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1.
Int J Nanomedicine ; 15: 8507-8517, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33154640

RESUMO

Purpose: The vaccine design has shifted from attenuated or inactivated whole pathogen vaccines to more pure and defined subunit vaccines. The purification of antigen proteins, especially the precise display of antigen regions, has become a key step affecting the effectiveness of subunit vaccines. Materials and Methods: This work presents the application of molecular docking for a peptide ligand designed for PCV2 Cap purification and assembly in one step. Based on the PCV2 Cap protein affinity peptide (L11-DYWWQSWE), the amino terminal of PCV2 Cap was covalently coupled with the polylactic acid-glycolic acid copolymer (PLGA) carboxyl terminal through the EDC/NHS method. Results: The PLGA had an average diameter of 106 nm. The average diameter increased to 122 nm after the PCV2 Cap protein conjugation, and the Zeta potential shifted from -13.7 mV to -9.6 mV, indicating that the PCV2 Cap protein stably binds to the PLGA. Compared with the free PCV2 Cap protein group, the neutralizing antibody titer was significantly increased on the 14th day after the PLGA-Cap immunization (P < 0.05). The neutralizing antibody level was extremely significant on the 28th day (P < 0.001). The CCK-8 analysis showed that PLGA-Cap had an obvious cytotoxic effect on RAW264.7 cells at the PLGA nanoparticle concentration up to 200 µg/mL but had no obvious cytotoxic effect on DC2.4 cells. Compared with the Cap protein group, the antigen-presenting cells had a stronger antigen uptake capacity and a higher fluorescence in the PLGA-Cap group. The immune effect showed that the level of the neutralizing antibody produced by this structure is much better than that of purified protein and helps improve the immune system response. Conclusion: This technology provides a potential new perspective for the rapid enrichment of the antigen protein with the affinity peptide ligand.


Assuntos
Proteínas do Capsídeo/imunologia , Circovirus/imunologia , Nanopartículas/química , Peptídeos/imunologia , Vacinas Virais/imunologia , Vacinas Virais/isolamento & purificação , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Células Apresentadoras de Antígenos/metabolismo , Sítios de Ligação , Linhagem Celular , Infecções por Circoviridae/imunologia , Citocinas/biossíntese , Inflamação/patologia , Ligantes , Camundongos , Simulação de Acoplamento Molecular , Nanopartículas/ultraestrutura , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química
2.
Nat Commun ; 11(1): 5253, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067459

RESUMO

Enterovirus 71 (EV71)-neutralizing antibodies correlate with protection and have potential as therapeutic agents. We isolate and characterize a panel of plasmablast-derived monoclonal antibodies from an infected child whose antibody response focuses on the plateau epitope near the icosahedral 3-fold axes. Eight of a total of 19 antibodies target this epitope and three of these potently neutralize the virus. Representative neutralizing antibodies 38-1-10A and 38-3-11A both confer effective protection against lethal EV71 challenge in hSCARB2-transgenic mice. The cryo-electron microscopy structures of the EV71 virion in complex with Fab fragments of these potent and protective antibodies reveal the details of a conserved epitope formed by residues in the BC and HI loops of VP2 and the BC and HI loops of VP3 spanning the region around the 3-fold axis. Remarkably, the two antibodies interact with the epitope in quite distinct ways. These plateau-binding antibodies provide templates for promising candidate therapeutics.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Enterovirus Humano A/imunologia , Infecções por Enterovirus/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Antivirais/química , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Enterovirus Humano A/química , Enterovirus Humano A/genética , Infecções por Enterovirus/virologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Testes de Neutralização
3.
Arch Virol ; 165(12): 2829-2835, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33000310

RESUMO

The swine pathogen porcine circovirus type 2 (PCV2) causes significant economic damage worldwide. The PCV2 capsid (CP) residues 169-STIDYFQPNNKR-180 have been identified as a decoy epitope that diverts the host immune response away from protective epitopes. However, the decoy epitope may include important linear or conformational protective epitopes against PCV2. In this study, we used the baculovirus system to express recombinant complete CP (1-233) and mutant CP (Δ169-180), in which the decoy epitope was deleted, and evaluated the immune response to these in mice. Immunization with mutant CP (Δ169-180) protein, which formed very low level of virus-like particles (VLPs), elicited significantly lower levels of PCV2 CP-specific IgG antibodies and a slightly lower neutralizing activity than immunization with the complete CP (1-233) protein. This finding suggests that the complete CP is important for efficient VLP assembly and induction of PCV2-specific IgG antibodies and neutralizing antibodies in mice. This study may provide useful information for next-generation vaccine design for PCV2 control.


