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1.
Front Immunol ; 13: 910136, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35911728

RESUMO

We assessed if immune responses are enhanced in CD-1 mice by heterologous vaccination with two different nucleic acid-based COVID-19 vaccines: a next-generation human adenovirus serotype 5 (hAd5)-vectored dual-antigen spike (S) and nucleocapsid (N) vaccine (AdS+N) and a self-amplifying and -adjuvanted S RNA vaccine (AAHI-SC2) delivered by a nanostructured lipid carrier. The AdS+N vaccine encodes S modified with a fusion motif to increase cell-surface expression and an N antigen modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal compartment and increase MHC class I and II stimulation potential. The S sequence in the AAHI-SC2 vaccine comprises the D614G mutation, two prolines to stabilize S in the prefusion conformation, and 3 glutamines in the furin cleavage region to confer protease resistance. CD-1 mice received vaccination by homologous and heterologous prime > boost combinations. Humoral responses to S were the highest with any regimen that included the AAHI-SC2 vaccine, and IgG bound to wild type and Delta (B.1.617.2) variant S1 at similar levels. An AAHI-SC2 prime followed by an AdS+N boost particularly enhanced CD4+ and CD8+ T-cell responses to both wild type and Delta S peptides relative to all other vaccine regimens. Sera from mice receiving AAHI-SC2 homologous or heterologous vaccination were found to be highly neutralizing for all pseudovirus strains tested: Wuhan, Beta, Delta, and Omicron strains. The findings here, taken in consideration with the availability of both vaccines in thermostable formulations, support the testing of heterologous vaccination by an AAHI-SC2 > AdS+N regimen in animal models of SARS-CoV-2 infection to assess its potential to provide increased protection against emerging SARS-CoV-2 variants particularly in regions of the world where the need for cold-chain storage has limited the distribution of other vaccines.


Assuntos
COVID-19 , Vacinas Virais , Animais , Anticorpos Neutralizantes , Antígenos Heterófilos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , DNA , Humanos , Camundongos , SARS-CoV-2 , Vacinação , Vacinas Sintéticas , Vacinas de mRNA
2.
Front Immunol ; 12: 729837, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34603305

RESUMO

We have developed a dual-antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike protein (S-Fusion) and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to increase the potential for MHC class II responses. The vaccine antigens are delivered by a human adenovirus serotype 5 platform, hAd5 [E1-, E2b-, E3-], previously demonstrated to be effective in the presence of Ad immunity. Vaccination of rhesus macaques with the hAd5 S-Fusion + N-ETSD vaccine by subcutaneous prime injection followed by two oral boosts elicited neutralizing anti-S IgG and T helper cell 1-biased T-cell responses to both S and N that protected the upper and lower respiratory tracts from high titer (1 x 106 TCID50) SARS-CoV-2 challenge. Notably, viral replication was inhibited within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge.


Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adenovírus Humanos/genética , Adenovírus Humanos/imunologia , Adenovírus Humanos/metabolismo , Administração Oral , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vacinas contra COVID-19/administração & dosagem , Citocinas/sangue , Imunização Secundária/métodos , Imunoglobulina G/sangue , Pulmão/virologia , Macaca mulatta , Nariz/virologia , Fosfoproteínas/imunologia , Domínios Proteicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinação , Replicação Viral/imunologia
3.
Sci Rep ; 11(1): 14917, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34290317

RESUMO

We have developed a COVID-19 vaccine, hAd5 S-Fusion + N-ETSD, that expresses SARS-CoV-2 spike (S) and nucleocapsid (N) proteins with modifications to increase immune responses delivered using a human adenovirus serotype 5 (hAd5) platform. Here, we demonstrate subcutaneous (SC) prime and SC boost vaccination of CD-1 mice with this dual-antigen vaccine elicits T-helper cell 1 (Th1) biased T-cell and humoral responses to both S and N that are greater than those seen with hAd5 S wild type delivering only unmodified S. We then compared SC to intranasal (IN) prime vaccination with SC or IN boosts and show that an IN prime with an IN boost is as effective at generating Th1 biased humoral responses as the other combinations tested, but an SC prime with an IN or SC boost elicits greater T cell responses. Finally, we used a combined SC plus IN (SC + IN) prime with or without a boost and found the SC + IN prime alone to be as effective in generating humoral and T-cell responses as the SC + IN prime with a boost. The finding that SC + IN prime-only delivery has the potential to provide broad immunity-including mucosal immunity-against SARS-CoV-2 supports further testing of this vaccine and delivery approach in animal models of viral challenge.


