytes, a rare peripheral immune cell type, to promote allergic inflammation through IL-8 production in a horse model of natural IgE-mediated allergy. We developed a mAb with confirmed specificity for both recombinant and native equine IL-8 for flow cytometric analysis. Equine IL-8 was produced by CD14+/MHC class II+/CD16- monocytes, including a subpopulation of IgE-binding monocytes, following stimulation with LPS. In addition, IgE cross-linking induced IL-8 production by both peripheral blood basophils and IgE-binding monocytes. IL-8 production was compared between healthy horses and those with a naturally occurring IgE-mediated skin allergy, Culicoides hypersensitivity. Allergic horses had significantly higher percentages of IL-8+ IgE-binding monocytes after IgE cross-linking. In contrast, frequencies of IL-8+ basophils after IgE cross-linking were similar in all horses, regardless of allergic disease, highlighting IgE-binding monocytes as a novel source of IL-8 during allergy. We concluded that IgE-binding monocytes from allergic individuals have an increased capacity for IL-8 production and likely contribute to the recruitment of innate immune cells during IgE-mediated allergy and promotion of inflammation during repeated allergen contact.


Assuntos
Alérgenos/imunologia , Ceratopogonidae/imunologia , Doenças dos Cavalos/imunologia , Hipersensibilidade/imunologia , Hipersensibilidade/veterinária , Imunoglobulina E/metabolismo , Interleucina-8/biossíntese , Monócitos/imunologia , Monócitos/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Basófilos/imunologia , Células CHO , Cricetulus , Doenças dos Cavalos/sangue , Cavalos , Hibridomas , Hipersensibilidade/sangue , Imunização/métodos , Interleucina-8/administração & dosagem , Interleucina-8/genética , Interleucina-8/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Transfecção
17.
Biochem Biophys Res Commun ; 558: 168-174, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33932776

RESUMO

Staphylococcal enterotoxins are one of the most important causative agents of food poisoning. These molecules function as both gastrointestinal toxins and superantigens (SAgs) which can simultaneously bind MHC-II and T cell receptor leading to a non-specific polyclonal T cell activation and massive proinflammatory cytokine release. Common symptoms include vomiting and diarrhea; however, in more severe cases, systemic dissemination may result in toxic shock syndrome and can be lethal in a few hours. Only small amounts of these heat-stable toxins are needed to cause the disease. Therefore, it is highly important to detect quickly low concentrations of SAgs in biological samples. In this work, we report a surface plasmon resonance (SPR)-based capture immunoassay for the detection of the SAg SEG. We analyzed the use of different amplification strategies. The SPR-based double-antibody sandwich approach could detect picomolar levels of SEG. The use of antibody-coated silica nanoparticles (AbSiNPs) as an alternative enhancing reagent also detected SEG in the picomolar range. Although AbSiNPs did not improve the limit of detection, for the same amount of SAg tested, AbSiNPs gave a higher response level than free antibodies. This work highlights the suitability of silica nanoparticles for signal amplification in SPR-based biosensors. Overall, SPR biosensors offer the capability for continuous real-time monitoring and high sensitivity that can be befitting for the detection of enterotoxins in food industries, laboratories and regulatory agencies.


Assuntos
Enterotoxinas/análise , Imunoensaio/métodos , Superantígenos/análise , Ressonância de Plasmônio de Superfície/métodos , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos , Técnicas Biossensoriais/métodos , Materiais Revestidos Biocompatíveis , Enterotoxinas/genética , Enterotoxinas/imunologia , Microbiologia de Alimentos , Humanos , Limite de Detecção , Nanopartículas , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Dióxido de Silício , Intoxicação Alimentar Estafilocócica/diagnóstico , Staphylococcus aureus/química , Staphylococcus aureus/genética , Staphylococcus aureus/imunologia , Superantígenos/genética , Superantígenos/imunologia
18.
Mol Immunol ; 135: 320-328, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33971510

RESUMO

Using antibody drug conjugates (ADC) which can exclusively bind to their target cells and upon internalization release their toxic agent, is one of the most effective methods for killing tumor cells. Therefore, increasing the internalization rate is an important factor for tumor treatment in this case. The aim of the present study was to develop a new variant of pertuzumab (an anti-ErbB2 humanized antibody) with higher internalization rate that can be a good candidate for the production of ADC. To this end, the Human Immunodeficiency Virus TAT Protein Transduction Domain (TAT-PTD) was replaced into the structure of the pertuzumab. At first, the best site in antibody heavy chain constant region for the replacement of TAT-PTD was predicted through computational methods. Then, the resulting recombinant antibody, of which TAT-PTD was located at amino acid position 130-140 and named Tatibody, was produced in CHO-S cell line. Finally, its physicochemical properties and biological activities were evaluated and compared with pertuzumab. Results showed that the binding ability of Tatibody to the ErbB2 receptor is similar to that of pertuzumab, but its internalization potency is 3.6 fold higher and can be used as a good candidate for ADC construction.