Assuntos
Proteínas do Capsídeo/imunologia , Circovirus/imunologia , Epitopos/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Animais , Anticorpos Neutralizantes/análise , Anticorpos Antivirais/análise , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Circovirus/genética , Epitopos/biossíntese , Epitopos/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Síndrome Definhante Multissistêmico de Suínos Desmamados/imunologia , Síndrome Definhante Multissistêmico de Suínos Desmamados/prevenção & controle , Suínos , Vacinação , Vacinas de Partículas Semelhantes a Vírus/genética
4.
Vet Res ; 51(1): 110, 2020 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-32883344

RESUMO

Canine parvovirus (CPV) can cause acute and highly contagious bloody enteritis in dog. To obtain antibodies against CPV, hens were immunized with virus-like particles (VLP) of CPV-VP2. The IgY single chain fragment variables (scFv) were generated by T7 phage display system and expressed in E. coli system. The titer of the primary scFv library reached to 1.5 × 106 pfu/mL, and 95% of the phages contained the target fragments. The CPV-VLP and CPV-VP2 protein showed similar reaction values to the purified scFv in the ELISA test, and the results of ELISA analysis using IgY-scFv toward CPV clinical samples were consistent with commercial immunochromatographic assay (ICA) and PCR detection, the scFv did not show cross reactivity with canine distemper virus (CDV) and canine coronavirus (CCV). IgY-scFv was successfully expressed in CRFK cells, and in the virus suppression assay, 55% of CPV infections were eliminated within 24 h. Docking results demonstrated that the number of amino acids of the binding sides between scFv and VP2 were AA37 and AA40, respectively. This study revealed the feasibility of a novel functional antibody fragment development strategy by generating diversified avian IgY-scFv libraries towards the pathogenic target of interest for both detection and therapeutic purposes in veterinary medicine.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Doenças do Cão/virologia , Imunoglobulinas/imunologia , Infecções por Parvoviridae/veterinária , Parvovirus Canino/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Galinhas/imunologia , Doenças do Cão/diagnóstico , Doenças do Cão/imunologia , Cães , Ensaio de Imunoadsorção Enzimática/veterinária , Feminino , Simulação de Acoplamento Molecular , Infecções por Parvoviridae/diagnóstico , Infecções por Parvoviridae/imunologia , Infecções por Parvoviridae/virologia , Anticorpos de Cadeia Única/genética
5.
Nat Commun ; 11(1): 4419, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887892

RESUMO

Echovirus 30 (E30), a serotype of Enterovirus B (EV-B), recently emerged as a major causative agent of aseptic meningitis worldwide. E30 is particularly devastating in the neonatal population and currently no vaccine or antiviral therapy is available. Here we characterize two highly potent E30-specific monoclonal antibodies, 6C5 and 4B10, which efficiently block binding of the virus to its attachment receptor CD55 and uncoating receptor FcRn. Combinations of 6C5 and 4B10 augment the sum of their individual anti-viral activities. High-resolution structures of E30-6C5-Fab and E30-4B10-Fab define the location and nature of epitopes targeted by the antibodies. 6C5 and 4B10 engage the capsid loci at the north rim of the canyon and in-canyon, respectively. Notably, these regions exhibit antigenic variability across EV-Bs, highlighting challenges in development of broad-spectrum antibodies. Our structures of these neutralizing antibodies of E30 are instructive for development of vaccines and therapeutics against EV-B infections.


Assuntos
Anticorpos Neutralizantes/ultraestrutura , Complexo Antígeno-Anticorpo/ultraestrutura , Proteínas do Capsídeo/imunologia , Enterovirus Humano B/imunologia , Animais , Anticorpos Monoclonais/ultraestrutura , Antígenos Virais , Antígenos CD55/imunologia , Microscopia Crioeletrônica , Epitopos/ultraestrutura , Humanos , Meningite Asséptica/virologia , Camundongos , Receptores Fc/imunologia , Sorogrupo
6.
Arch Virol ; 165(10): 2301-2309, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32757056