Assuntos
Vacinas contra COVID-19/administração & dosagem , COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Adenoviridae/genética , Administração Intranasal , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , Feminino , Vetores Genéticos , Hipodermóclise , Imunidade Celular/imunologia , Imunidade nas Mucosas/imunologia , Imunização Secundária , Camundongos , Camundongos Endogâmicos , Vacinação/métodos
4.
Oncotarget ; 6(31): 31344-59, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26374823

RESUMO

Phenotypic heterogeneity of human carcinoma lesions, including heterogeneity in expression of tumor-associated antigens (TAAs), is a well-established phenomenon. Carcinoembryonic antigen (CEA), MUC1, and brachyury are diverse TAAs, each of which is expressed on a wide range of human tumors. We have previously reported on a novel adenovirus serotype 5 (Ad5) vector gene delivery platform (Ad5 [E1-, E2b-]) in which regions of the early 1 (E1), early 2 (E2b), and early 3 (E3) genes have been deleted. The unique deletions in this platform result in a dramatic decrease in late gene expression, leading to a marked reduction in host immune response to the vector. Ad5 [E1-, E2b-]-CEA vaccine (ETBX-011) has been employed in clinical studies as an active vaccine to induce immune responses to CEA in metastatic colorectal cancer patients. We report here the development of novel recombinant Ad5 [E1-, E2b-]-brachyury and-MUC1 vaccine constructs, each capable of activating antigen-specific human T cells in vitro and inducing antigen-specific CD4+ and CD8+ T cells in vaccinated mice. We also describe the use of a combination of the three vaccines (designated Tri-Ad5) of Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-brachyury and Ad5 [E1-, E2b-]-MUC1, and demonstrate that there is minimal to no "antigenic competition" in in vitro studies of human dendritic cells, or in murine vaccination studies. The studies reported herein support the rationale for the application of Tri-Ad5 as a therapeutic modality to induce immune responses to a diverse range of human TAAs for potential clinical studies.


Assuntos
Adenoviridae/genética , Vacinas contra Adenovirus/uso terapêutico , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/uso terapêutico , Imunoterapia , Neoplasias/terapia , Proteínas E1 de Adenovirus/genética , Proteínas E1 de Adenovirus/imunologia , Proteínas E2 de Adenovirus/genética , Proteínas E2 de Adenovirus/imunologia , Animais , Antígenos de Neoplasias/genética , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/imunologia , Células Dendríticas/imunologia , Feminino , Citometria de Fluxo , Vetores Genéticos/administração & dosagem , Humanos , Imunização , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T/imunologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Cancer Immunol Immunother ; 64(8): 977-87, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25956394

RESUMO

A phase 1/2 clinical trial evaluating dosing, safety, immunogenicity, and overall survival on metastatic colorectal cancer (mCRC) patients after immunotherapy with an advanced-generation Ad5 [E1-, E2b-]-CEA(6D) vaccine was performed. We report our extended observations on long-term overall survival and further immune analyses on a subset of treated patients including assessment of cytolytic T cell responses, T regulatory (Treg) to T effector (Teff) cell ratios, flow cytometry on peripheral blood mononuclear cells (PBMCs), and determination of HLA-A2 status. An overall survival of 20 % (median survival 11 months) was observed during long-term follow-up, and no long-term adverse effects were reported. Cytolytic T cell responses increased after immunizations, and cell-mediated immune (CMI) responses were induced whether or not patients were HLA-A2 positive or Ad5 immune. PBMC samples from a small subset of patients were available for follow-up immune analyses. It was observed that the levels of carcinoembryonic antigen (CEA)-specific CMI activity decreased from their peak values during follow-up in five patients analyzed. Preliminary results revealed that activated CD4+ and CD8+ T cells were detected in a post-immunization sample exhibiting high CMI activity. Treg to Teff cell ratios were assessed, and samples from three of five patients exhibited a decrease in Treg to Teff cell ratio during the treatment protocol. Based upon the favorable safety and immunogenicity data obtained, we plan to perform an extensive immunologic and survival analysis on mCRC patients to be enrolled in a randomized/controlled clinical trial that investigates Ad5 [E1-, E2b-]-CEA(6D) as a single agent with booster immunizations.


Assuntos
Vacinas Anticâncer/uso terapêutico , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/terapia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Adenoviridae , Proteínas E1 de Adenovirus/genética , Proteínas E2 de Adenovirus/genética , Adulto , Idoso , Vacinas Anticâncer/imunologia , Células Cultivadas , Neoplasias Colorretais/patologia , Citotoxicidade Imunológica , Feminino , Seguimentos , Humanos , Imunização , Interferon gama/metabolismo , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Deleção de Sequência/genética , Análise de Sobrevida
6.
J Mol Biol ; 386(3): 717-32, 2009 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-19150361

