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1.
Inflamm Res ; 50(8): 400-8, 2001 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-11556520

RESUMO

OBJECTIVE AND DESIGN: We have evaluated the effects of the broad-spectrum cysteine protease inhibitor E64 on allergic lung inflammation in the mouse ovalbumin model of human asthma. We have also characterised membrane-associated cathepsin enzyme activity on a range of cell types. MATERIALS: Balb/C mice, E64 and CA074, various cell lines. TREATMENT: E64 was administered by subcutaneous minipump into ovalbumin-sensitised mice prior to intranasal ovalbumin challenge. The effect of E64 on ovalbumin-induced inflammation in vivo and ovalbumin-specific T cell proliferation in vitro and ex vivo was examined. Membrane-associated cathepsin activity on various cell types was measured. RESULTS: E64 treatment (0.36-0.48 mg/day) led to a significant reduction in eosinophil numbers and lung weights in the mouse model. Histological examination of lungs confirmed the anti-inflammatory effect. E64 greatly reduced ovalbumin-specific T cell numbers in the lymph nodes draining the lung following intranasal challenge whilst an accumulation of these T cells was found in the 'priming' lymph nodes. An analysis of various cells involved in lymphocyte priming and migration revealed that monocytes, dendritic cells and endothelial cells express high levels of membrane-associated cathepsin B activity. CONCLUSIONS: Since E64 is not cell permeable and does not inhibit antigen-induced T cell proliferation in vitro or in vivo, the data indicate that membrane-associated cysteine proteases, possibly cathepsin B, may regulate T lymphocyte migration in vivo.


Assuntos
Alérgenos/farmacologia , Inibidores de Cisteína Proteinase/uso terapêutico , Pneumonia/tratamento farmacológico , Linfócitos T/efeitos dos fármacos , Animais , Catepsina B/metabolismo , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Feminino , Células-Tronco Hematopoéticas/efeitos dos fármacos , Leucina/análogos & derivados , Leucina/uso terapêutico , Pulmão/enzimologia , Pulmão/patologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Pneumonia/imunologia , Pneumonia/patologia
2.
Eur J Immunol ; 31(2): 440-9, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11180108

RESUMO

Engineered MHC-peptide targets capable of inducing recognition by CTL may prove useful in designing vaccines for infectious disease and cancer. We tested whether peptides directly linked to beta2-microglobulin (beta2m) could complex with human HLA class I heavy chain, and could be recognized by human CTL, both as soluble reagents and as cell surface constituents. An HLA-A2-restricted peptide epitope was physically linked to the N terminus of human beta2m. This fusion protein refolded efficiently in vitro with HLA-A2 heavy chain, and when multimerized, the resultant complexes ("fusamers") bound specifically to appropriate CTL clones. These fused peptide/MHC complexes were as efficient as standard tetrameric peptide/MHC complexes in recognizing antigen-specific CTL. When the fusion protein was delivered to target cells using a retroviral vector, these cells were recognized and killed by appropriate CTL clones. Efficient sensitization to CTL lysis was achieved in TAP-negative and beta2m-negative cell lines, as well as in unmutated B cell lines, proving that such constructs may be effective in inducing CTL even when the MHC class I pathway has been disrupted. Specific peptides covalently linked to beta2m and delivered via retroviral vectors may be useful reagents for in vivo priming of CTL against epitopes of clinical relevance.


Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/fisiologia , Neoplasias/terapia , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Citotóxicos/imunologia , Microglobulina beta-2/imunologia , Linhagem Celular , Citotoxicidade Imunológica , Terapia Genética , Antígenos de Histocompatibilidade Classe I/química , Humanos , Neoplasias/imunologia , Retroviridae/genética , Microglobulina beta-2/genética
3.
Vaccine ; 18(17): 1863-9, 2000 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-10699335

RESUMO

We characterized the immunogenicity of the hybrid Ty-virus-like carrying the CD8(+) T cell epitope (SYVPSAEQI) of the circumsporozoite (CS) protein of Plasmodium yoelii (TyCS-VLP), a rodent malaria parasite. Balb/c mice were immunized with hybrid TyCS-VLP, and their CS-specific CD8(+) T cell response was quantitatively evaluated with the ELISPOT assay, based on the enumeration of epitope specific gamma-interferon secreting CD8(+) T cell. A single immunization with the TyCS-VLP by a variety of routes and doses indicated that the maximal response occurred in mice, which were immunized with 50 micrograms of these particles, administered via intramuscular. Combined immunization of mice with this TyCS-VLP followed by recombinant vaccinia virus expressing the entire P. yoelii CS protein (VacPyCS) or irradiated sporozoites, induced high levels of IFN-gamma-producing cells. The immunization regime, priming with TyCS-VLP and boosting with VacPyCS generated a potent protective immune response, which strongly inhibited P. yoelii liver stages development and protected 62% of the mice against a subsequent live P. yoelii sporozoite challenge.


