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1.
J Am Chem Soc ; 140(48): 16596-16609, 2018 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-30398345

RESUMO

Human mucin-1 (MUC1) is a highly attractive antigen for the development of anticancer vaccines. However, in human clinical trials of multiple MUC1 based vaccines, despite the generation of anti-MUC1 antibodies, the antibodies often failed to exhibit much binding to tumor presumably due to the challenges in inducing protective immune responses in the immunotolerant environment. To design effective MUC1 based vaccines functioning in immunotolerant hosts, vaccine constructs were first synthesized by covalently linking the powerful bacteriophage Qß carrier with MUC1 glycopeptides containing 20-22 amino acid residues covering one full length of the tandem repeat region of MUC1. However, IgG antibodies elicited by these first generation constructs in tolerant human MUC1 transgenic (Tg) mice did not bind tumor cells strongly. To overcome this, a peptide array has been synthesized. By profiling binding selectivities of antibodies, the long MUC1 glycopeptide was found to contain immunodominant but nonprotective epitopes. Critical insights were obtained into the identity of the key protective epitope. Redesign of the vaccine focusing on the protective epitope led to a new Qß-MUC1 construct, which was capable of inducing higher levels of anti-MUC1 IgG antibodies in MUC1.Tg mice to react strongly with and kill a wide range of tumor cells compared to the construct containing the gold standard protein carrier, i.e., keyhole limpet hemocyanin. Vaccination with this new Qß-MUC1 conjugate led to significant protection of MUC1.Tg mice in both metastatic and solid tumor models. The antibodies exhibited remarkable selectivities toward human breast cancer tissues, suggesting its high translational potential.


Assuntos
Neoplasias da Mama/terapia , Vacinas Anticâncer/uso terapêutico , Epitopos/imunologia , Mucina-1/imunologia , Allolevivirus/química , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Neoplasias da Mama/imunologia , Vacinas Anticâncer/imunologia , Feminino , Gastrópodes/química , Hemocianinas/síntese química , Hemocianinas/química , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mucina-1/química , Mucina-1/genética , Metástase Neoplásica/imunologia , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Proteínas Virais/síntese química , Proteínas Virais/química
2.
Nat Chem Biol ; 12(5): 353-360, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26999782

RESUMO

Membrane proteins are assembled through balanced interactions among proteins, lipids and water. Studying their folding while maintaining the native lipid environment is necessary but challenging. Here we present methods for analyzing key elements of membrane protein folding including thermodynamic stability, compactness of the unfolded state and folding cooperativity under native conditions. The methods are based on steric trapping, which couples the unfolding of a doubly biotinylated protein to the binding of monovalent streptavidin (mSA). We further advanced this technology for general application by developing versatile biotin probes possessing spectroscopic reporters that are sensitized by mSA binding or protein unfolding. By applying these methods to the Escherichia coli intramembrane protease GlpG, we elucidated a widely unraveled unfolded state, subglobal unfolding of the region encompassing the active site, and a network of cooperative and localized interactions to maintain stability. These findings provide crucial insights into the folding energy landscape of membrane proteins.


Assuntos
Proteínas de Ligação a DNA/química , Endopeptidases/química , Proteínas de Escherichia coli/química , Proteínas de Membrana/química , Biotina , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Análise Espectral
3.
Cancer Immunol Immunother ; 66(4): 451-460, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28011995