RESUMO

Ferroportin is a multipass membrane protein that serves as an iron exporter in many vertebrate cell types. Ferroportin-mediated iron export is controlled by the hormone hepcidin, which binds ferroportin, causing its internalization and degradation. Mutations in ferroportin cause a form of the iron overload hereditary disease hemochromatosis. Relatively little is known about ferroportin's properties or the mechanism by which mutations cause disease. In this study, we expressed and purified human ferroportin to characterize its biochemical/biophysical properties in solution and conducted cell biological studies in mammalian cells. We found that purified detergent-solubilized ferroportin is a well-folded monomer that binds hepcidin. In cell membranes, the N- and C-termini were both cytosolic, implying an even number of transmembrane regions, and ferroportin was mainly localized to the plasma membrane. Hepcidin addition resulted in a redistribution of ferroportin to intracellular compartments that labeled with early endosomal and lysosomal, but not Golgi, markers and that trafficked along microtubules. An analysis of 16 disease-related ferroportin mutants revealed that all were expressed and trafficked to the plasma membrane but that some were resistant to hepcidin-induced internalization. The characterizations reported here form a basis upon which models for ferroportin's role in regulating iron homeostasis in health and disease can be interpreted.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Animais , Linhagem Celular , Membrana Celular/química , Endossomos/química , Hepcidinas , Humanos , Lisossomos/química , Modelos Biológicos , Modelos Moleculares , Peso Molecular , Proteínas Mutantes/metabolismo , Ligação Proteica , Transporte Proteico
7.
Biochemistry ; 46(2): 436-47, 2007 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-17209554

RESUMO

The iron-dependent regulator IdeR is a key transcriptional regulator of iron uptake in Mycobacterium tuberculosis. In order to increase our insight into the role of the SH3-like third domain of this essential regulator, the metal-binding and DNA-binding properties of two-domain IdeR (2D-IdeR) whose SH3-like domain has been truncated were characterized. The equilibrium dissociation constants for Co2+ and Ni2+ activation of 2D-IdeR for binding to the fxbA operator and the DNA-binding affinities of 2D-IdeR in the presence of excess metal ions were estimated using fluorescence spectroscopy. 2D-IdeR binds to fxbA operator DNA with similar affinity as full-length IdeR in the presence of excess metal ion. However, the Ni2+ concentrations required to activate 2D-IdeR for DNA binding appear to be smaller than that for full-length IdeR while the concentration of Co2+ required for activation remains the same. We have determined the crystal structures of Ni2+-activated 2D-IdeR at 1.96 A resolution and its double dimer complex with the mbtA-mbtB operator DNA in two crystal forms at 2.4 A and 2.6 A, the highest resolutions for DNA complexes for any structures of iron-dependent regulator family members so far. The 2D-IdeR-DNA complex structures confirm the specificity of Ser37 and Pro39 for thymine bases and suggest preferential contacts of Gln43 to cytosine bases of the DNA. In addition, our 2D-IdeR structures reveal a remarkable property of the TEV cleavage sequence remaining after removal of the C-terminal His6. This C-terminal tail promotes crystal contacts by forming a beta-sheet with the corresponding tail of neighboring subunits in two unrelated structures of 2D-IdeR, one with and one without DNA. The contact-promoting properties of this C-terminal TEV cleavage sequence may be beneficial for crystallizing other proteins.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Dimerização , Cinética , Substâncias Macromoleculares , Metais/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/genética , Conformação de Ácido Nucleico , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética
8.
J Mol Biol ; 354(3): 630-41, 2005 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-16246368

RESUMO

On encountering low oxygen conditions, DosR activates the transcription of 47 genes, promoting long-term survival of Mycobacterium tuberculosis in a non-replicating state. Here, we report the crystal structures of the DosR C-terminal domain and its complex with a consensus DNA sequence of the hypoxia-induced gene promoter. The DosR C-terminal domain contains four alpha-helices and forms tetramers consisting of two dimers with non-intersecting dyads. In the DNA-bound structure, each DosR C-terminal domain in a dimer places its DNA-binding helix deep into the major groove, causing two bends in the DNA. DosR makes numerous protein-DNA base contacts using only three amino acid residues per subunit: Lys179, Lys182, and Asn183. The DosR tetramer is unique among response regulators with known structures.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/fisiologia , Oxigênio/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cristalografia por Raios X , DNA Bacteriano/química , DNA Bacteriano/genética , Dimerização , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Hipóxia/metabolismo , Hipóxia/microbiologia , Modelos Moleculares , Dados de Sequência Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Conformação de Ácido Nucleico , Oxigênio/farmacologia , Ligação Proteica , Estrutura Quaternária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Ativação Transcricional
9.
J Mol Biol ; 338(3): 585-96, 2004 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-15081815