RESUMO

Porcine circovirus type 2 (PCV2) is a major pathogen associated with swine diseases. It is the smallest single-stranded DNA virus, and its genome contains four major open reading frames (ORFs). ORF2 encodes the major structural protein Cap, which can self-assemble into virus-like particles (VLPs) in vitro and contains the primary antigenic determinants. In this study, we developed a high-efficiency method for obtaining VLPs and optimized the purification conditions. In this method, we expressed the protein Cap with a 6× His tag using baculovirus-infected silkworm larvae as well as the E. coli BL21(DE3) prokaryotic expression system. The PCV2 Cap proteins produced by the silkworm larvae and E. coli BL21(DE3) were purified. Cap proteins purified from silkworm larvae self-assembled into VLPs in vitro, while the Cap proteins purified from bacteria were unable to self-assemble. Transmission electron microscopy confirmed the self-assembly of VLPs. The immunogenicity of the VLPs produced using the baculovirus system was demonstrated using an enzyme-linked immunosorbent assay (ELISA). Furthermore, the purification process was optimized. The results demonstrated that the expression system using baculovirus-infected silkworm larvae is a good choice for obtaining VLPs of PCV2 and has potential for the development of a low-cost and efficient vaccine.


Assuntos
Anticorpos Antivirais/biossíntese , Baculoviridae/genética , Bombyx/virologia , Proteínas do Capsídeo/imunologia , Circovirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas Virais/biossíntese , Animais , Antígenos Virais/química , Antígenos Virais/imunologia , Baculoviridae/imunologia , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/virologia , Circovirus/genética , Epitopos/química , Epitopos/imunologia , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histidina/genética , Histidina/imunologia , Soros Imunes/química , Imunogenicidade da Vacina , Larva/virologia , Camundongos , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/virologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/isolamento & purificação
7.
Arch Virol ; 165(10): 2367-2372, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32757058

RESUMO

Mammalian orthoreoviruses (MRVs) infect almost all mammals, and there are some reports on MRVs in China. In this study, a novel strain was identified, which was designated as HLJYC2017. The results of genetic analysis showed that MRV HLJYC2017 is a reassortant strain. According to biological information analysis, different serotypes of MRV contain specific amino acid insertions and deletions in the σ1 protein. Neutralizing antibody epitope analysis revealed partial cross-protection among MRV1, MRV2, and MRV3 isolates from China. L3 gene recombination in MRV was identified for the first time in this study. The results of this study provide valuable information on MRV reassortment and evolution.


Assuntos
Antígenos Virais/genética , Proteínas do Capsídeo/genética , Orthoreovirus de Mamíferos/genética , Vírus Reordenados/genética , Infecções por Reoviridae/epidemiologia , Infecções por Reoviridae/veterinária , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , China/epidemiologia , Quirópteros , Cervos , Fezes/virologia , Expressão Gênica , Mutação INDEL , Camundongos , Epidemiologia Molecular , Orthoreovirus de Mamíferos/classificação , Orthoreovirus de Mamíferos/imunologia , Orthoreovirus de Mamíferos/isolamento & purificação , Filogenia , RNA Viral/genética , Vírus Reordenados/classificação , Vírus Reordenados/imunologia , Vírus Reordenados/isolamento & purificação , Infecções por Reoviridae/imunologia , Infecções por Reoviridae/virologia , Sorogrupo , Suínos
8.
PLoS Pathog ; 16(8): e1008732, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32750093

RESUMO

Rotavirus is a major cause of gastroenteritis in children, with infection typically inducing high levels of protective antibodies. Antibodies targeting the middle capsid protein VP6 are particularly abundant, and as VP6 is only exposed inside cells, neutralisation must be post-entry. However, while a system of poly immune globulin receptor (pIgR) transcytosis has been proposed for anti-VP6 IgAs, the mechanism by which VP6-specific IgG mediates protection remains less clear. We have developed an intracellular neutralisation assay to examine how antibodies neutralise rotavirus inside cells, enabling comparison between IgG and IgA isotypes. Unexpectedly we found that neutralisation by VP6-specific IgG was much more efficient than by VP6-specific IgA. This observation was highly dependent on the activity of the cytosolic antibody receptor TRIM21 and was confirmed using an in vivo model of murine rotavirus infection. Furthermore, mice deficient in only IgG and not other antibody isotypes had a serious deficit in intracellular antibody-mediated protection. The finding that VP6-specific IgG protect mice against rotavirus infection has important implications for rotavirus vaccination. Current assays determine protection in humans predominantly by measuring rotavirus-specific IgA titres. Measurements of VP6-specific IgG may add to existing mechanistic correlates of protection.