RESUMO

Cytotoxic T lymphocyte (CTL) can have remarkable abilities to kill tumor cells. However, the establishment of successful CTL-based anticancer therapy has met with many challenges. Within tumor cells, there exist subpopulations with low or no expression of the targeted antigen (termed as antigen-loss variants). In addition, tumor cells can downregulate the levels of major histocompatibility complex class I (MHC-I) molecules on cell surface due to immune pressure. As a result, some tumor cells can escape the immune pressure bestowed by CTLs, resulting in treatment failure. To address these difficulties, a new approach is developed to deliver foreign high-affinity CTL epitopes to tumor tissues utilizing pH-responsive "smart" microparticles (MPs). These MPs could encapsulate CTL peptide epitope, release the peptide under acidic condition encountered in tumor tissues and enhance CTL activation. Mice bearing pre-established tumor as "antigen-loss variant" solid tumor models were administered intratumorally with MPs containing the CTL peptide, which showed 100% survival following the treatment. In contrast, all control mice died from tumor. Significant protection from tumor-induced death was also observed with systemic administration of CTL peptide-MPs. The therapeutic efficacy can be attributed to enhanced delivery of the epitope to tumor tissues, presentation of the epitope by tumor cells as well as tumor stromal cells and/or generation of epitope-specific CTLs by the peptide-containing MPs. These findings offer a promising new direction for treating established solid tumor using CTL therapy.


Assuntos
Adenocarcinoma/terapia , Epitopos de Linfócito T/administração & dosagem , Imunoterapia/métodos , Lipossomos/administração & dosagem , Linfoma/terapia , Peptídeos/administração & dosagem , Linfócitos T Citotóxicos/imunologia , Adenocarcinoma/imunologia , Animais , Linhagem Celular Tumoral , Epitopos de Linfócito T/química , Feminino , Humanos , Concentração de Íons de Hidrogênio , Lipossomos/química , Ativação Linfocitária , Linfoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias Experimentais , Peptídeos/química , Evasão Tumoral
4.
Vet Microbiol ; 291: 110033, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38432077

RESUMO

The genomes of many pathogens contain high-CpG content, which is less common in most vertebrate host genomes. Such a distinct di-nucleotide composition in a non-self invader constitutes a special feature recognized by its host's immune system. The zinc-finger antiviral protein (ZAP) is part of the pattern recognition receptors (PRRs) that recognize CpG-rich viral RNA and subsequently initiate RNA degradation as an antiviral defense measure. To counteract such ZAP-mediated restriction, some viruses evolve to either suppress the CpG content in their genome or produce an antagonistic factor to evade ZAP sensing. We have previously shown that a coronavirus, Porcine epidermic diarrhea virus (PEDV), employs its nucleocapsid protein (PEDV-N) to suppress the ZAP-dependent antiviral activity. Here, we propose a mechanism by which PEDV-N suppresses ZAP function by interfering with the interaction between ZAP and its essential cofactor, Tripartite motif-containing protein 25 (TRIM25). PEDV-N was found to interact with ZAP through its N-terminal domain and with TRIM25 through its C-terminal domain. We showed that PEDV-N and ZAP compete for binding to the SPla and the RYanodine Receptor (SPRY) domain of TRIM25, resulting in PEDV-N preventing TRIM25 from interacting with and promoting ZAP. Our result also showed that the presence of PEDV-N in the complex reduces the E3 ligase activity of TRIM25 on ZAP, which is required for the antiviral activity of ZAP. The host-pathogen interaction mechanism presented herein provides an insight into the new function of this abundant and versatile viral protein from a coronavirus which could be a key target for development of antiviral interventions.


Assuntos
Ubiquitina-Proteína Ligases , Vírus , Animais , Suínos , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Antivirais/farmacologia , Antivirais/metabolismo , Nucleocapsídeo , Zinco
5.
PeerJ ; 11: e14918, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36883057