RESUMO

The terminal branch of the general secretion pathway (Gsp or type II secretion system) is used by several pathogenic bacteria for the secretion of their virulence factors across the outer membrane. In these secretion systems, a complex of 12-15 Gsp proteins spans from the pore in the outer membrane via several associated signal or energy-transducing proteins in the inner membrane to a regulating ATPase in the cytosol. The human pathogen Vibrio cholerae uses such a system, called the Eps system, for the export of the cholera toxin and other virulence factors from its periplasm into the lumen of the gastrointestinal tract of the host. Here, we report the atomic structure of the periplasmic domain of the EpsM protein from V.cholerae, which is a part of the interface between the regulating part and the rest of the Eps system. The crystal structure was determined by Se-Met MAD phasing and the model was refined to 1.7A resolution. The monomer consists of two alphabetabeta-subdomains forming a sandwich of two alpha-helices and a four-stranded antiparallel beta-sheet. In the dimer, a deep cleft with a polar rim and a hydrophobic bottom made by conserved residues is located between the monomers. This cleft contains an extra electron density suggesting that this region might serve as a binding site of an unknown ligand or part of a protein partner. Unexpectedly, the fold of the periplasmic domain of EpsM is an undescribed circular permutation of the ferredoxin fold.


Assuntos
Proteínas de Bactérias/química , Proteínas de Membrana/química , Vibrio cholerae/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Cromatografia em Gel , Sequência Conservada , Proteínas de Membrana/genética , Dados de Sequência Molecular , Família Multigênica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Vibrio cholerae/genética
10.
Biochemistry ; 42(51): 15197-207, 2003 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-14690430

RESUMO

The dual specificity mitogen-activated protein kinase phosphatase MKP3 downregulates mitogenic signaling through dephosphorylation of extracellular signal-regulated kinase (ERK). Like other MKPs, MKP3 consists of a noncatalytic N-terminal domain and a catalytic C-terminal domain. ERK binding to the N-terminal noncatalytic domain of MKP3 has been shown to increase (up to 100-fold) the catalytic activity of MKP3 toward small artificial substrates. Here, we address the function of the N-terminal domain of MKP3 in either inter- or intramolecular dephosphorylation of pERK (phosphorylated ERK) and the stoichiometry of the MKP3/pERK Michaelis complex. These are important mechanistic distinctions given the observation that ERK exists in a monomer/dimer equilibrium that is shifted toward the dimer when phosphorylated and given that MKP3 undergoes catalytic activation toward other substrates when bound to ERK. Wild-type and engineered mutants of ERK and MKP3, binding analyses, reaction kinetics, and chemical cross-linking studies were used to demonstrate that the monomer of MKP3 binds to the monomeric form of pERK and that MKP3 within the resulting heterodimer performs intramolecular dephosphorylation of pERK. This study provides the first direct evidence that MKP3 utilizes intramolecular dephosphorylation between a complex consisting of one molecule each of MKP3 and ERK. Catalytic activation and substrate tethering by MKP3 lead to a >or=4000-fold rate enhancement (k(cat)/K(m)) for dephosphorylation of pERK.


Assuntos
Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Tirosina Fosfatases/química , Proteínas Tirosina Fosfatases/metabolismo , Ligação Competitiva/genética , Catálise , Fosfatase 3 de Especificidade Dupla , Fosfatase 6 de Especificidade Dupla , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Proteínas Tirosina Fosfatases/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato/genética , Vaccinia virus/enzimologia , Vaccinia virus/genética
11.
Biochemistry ; 41(9): 3009-17, 2002 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-11863439

RESUMO

Human VHR (vaccinia H1 related phosphatase) is a member of the dual-specificity phosphatases (DSPs) that often act on bisphosphorylated protein substrates. Unlike most DSPs, VHR displays a strong preference for dephosphorylating phosphotyrosine residues over phosphothreonine residues. Here we describe the 2.75 A crystal structure of the C124S inactive VHR mutant in complex with a bisphosphorylated peptide corresponding to the MAP kinase activation lip. This structure and subsequent biochemical studies revealed the basis for the strong preference for hydrolyzing phosphotyrosine within bisphosphorylated substrates containing -pTXpY-. In the structure, the two phospho residues are oriented into distinct pockets; the phosphotyrosine is bound in the exposed yet deep active site cleft while the phosphothreonine is loosely tethered into a nearby basic pocket containing Arg(158). As this structure is the first substrate-enzyme complex reported for the DSP family of enzymes, these results provide the first glimpse into how DSPs bind their protein substrates.


Assuntos
Proteínas Quinases Ativadas por Mitógeno/química , Proteínas Tirosina Fosfatases/química , Sítios de Ligação , Cristalografia por Raios X , Fosfatase 3 de Especificidade Dupla , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Moleculares , Peptídeos/química , Fosforilação , Fosfotreonina/química , Conformação Proteica , Proteínas Tirosina Fosfatases/metabolismo , Especificidade por Substrato
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