Assuntos
Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Imunoglobulina G/imunologia , Infecções por Rotavirus/imunologia , Rotavirus/fisiologia , Animais , Antígenos Virais/genética , Proteínas do Capsídeo/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Rotavirus/genética , Infecções por Rotavirus/virologia , Especificidade da Espécie
9.
Nat Commun ; 11(1): 3849, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737300

RESUMO

Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr Virus (EBV) establish life-long infections and are associated with malignancies. Striking geographic variation in incidence and the fact that virus alone is insufficient to cause disease, suggests other co-factors are involved. Here we present epidemiological analysis and genome-wide association study (GWAS) in 4365 individuals from an African population cohort, to assess the influence of host genetic and non-genetic factors on virus antibody responses. EBV/KSHV co-infection (OR = 5.71(1.58-7.12)), HIV positivity (OR = 2.22(1.32-3.73)) and living in a more rural area (OR = 1.38(1.01-1.89)) are strongly associated with immunogenicity. GWAS reveals associations with KSHV antibody response in the HLA-B/C region (p = 6.64 × 10-09). For EBV, associations are identified for VCA (rs71542439, p = 1.15 × 10-12). Human leucocyte antigen (HLA) and trans-ancestry fine-mapping substantiate that distinct variants in HLA-DQA1 (p = 5.24 × 10-44) are driving associations for EBNA-1 in Africa. This study highlights complex interactions between KSHV and EBV, in addition to distinct genetic architectures resulting in important differences in pathogenesis and transmission.


Assuntos
Anticorpos Antivirais/biossíntese , Resistência à Doença/genética , Infecções por Vírus Epstein-Barr/genética , Infecções por Henipavirus/genética , Interações Hospedeiro-Patógeno/genética , Sarcoma de Kaposi/genética , Adolescente , Adulto , Antígenos Virais/genética , Antígenos Virais/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Coinfecção , Infecções por Vírus Epstein-Barr/epidemiologia , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/virologia , Antígenos Nucleares do Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr/imunologia , Feminino , Expressão Gênica , Estudo de Associação Genômica Ampla , HIV/genética , HIV/imunologia , HIV/patogenicidade , Cadeias alfa de HLA-DQ/genética , Cadeias alfa de HLA-DQ/imunologia , Infecções por Henipavirus/epidemiologia , Infecções por Henipavirus/imunologia , Infecções por Henipavirus/virologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/imunologia , Herpesvirus Humano 4/patogenicidade , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/imunologia , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno/imunologia , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , População Rural , Sarcoma de Kaposi/epidemiologia , Sarcoma de Kaposi/imunologia , Sarcoma de Kaposi/virologia , Uganda/epidemiologia , População Urbana
10.
Arch Virol ; 165(9): 2065-2071, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32613291

RESUMO

Mink enteritis virus (MEV) is a parvovirus that causes acute enteritis in mink. The capsid protein VP2 of MEV is a major immunogenicity that is important for disease prevention. In this study, this protein was expressed in Spodoptera frugiperda 9 cells using a recombinant baculovirus system and was observed to self-assemble into virus-like particles (VLPs) with a high hemagglutination (HA) titer (1:216). A single-dose injection of VLPs (HA titer, 1:256) resulted in complete protection of mink against virulent MEV challenge for at least 180 days. These data suggest that these MEV VLPs could be used as a vaccine for the prevention of viral enteritis in mink.


Assuntos
Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Enterite Viral do Vison/prevenção & controle , Vírus da Enterite do Vison/imunologia , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Proteínas do Capsídeo/administração & dosagem , Expressão Gênica , Vison/imunologia , Vison/virologia , Enterite Viral do Vison/imunologia , Enterite Viral do Vison/virologia , Vírus da Enterite do Vison/genética , Vírus da Enterite do Vison/patogenicidade , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Células Sf9 , Spodoptera , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/imunologia , Virulência
11.
Nat Commun ; 11(1): 2841, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503989

RESUMO

The capsid of human papillomavirus (HPV) spontaneously arranges into a T = 7 icosahedral particle with 72 L1 pentameric capsomeres associating via disulfide bonds between Cys175 and Cys428. Here, we design a capsomere-hybrid virus-like particle (chVLP) to accommodate multiple types of L1 pentamers by the reciprocal assembly of single C175A and C428A L1 mutants, either of which alone encumbers L1 pentamer particle self-assembly. We show that co-assembly between any pair of C175A and C428A mutants across at least nine HPV genotypes occurs at a preferred equal molar stoichiometry, irrespective of the type or number of L1 sequences. A nine-valent chVLP vaccine-formed through the structural clustering of HPV epitopes-confers neutralization titers that are comparable with that of Gardasil 9 and elicits minor cross-neutralizing antibodies against some heterologous HPV types. These findings may pave the way for a new vaccine design that targets multiple pathogenic variants or cancer cells bearing diverse neoantigens.