RESUMO

Alveolar macrophages are tissue-resident immune cells that protect epithelial cells in the alveoli from invasion by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, the interaction between macrophages and SARS-CoV-2 is inevitable. However, little is known about the role of macrophages in SARS-CoV-2 infection. Here, we generated macrophages from human induced pluripotent stem cells (hiPSCs) to investigate the susceptibility of hiPSC-derived macrophages (iMΦ) to the authentic SARS-CoV-2 Delta (B.1.617.2) and Omicron (B.1.1.529) variants as well as their gene expression profiles of proinflammatory cytokines during infection. With undetectable angiotensin-converting enzyme 2 (ACE2) mRNA and protein expression, iMΦ were susceptible to productive infection with the Delta variant, whereas infection of iMΦ with the Omicron variant was abortive. Interestingly, Delta induced cell-cell fusion or syncytia formation in iMΦ, which was not observed in Omicron-infected cells. However, iMΦ expressed moderate levels of proinflammatory cytokine genes in response to SARS-CoV-2 infection, in contrast to strong upregulation of these cytokine genes in response to polarization by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Overall, our findings indicate that the SARS-CoV-2 Delta variant can replicate and cause syncytia formation in macrophages, suggesting that the Delta variant can enter cells with undetectable ACE2 levels and exhibit greater fusogenicity.


Assuntos
COVID-19 , Células Gigantes , Células-Tronco Pluripotentes Induzidas , Humanos , Enzima de Conversão de Angiotensina 2/genética , COVID-19/virologia , Citocinas/genética , Macrófagos , SARS-CoV-2/genética
6.
Front Microbiol ; 13: 975632, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36160209

RESUMO

Coronaviruses have long posed a major threat not only to human health but also to agriculture. Outbreaks of an animal coronavirus such as porcine epidemic diarrhea virus (PEDV) can cause up-to-100% mortality in suckling piglets, resulting in devastating effects on the livestock industry. Understanding how the virus evades its host's defense can help us better manage the infection. Zinc-finger antiviral protein (ZAP) is an important class of host antiviral factors against a variety of viruses, including the human coronavirus. In this study, we have shown that a representative porcine coronavirus, PEDV, can be suppressed by endogenous or porcine-cell-derived ZAP in VeroE6 cells. An uneven distribution pattern of CpG dinucleotides in the viral genome is one of the factors contributing to suppression, as an increase in CpG content in the nucleocapsid (N) gene renders the virus more susceptible to ZAP. Our study revealed that the virus uses its own nucleocapsid protein (pCoV-N) to interact with ZAP and counteract the activity of ZAP. The insights into coronavirus-host interactions shown in this work could be used in the design and development of modern vaccines and antiviral agents for the next pandemic.

7.
ACS Chem Biol ; 17(11): 3047-3058, 2022 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-35142488

RESUMO

Vaccines are critical tools to treat and prevent diseases. For an effective conjugate vaccine, the carrier is crucial, but few carriers are available for clinical applications. In addition, a drawback of current protein carriers is that high levels of antibodies against the carrier are induced by the conjugate vaccine, which are known to interfere with the immune responses against the target antigen. To overcome these challenges, we obtained the near atomic resolution crystal structure of an emerging protein carrier, i.e., the bacteriophage Qß virus like particle. On the basis of the detailed structural information, novel mutants of bacteriophage Qß (mQß) have been designed, which upon conjugation with tumor associated carbohydrate antigens (TACAs), a class of important tumor antigens, elicited powerful anti-TACA IgG responses and yet produced lower levels of anticarrier antibodies as compared to those from the wild type Qß-TACA conjugates. In a therapeutic model against an aggressive breast cancer in mice, 100% unimmunized mice succumbed to tumors in just 12 days even with chemotherapy. In contrast, 80% of mice immunized with the mQß-TACA conjugate were completely free from tumors. Besides TACAs, to aid in the development of vaccines to protect against COVID-19, the mQß based conjugate vaccine has been shown to induce high levels of IgG antibodies against peptide antigens from the SARS-CoV-2 virus, demonstrating its generality. Thus, mQß is a promising next-generation carrier platform for conjugate vaccines, and structure-based rational design is a powerful strategy to develop new vaccine carriers.