Assuntos
Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proteínas Recombinantes/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Animais , Anticorpos Monoclonais Humanizados/imunologia , Afinidade de Anticorpos/imunologia , Células CHO , Linhagem Celular Tumoral , Cricetulus , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Simulação de Acoplamento Molecular , Conformação Proteica , Transporte Proteico/genética , Transporte Proteico/fisiologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/imunologia , Proteínas Recombinantes/imunologia
19.
Mol Immunol ; 135: 373-387, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34020083

RESUMO

Vibrio parahaemolyticus causes vibriosis in wide range of marine organisms, and is responsible for food borne illnesses in humans through consumption of contaminated uncooked/partially cooked seafood. Continued and widespread antibiotics usage to increase the productivity has led to antibiotics resistance development. This has necessitated the need to develop alternative methods to control its infection. Use of safe and effective vaccines against the virulence factors not only protects from infection, it also minimizes antibiotic usage. The colonization of V. parahaemolyticus in the host and disease development requires several adhesins present on the cell surface, and thereby make them attractive vaccine candidates. V. parahaemolyticus produces extracellular type 1 fimbriae that have been shown to play a role in adhesion, biofilm formation and virulence. FimH is one of the minor components of the type 1 fimbriae occurring on its very tip. Being present on the cell surface, it is highly immunogenic, and can be targeted as a potential vaccine candidate. The present study describes the immunogenic and vaccine potential of recombinant V. parahaemolyticus FimH (rVpFimH) expressed in E. coli. Immunization of BALB/c mice with the rVpFimH elicited a strong mixed immune response, T-cell memory (evidenced by antibody isotyping, cytokine profiling and T-cell proliferation assay), and agglutination positive antibodies. FACS analysis and immunogold labeling showed that the polyclonal anti-rVpFimH antibodies were able to recognize the FimH on V. parahaemolyticus cells. In vivo challenge of the rVpFimH-immunized mice with 2×LD50 dose of live bacteria showed one hundred percent survival. Thus, our findings clearly demonstrate the potential of FimH as an effective vaccine candidate against V. parahaemolyticus.


Assuntos
Adesinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Proteínas de Fímbrias/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Vibrioses/prevenção & controle , Vibrio parahaemolyticus/imunologia , Adesinas Bacterianas/genética , Animais , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Biofilmes/crescimento & desenvolvimento , Modelos Animais de Doenças , Proteínas de Fímbrias/genética , Doenças Transmitidas por Alimentos/microbiologia , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Alimentos Crus/microbiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alimentos Marinhos/microbiologia , Vibrioses/imunologia , Vibrio parahaemolyticus/genética , Virulência , Fatores de Virulência/genética , Fatores de Virulência/imunologia
20.
Acta Biochim Biophys Sin (Shanghai) ; 53(6): 707-718, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-33963824

RESUMO

The major etiologic agent that causes acute gastroenteritis worldwide in young animals and children is Group A rotavirus. Currently, commercially available vaccines do not often prevent porcine rotavirus (PRV) infection. In this study, we evaluated the efficacy of oral recombinant Lactobacillus vaccine against PRV in a mouse model. Lactobacillus plantarum NC8 was used as the host strain, and bacterial vectors were constructed, because the NC8 isolated has shown the capability to survive gastric transit and to colonize the intestinal tract of humans and other mammals. To explore the immunological mechanisms, lactic acid bacterial vectors were used to express VP7 antigen from PRV. We constructed an L. plantarum strain with surface-displayed VP7, named NC8-pSIP409-pgsA-VP7-DCpep. The expressed recombinant protein had a molecular weight of ∼37 kDa. The strain was used to immunize BALB/c mice to evaluate their immunomodulatory characteristics. Mice were orally immunized with recombinant L. plantarum NC8-pSIP409-pgsA-VP7-DCpep at a dose of 2 × 109 colony forming units/200 µl. The results showed that NC8-pSIP409-pgsA-VP7-DCpep significantly stimulated the differentiation of dendritic cells (DCs) in Peyer's patches (PPs) and increased the serum levels of IL-4 and IFN-γ, as measured by enzyme-linked immunosorbent assay in mice treated with NC8-pSIP409-pgsA-VP7-DCpep. Compared to the empty vector group, NC8-pSIP409-pgsA-VP7-DCpep significantly increased the production of B220+ B cells in mesenteric lymph nodes (MLNs) and PPs and also increased the titer levels of the VP7-specific antibodies, including IgG and sIgA. The administration of NC8-pSIP409-pgsA-VP7-DCpep mediated relatively broad cellular responses. This study reveals that clear alternatives exist for PRV control strategies and provides information on PRV infection.


Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Engenharia Genética/métodos , Imunização/métodos , Imunogenicidade da Vacina , Lactobacillus plantarum/genética , Lactobacillus plantarum/metabolismo , Vacinas Sintéticas/administração & dosagem , Animais , Antígenos Heterófilos/genética , Antígenos Heterófilos/imunologia , Antígenos Heterófilos/metabolismo , Antígenos Virais/metabolismo , Linfócitos B/imunologia , Proteínas do Capsídeo/metabolismo , Citocinas/sangue , Feminino , Genes Virais , Imunoglobulina A Secretora/imunologia , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Rotavirus/imunologia , Rotavirus/metabolismo , Suínos , Vacinas Sintéticas/imunologia
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