Assuntos
Proteínas do Capsídeo/imunologia , Neoplasias/terapia , Papillomaviridae/imunologia , Infecções por Papillomavirus/terapia , Vacinas contra Papillomavirus/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/administração & dosagem , Proteínas do Capsídeo/genética , Desenho de Fármacos , Epitopos/genética , Epitopos/imunologia , Feminino , Humanos , Imunogenicidade da Vacina , Camundongos , Modelos Animais , Mutação , Neoplasias/virologia , Testes de Neutralização , Papillomaviridae/genética , Infecções por Papillomavirus/virologia , Vacinas contra Papillomavirus/administração & dosagem , Vacinas contra Papillomavirus/genética , Multimerização Proteica/genética , Multimerização Proteica/imunologia , Vacinas de Partículas Semelhantes a Vírus/administração & dosagem , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia
12.
PLoS Pathog ; 16(5): e1008517, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365139

RESUMO

Ross River fever is a mosquito-transmitted viral disease that is endemic to Australia and the surrounding Pacific Islands. Ross River virus (RRV) belongs to the arthritogenic group of alphaviruses, which largely cause disease characterized by debilitating polyarthritis, rash, and fever. There is no specific treatment or licensed vaccine available, and the mechanisms of protective humoral immunity in humans are poorly understood. Here, we describe naturally occurring human mAbs specific to RRV, isolated from subjects with a prior natural infection. These mAbs potently neutralize RRV infectivity in cell culture and block infection through multiple mechanisms, including prevention of viral attachment, entry, and fusion. Some of the most potently neutralizing mAbs inhibited binding of RRV to Mxra8, a recently discovered alpahvirus receptor. Epitope mapping studies identified the A and B domains of the RRV E2 protein as the major antigenic sites for the human neutralizing antibody response. In experiments in mice, these mAbs were protective against cinical disease and reduced viral burden in multiple tissues, suggesting a potential therapeutic use for humans.


Assuntos
Infecções por Alphavirus/prevenção & controle , Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Proteínas do Capsídeo/imunologia , Epitopos/imunologia , Vírus do Rio Ross/imunologia , Proteínas do Envelope Viral/imunologia , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/patologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/farmacologia , Chlorocebus aethiops , Feminino , Humanos , Camundongos , Pessoa de Meia-Idade , Células Vero
13.
Mol Biol (Mosk) ; 54(2): 278-284, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32392197

RESUMO

Thanks to their strong immunostimulating properties and safety for humans, plant viruses represent an appropriate basis for the design of novel vaccines. The coat protein of Alternanthera mosaic virus can form virus-like particles that are stable under physiological conditions and have adjuvant properties. This work presents a recombinant human rotavirus A antigen based on the epitope of rotavirus structural protein VP6, using Alternanthera mosaic virus coat protein as a carrier. An expression vector containing the gene of Alternanthera mosaic virus (MU strain) coat protein fused to the epitope of rotavirus protein VP6 was designed. Immunoblot analysis showed that the chimeric protein was effectively recognized by commercial polyclonal antibodies to rotavirus and therefore is a suitable candidate for development of a vaccine prototype. Interaction of the chimeric recombinant protein with the native coat protein of Alternanthera mosaic virus and its RNA resulted in the formation of ribonucleoprotein complexes that were recognized by anti-rotavirus antibodies.


Assuntos
Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Potexvirus/imunologia , Rotavirus/imunologia , Anticorpos Antivirais , Humanos , Proteínas Recombinantes/imunologia
14.
Mol Immunol ; 123: 26-31, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32388275

RESUMO

Nanoparticles are highly immunogenic due to the multivalent, repetitive antigen expression and direct activation of antigen presenting cells (APCs), key players of adaptive immune responses. Different virus-like particles (VLPs) have been used as display platforms to amplify immune responses to biologically relevant, but poorly immunogenic foreign antigens. A candidate platform based on rotavirus (RV) inner-capsid protein VP6 oligomers, such as nanotubes (T-VP6) and nanospheres (S-VP6), is also considered. Different VP6 nanostructures were compared for internalization and antigen presentation by the APCs. We found, that a lack of a high-order structures, T-VP6 and S-VP6, did not negatively affect VP6 uptake and presentation by murine bone-marrow derived dendritic cells (BMDCs) in vitro. Furthermore, T-VP6 was preferable to norovirus (NoV) VLPs for BMDC internalization resulting in significantly higher VP6-specific immune responses when T-VP6 and NoV VLP pulsed BMDCs were transferred to syngeneic mice. These results support the use of different VP6 nanostructures as foreign antigen delivery platforms.