Assuntos
COVID-19 , Neoplasias , Camundongos , Animais , Vacinas Conjugadas , SARS-CoV-2 , Allolevivirus/química , Antígenos Glicosídicos Associados a Tumores , Imunoglobulina G , Neoplasias/terapia
8.
Virology ; 540: 45-56, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31756532

RESUMO

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV) share tropism for swine intestinal epithelial cells. Whether mixing of viral components during co-infection alters pathogenic outcomes or viral replication is not known. In this study, we investigated how different coronavirus nucleocapsid (CoV N) proteins interact and affect PEDV replication. We found that PDCoV N and TGEV N can competitively interact with PEDV N. However, the presence of PDCoV or TGEV N led to very different outcomes on PEDV replication. While PDCoV N significantly suppresses PEDV replication, overexpression of TGEV N, like that of PEDV N, increases production of PEDV RNA and virions. Despite partial interchangeability in nucleocapsid oligomerization and viral RNA synthesis, endogenous PEDV N cannot be replaced in the production of infectious PEDV particles. Results from this study give insights into functional compatibilities and evolutionary relationship between CoV viral proteins during viral co-infection and co-evolution.


Assuntos
Interações Microbianas , Proteínas do Nucleocapsídeo/metabolismo , Vírus da Diarreia Epidêmica Suína/crescimento & desenvolvimento , Animais , Chlorocebus aethiops , Coinfecção/virologia , Coronavirus/crescimento & desenvolvimento , Proteínas do Nucleocapsídeo de Coronavírus , Células Epiteliais/virologia , Células HEK293 , Humanos , Vírus da Gastroenterite Transmissível/crescimento & desenvolvimento , Células Vero
9.
PLoS One ; 14(3): e0212632, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30840701

RESUMO

More recently emerging strains of porcine epidemic diarrhea virus (PEDV) cause severe diarrhea and especially high mortality rates in infected piglets, leading to substantial economic loss to worldwide swine industry. These outbreaks urgently call for updated and effective PEDV vaccines. Better understanding in PEDV biology and improvement in technological platforms for virus production can immensely assist and accelerate PEDV vaccine development. In this study, we explored the ability of PEDV nucleocapsid (N) protein in improving viral yields in cell culture systems. We demonstrated that PEDV N expression positively affected both recovery of PEDV from infectious clones and PEDV propagation in cell culture. Compared to Vero E6 cells, Vero E6 cells expressing PEDV N could accelerate growth of a slow-growing PEDV strain to higher peak titers by 12 hours or enhance the yield of a vaccine candidate strain by two orders of magnitude. Interestingly, PEDV N also slightly enhances replication of porcine reproductive and respiratory virus, a PEDV relative in the Nidovirales order. These results solidify the importance of N in PEDV recovery and propagation and suggest a potentially useful consideration in designing vaccine production platforms for PEDV or closely related pathogens.


Assuntos
Proteínas do Nucleocapsídeo/biossíntese , Vírus da Diarreia Epidêmica Suína/crescimento & desenvolvimento , Animais , Chlorocebus aethiops , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/veterinária , Suínos , Doenças dos Suínos/metabolismo , Doenças dos Suínos/virologia , Células Vero
10.
ACS Chem Biol ; 13(6): 1668-1676, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29782143

RESUMO

Mucin-1 (MUC1) is one of the top ranked tumor associated antigens. In order to generate effective anti-MUC1 immune responses as potential anticancer vaccines, MUC1 peptides and glycopeptides have been covalently conjugated to bacteriophage Qß. Immunization of mice with these constructs led to highly potent antibody responses with IgG titers over one million, which are among the highest anti-MUC1 IgG titers reported to date. Furthermore, the high IgG antibody levels persisted for more than six months. The constructs also elicited MUC1 specific cytotoxic T cells, which can selectively kill MUC1 positive tumor cells. The unique abilities of Qß-MUC1 conjugates to powerfully induce both antibody and cytotoxic T cell immunity targeting tumor cells bode well for future translation of the constructs as anticancer vaccines.


Assuntos
Bacteriófagos/imunologia , Vacinas Anticâncer/imunologia , Imunidade Humoral/imunologia , Imunoglobulina G/imunologia , Ativação Linfocitária/imunologia , Mucina-1/imunologia , Sequência de Aminoácidos , Animais , Formação de Anticorpos/imunologia , Bacteriófagos/química , Vacinas Anticâncer/síntese química , Linhagem Celular Tumoral , Humanos , Imunização , Linfoma/imunologia , Camundongos Endogâmicos C57BL , Análise em Microsséries , Mucina-1/química , Linfócitos T Citotóxicos/imunologia , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologia
12.
Methods Enzymol ; 597: 359-376, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28935111

RESUMO

Tumor-associated carbohydrate antigens (TACAs) are attractive targets for anticancer vaccine development. Due to the low immunogenicity of TACAs, a powerful carrier system is needed to boost immune responses. Virus-like particles (VLPs) are an exciting platform for delivering TACAs to the immune system. The high symmetry of VLPs enables the display of TACAs in an organized manner, which in turn can potently activate antibody secreting B cells, eliciting high titers of antiglycan IgG antibodies. In this chapter, the protocol for conjugating a prototypical TACA, the Tn antigen to a VLP, bacteriophage Qß, is presented. On an average around 370 copies of Tn can be attached to each Qß capsid. Immunization of mice with Qß-Tn conjugate leads to over two orders of magnitude higher IgG antibodies compared to control mice receiving Qß only without the Tn antigen. Antibodies induced by Qß-Tn recognize Tn-expressing tumor cells strongly and protect mice from tumor-induced death. The techniques for evaluating antibody titers by enzyme-linked immunosorbent assay, antibody binding to tumor cells by flow cytometry, and the protection efficacy of the vaccine in a therapeutic model of tumor are discussed in this chapter.


Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Neoplasias/terapia , Vacinas Conjugadas/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Allolevivirus/imunologia , Animais , Antígenos Glicosídicos Associados a Tumores/uso terapêutico , Linfócitos B/imunologia , Biomarcadores Tumorais/imunologia , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Imunização/métodos , Camundongos , Neoplasias/imunologia , Vacinas Conjugadas/uso terapêutico , Vacinas de Partículas Semelhantes a Vírus/uso terapêutico
13.
ACS Appl Mater Interfaces ; 7(31): 17535-44, 2015 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-26200668

RESUMO

Although iron oxide magnetic nanoparticles (NPs) have been widely utilized in molecular imaging and drug delivery studies, they have not been evaluated as carriers for glycoconjugate-based anticancer vaccines. Tumor-associated carbohydrate antigens (TACAs) are attractive targets for the development of anticancer vaccines. Due to the weak immunogenicity of these antigens, it is highly challenging to elicit strong anti-TACA immune responses. With their high biocompatibilities and large surface areas, magnetic NPs were synthesized for TACA delivery. The magnetic NPs were coated with phospholipid-functionalized TACA glycopeptides through hydrophobic-hydrophobic interactions without the need for any covalent linkages. Multiple copies of glycopeptides were presented on NPs, potentially leading to enhanced interactions with antibody-secreting B cells through multivalent binding. Mice immunized with the NPs generated strong antibody responses, and the glycopeptide structures important for high antibody titers were identified. The antibodies produced were capable of recognizing both mouse and human tumor cells expressing the glycopeptide, resulting in tumor cell death through complement-mediated cytotoxicities. These results demonstrate that magnetic NPs can be a new and simple platform for multivalently displaying TACA and boosting anti-TACA immune responses without the need for a typical protein carrier.


Assuntos
Vacinas Anticâncer/imunologia , Compostos Férricos/química , Glicoconjugados/química , Lipopeptídeos/química , Nanopartículas de Magnetita/química , Vacinas Sintéticas/imunologia , Animais , Anticorpos/imunologia , Antígenos Glicosídicos Associados a Tumores/química , Antígenos Glicosídicos Associados a Tumores/imunologia , Materiais Biocompatíveis/química , Ativação do Complemento , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Difusão Dinâmica da Luz , Glicopeptídeos/imunologia , Humanos , Células MCF-7 , Camundongos , Mucina-1/imunologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
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