Assuntos
Apresentação do Antígeno , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Rotavirus/fisiologia , Animais , Formação de Anticorpos , Antígenos Virais/química , Proteínas do Capsídeo/química , Células Cultivadas , Células Dendríticas/virologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Nanoestruturas/química , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Rotavirus/metabolismo , Internalização do Vírus
15.
Medicine (Baltimore) ; 99(15): e19794, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32282743

RESUMO

RATIONALE: Multiple evanescent white dot syndrome (MEWDS) is a self-limited multifocal chorioretinopathy that typically affects otherwise healthy young females in the second to fourth decades of life. Current understanding of the pathophysiology of MEWDS is still limited. One of the possible underlying causes is an infectious etiology. PATIENT CONCERNS: A 24-year-old female with recurrent episodes of typical MEWDS ocular manifestation was observed over 2 years. Viral-specific antibody serologic tests showed evidence of exposure to the Herpesviridae family during the acute stage of MEWDS in the first and recurrent episodes. DIAGNOSES: MEWDS was diagnosed by the clinical findings and ancillary testing results of fundus photography, optical coherence tomography, fluorescein angiography, indocyanine green angiography and electroretinogram. The laboratory serology data was positive for varicella-zoster virus (VZV) immunoglobulin M (IgM) in the first episode and exhibited high Epstein-Barr virus (EBV) elevated immunoglobulin G (IgG) titer in the recurrent episode. INTERVENTIONS: Due to the self-limited nature of MEWDS, we observed the clinical course without intervention. OUTCOMES: During acute onset of MEWDS, serologic data for VZV IgM antibody was positive in the first episode. Two years later, the patient had recurrent episodes of MEWDS in the contralateral eye. Serologic study showed highly elevated IgG titer (1:160) of Epstein-Barr virus capsid antigen (EB-VCA) in the acute stage. The follow-up paired serum virus serology test showed that the prior EB-VCA IgG titer decreased fourfold to 1:40 in the recovery stage. LESSONS: Recurrence of MEWDS may be associated with acute systemic infection of the Herpesviridae family or virus-induced autoimmune inflammatory reaction.


Assuntos
Infecções por Herpesviridae/complicações , Herpesviridae/imunologia , Doenças Retinianas/virologia , Síndromes do Ponto Branco/virologia , Angiografia/métodos , Antígenos Virais/imunologia , Grupo com Ancestrais do Continente Asiático/etnologia , Proteínas do Capsídeo/imunologia , Eletrorretinografia/métodos , Feminino , Angiofluoresceinografia/métodos , Fundo de Olho , Infecções por Herpesviridae/virologia , Herpesvirus Humano 3/imunologia , Herpesvirus Humano 4/imunologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Verde de Indocianina/administração & dosagem , Recidiva , Doenças Retinianas/diagnóstico por imagem , Doenças Retinianas/patologia , Tomografia de Coerência Óptica/métodos , Síndromes do Ponto Branco/diagnóstico por imagem , Síndromes do Ponto Branco/etiologia , Adulto Jovem
16.
J Virol ; 94(12)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32238587

RESUMO

Cyclic GMP-AMP synthase (cGAS) senses double-stranded DNA and synthesizes the second messenger cyclic GMP-AMP (cGAMP), which binds to mediator of IRF3 activation (MITA) and initiates MITA-mediated signaling, leading to induction of type I interferons (IFNs) and other antiviral effectors. Human cytomegalovirus (HCMV), a widespread and opportunistic pathogen, antagonizes the host antiviral immune response to establish latent infection. Here, we identified HCMV tegument protein UL94 as an inhibitor of the cGAS-MITA-mediated antiviral response. Ectopic expression of UL94 impaired cytosolic double-stranded DNA (dsDNA)- and DNA virus-triggered induction of type I IFNs and enhanced viral replication. Conversely, UL94 deficiency potentiated HCMV-induced transcription of type I IFNs and downstream antiviral effectors and impaired viral replication. UL94 interacted with MITA, disrupted the dimerization and translocation of MITA, and impaired the recruitment of TBK1 to the MITA signalsome. These results suggest that UL94 plays an important role in the immune evasion of HCMV.IMPORTANCE Human cytomegalovirus (HCMV), a large double-stranded DNA (dsDNA) virus, encodes more than 200 viral proteins. HCMV infection causes irreversible abnormalities of the central nervous system in newborns and severe syndromes in organ transplantation patients or AIDS patients. It has been demonstrated that HCMV has evolved multiple immune evasion strategies to establish latent infection. Previous studies pay more attention to the mechanism by which HCMV evades immune response in the early phase of infection. In this study, we identified UL94 as a negative regulator of the innate immune response, which functions in the late phase of HCMV infection.


Assuntos
Proteínas do Capsídeo/imunologia , Citomegalovirus/imunologia , Genoma Viral , Evasão da Resposta Imune , Proteínas de Membrana/imunologia , Proteínas Serina-Treonina Quinases/imunologia , RNA Interferente Pequeno/genética , Proteínas do Capsídeo/genética , Núcleo Celular/imunologia , Núcleo Celular/virologia , GMP Cíclico/imunologia , GMP Cíclico/metabolismo , Citomegalovirus/genética , Citomegalovirus/crescimento & desenvolvimento , Citosol/imunologia , Citosol/virologia , DNA/imunologia , DNA/metabolismo , Fibroblastos/imunologia , Fibroblastos/virologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Proteínas de Membrana/genética , Cultura Primária de Células , Ligação Proteica , Multimerização Proteica , Transporte Proteico , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/imunologia , Transdução de Sinais , Sequenciamento Completo do Exoma
17.
Nat Commun ; 11(1): 1985, 2020 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-32332752

RESUMO

The unsatisfactory response rate of immune checkpoint blockade (ICB) immunotherapy severely limits its clinical application as a tumor therapy. Here, we generate a vaccine-based nanosystem by integrating siRNA for Cd274 into the commercial human papillomavirus (HPV) L1 (HPV16 L1) protein. This nanosystem has good biosafety and enhances the therapeutic response rate of anti-tumor immunotherapy. The HPV16 L1 protein activates innate immunity through the type I interferon pathway and exhibits an efficient anti-cancer effect when cooperating with ICB therapy. For both resectable and unresectable breast tumors, the nanosystem decreases 71% tumor recurrence and extends progression-free survival by 67%. Most importantly, the nanosystem successfully induces high response rates in various genetically modified breast cancer models with different antigen loads. The strong immune stimulation elicited by this vaccine-based nanosystem might constitute an approach to significantly improve current ICB immunotherapy.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Neoplasias da Mama/terapia , Vacinas Anticâncer/administração & dosagem , Imunoterapia/métodos , Nanopartículas/administração & dosagem , Animais , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Vacinas Anticâncer/genética , Vacinas Anticâncer/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/imunologia , Humanos , Imunidade Inata/genética , Camundongos , Recidiva Local de Neoplasia , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Intervalo Livre de Progressão , RNA Interferente Pequeno/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
18.
PLoS One ; 15(4): e0229196, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32294099

RESUMO

Citrus mosaic virus (CiMV) is one of the causal viruses of citrus mosaic disease in satsuma mandarins (Citrus unshiu). Prompt detection of trees infected with citrus mosaic disease is important for preventing the spread of this disease. Although rabbit monoclonal antibodies (mAbs) exhibit high specificity and affinity, their applicability is limited by technical difficulties associated with the hybridoma-based technology used for raising these mAbs. Here, we demonstrate a feasible CiMV detection system using a specific rabbit mAb against CiMV coat protein. A conserved peptide fragment of the small subunit of CiMV coat protein was designed and used to immunize rabbits. Antigen-specific antibody-producing cells were identified by the immunospot array assay on a chip method. After cloning of variable regions in heavy or light chain by RT-PCR from these cells, a gene set of 33 mAbs was constructed and these mAbs were produced using Expi293F cells. Screening with the AlphaScreen system revealed eight mAbs exhibiting strong interaction with the antigen peptide. From subsequent sequence analysis, they were grouped into three mAbs denoted as No. 4, 9, and 20. Surface plasmon resonance analysis demonstrated that the affinity of these mAbs for the antigen peptide ranged from 8.7 × 10-10 to 5.5 × 10-11 M. In addition to CiMV, mAb No. 9 and 20 could detect CiMV-related viruses in leaf extracts by ELISA. Further, mAb No. 20 showed a high sensitivity to CiMV and CiMV-related viruses, simply by dot blot analysis. The anti-CiMV rabbit mAbs obtained in this study are envisioned to be extremely useful for practical applications of CiMV detection, such as in a virus detection kit.


Assuntos
Anticorpos Monoclonais/biossíntese , Citrus/virologia , Vírus do Mosaico/isolamento & purificação , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Especificidade de Anticorpos/imunologia , Proteínas do Capsídeo/imunologia , Cinética , Folhas de Planta/virologia , Coelhos
19.
Vet Immunol Immunopathol ; 223: 110034, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32278900

RESUMO

Porcine circovirus type 2 (PCV2) has one of the highest evolutionary rates among DNA viruses. Traditionally, PCV2 vaccines have been based on the 2a genotype as this was the first genotype discovered. Today, eight genotypes of PCV2 viruses have been identified, and, taken together with the rapid evolutionary rate, propensity to recombine, and high rate of vaccination, further variation in PCV2 is expected. For these reasons, there is a growing genetic gap between available vaccines and field strains. When selecting vaccines, it is important to consider vaccines that contain T cell epitopes that are well-matched to the circulating strains. To quantify the relatedness between PCV2 vaccines and field strains, we predicted and compared their T cell epitope content and calculated Epitope Content Comparison (EpiCC) scores using established in silico tools. T cell epitopes predicted to bind common class I and class II swine leukocyte antigen (SLA) alleles were identified from two major structural proteins, the capsid (encoded by ORF2) and the replicase (encoded by ORF1). The T cell epitope content of three commercial PCV2a-based vaccines (a baculovirus expressed PCV2a ORF2 [VacAlt], a PCV1-PCV2a chimeric virus vaccine [VacA] and a combination cPCV2a-cPCV2b chimeric virus vaccine [VacAB]) and an experimental PCV2b ORF2-based chimeric virus vaccine [VacB] (Table 1), were compared to that of 161 PCV2 field strains (representing genotypes a-f). The T cell epitope content and conservation between vaccine and field strains varied. While all vaccine strains provided broad coverage of the field strains including heterologous genotypes, none of the vaccines covered all the putative T cell epitopes identified in the field strains. PCV2a-based vaccine strains generally scored higher in terms of conserved epitope content against PCV2a field isolates but were not identical. The PCV2b-based vaccine strain had higher scores against PCV2b and PCV2d field strains. The combination PCV2a-PCV2b vaccine (VacAB) had, on average, the highest EpiCC score. PCV2 continues to evolve and EpiCC analysis provides a new tool to assess the possible impact of virus genetic divergence on T cell epitope coverage of vaccine strains. Given that multiple genotypes are currently found and may co-exist on farms, this analysis suggests that a combination of PCV2a and PCV2b vaccine strains may be required to provide optimal coverage of current and future field isolates.


Assuntos
Infecções por Circoviridae/veterinária , Circovirus/imunologia , Epitopos de Linfócito T/genética , Vacinas Virais/genética , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/imunologia , Infecções por Circoviridae/prevenção & controle , Circovirus/genética , Simulação por Computador , Epitopos de Linfócito T/imunologia , Genótipo , Imunidade Celular , Suínos , Doenças dos Suínos/imunologia
20.
Gene ; 747: 144682, 2020 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-32304786

RESUMO

Human Papillomavirus 16 (HPV16) is the most oncogenic HPV and the most associated genotype with cervical cancer development and progression. Currently, all developed vaccines are targeting HPV16 and were designed based on the major L1 capsid protein. Thus, evaluation of the diversity of HPV16 L1 sequence, mainly in the antigenic regions, will be of a great interest to assess the efficacy of the prophylactic vaccines and to predict the impact of genetic variations in these regions on the vaccination-induced immunity. A total of 377 HPV16 L1 sequences, published in public domain GenBank database, from the Americas, Africa, Asia, and Europe were collected and assembled. A total of 626 mutation events affecting 83 distinct nucleotides into the five antigenic regions of L1 gene of HPV16 were reported, and most SNPs were located in DE (27.38%, 23/83) and FG (31%, 26/83) loops. Overall, 4 mutations were frequently found in HPV16 sequences: T176N and N181T in EF loop; A266T in the FG loop and T353P/I/N HI loop. Of particular interest, some SNPs are ubiquitous and were found in all populations whereas others were population specific and their presence was limited to one or 2 at the maximum. Association between mutations in the antigenic regions and ethnicity was also investigated and showed that mutations in BC and DE loops were present with no significant difference in sequences from Europe, Asia, America and Africa. However, most mutations in FG loop are reported in sequences from European cases and are less pronounced in cases from America and Asia, whereas mutations EF and HI loops prevail in Asian cases. These data highlight a high number of variant amino acid residues that could affect the vaccination-induced immunity and impact the effectiveness of the prophylactic vaccination to fight against HPV, warranting the need of further investigation for vaccines and natural history studies of HPV16.


Assuntos
Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Variação Genética , Papillomavirus Humano 16/genética , Imunidade , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Vacinação , Aminoácidos/genética , Antígenos Virais/imunologia , Sequência de Bases , Grupos Étnicos/genética , Humanos , Modelos Moleculares , Mutação/genética , Filogenia , Polimorfismo de Nucleotídeo Único/genética
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