Assuntos
Antígenos de Protozoários/imunologia , Vacinas Antimaláricas/imunologia , Malária/prevenção & controle , Plasmodium yoelii/imunologia , Proteínas de Protozoários/imunologia , Retroelementos/imunologia , Vaccinia virus/imunologia , Animais , Antígenos de Protozoários/genética , Linfócitos T CD8-Positivos/imunologia , Relação Dose-Resposta Imunológica , Vias de Administração de Medicamentos , Epitopos/genética , Epitopos/imunologia , Feminino , Imunização Secundária , Memória Imunológica , Interferon gama/biossíntese , Fígado/imunologia , Fígado/metabolismo , Fígado/parasitologia , Malária/imunologia , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/genética , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium yoelii/genética , Plasmodium yoelii/crescimento & desenvolvimento , Proteínas de Protozoários/genética , RNA Ribossômico/biossíntese , Retroelementos/genética , Baço/citologia , Baço/imunologia , Baço/metabolismo , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Vaccinia virus/genética
4.
Int Immunol ; 11(12): 1927-34, 1999 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-10590258

RESUMO

It has been previously demonstrated that hybrid Ty virus-like particles (VLP) prime effective CD8(+) and CD4(+) T cell responses. In this study, we investigated the effect of treating mice with Ty VLP carrying the immunodominant epitope of Der p 1 after sensitizing them to the group 1 allergen of the house dust mite Dermatophagoides pteronyssinus (Der p 1), under conditions that induce T(h)2 immunity. We show that i.p. treatment with the hybrid VLP abrogated allergen-specific IL-5 production and reduced allergen-specific cell proliferation. This suppression of the response was mediated by CD4(+) T cells and was not accompanied by an increase in IFN-gamma production.


Assuntos
Alérgenos/imunologia , Linfócitos T CD4-Positivos/fisiologia , Epitopos de Linfócito T , Glicoproteínas/imunologia , Epitopos Imunodominantes/imunologia , Interferon gama/biossíntese , Interleucina-5/biossíntese , Animais , Antígenos de Dermatophagoides , Células Cultivadas , Regulação para Baixo , Feminino , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Ácaros/imunologia , Fragmentos de Peptídeos/farmacologia
5.
J Med Virol ; 59(1): 78-83, 1999 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10440812

RESUMO

Yeast Ty virus-like particles (VLPs) containing viral protein inserts have previously been shown to be potent immunogens, inducing both humoral and cell mediated immunity (CMI). The antigenicity of hybrid VLPs containing fragments of the varicella-zoster virus (VZV) gE protein or the assembly protein (AP) was assessed by lymphocyte proliferation. Peripheral blood mononuclear cells (PBMCs) from patients with a recent natural VZV infection were stimulated in vitro with VZV-VLPs together with control antigens. PBMC samples from both varicella (85%) and zoster (75%) patients proliferated in responses to at least one of the gE VZV-VLPs. As reported for the first time, VZV specific lymphocyte responses were also identified towards the VZV AP in two varicella and two zoster patient samples. The results demonstrate specific CMI recognition of the VZV gE fragments tested and the VZV AP delivered in the form of recombinant Ty-VLPs, and highlights their potential use as a recombinant antigen delivery system for vaccination.


Assuntos
Herpesvirus Humano 3/imunologia , Linfócitos T/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas Virais/imunologia , Vírion/imunologia , Adulto , Antígenos Virais/imunologia , Varicela/imunologia , Elementos de DNA Transponíveis/genética , Herpes Zoster/imunologia , Herpesvirus Humano 3/química , Humanos , Ativação Linfocitária , Pessoa de Meia-Idade , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/genética , Proteínas Virais/genética , Vírion/genética , Montagem de Vírus , Leveduras/genética
6.
Nat Biotechnol ; 15(12): 1280-4, 1997 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9359112

RESUMO

Ty virus-like particles consist of a single protein species that can be produced in yeast. Recombinant Ty-VLPs carrying a string of up to 15 defined cytotoxic T lymphocyte (CTL) epitopes from Plasmodium species prime protective CTL responses in mice following a single administration without adjuvant. Effective processing of epitopes from the string was demonstrated in vitro and in vivo and was not affected by flanking sequences.


Assuntos
Epitopos/química , Vacinas Antimaláricas/química , Plasmodium falciparum/imunologia , Sequência de Aminoácidos , Animais , Epitopos/imunologia , Feminino , Humanos , Vacinas Antimaláricas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Plasmodium berghei/imunologia , Linfócitos T Citotóxicos/imunologia
7.
Virus Res ; 49(2): 187-91, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9213393

RESUMO

The majority of condylomata acuminata (anogenital warts) are caused by infection with Human Papilloma Virus type 6 (HPV-6). We have sequenced the HPV-6 early genes, E1-E4, E6 and E7 from wart biopsy DNA samples sourced from the UK and USA and derived a consensus sequence for these genes and the proteins they encode. When compared to the prototype HPV-6b sequence, published over 12 years ago, the E1-E4 consensus sequence showed 3/91 (3.3%) amino acid changes, the E6 consensus sequence showed 1/150 (0.7%) changes and the E7 consensus sequence showed 1/98 (1.0%) changes. Since many of the early gene sequences from biopsy material were more similar to the HPV-6a subtype than HPV-6b, this data supports the use of HPV-6a as the HPV-6 prototype.


Assuntos
Deleção de Genes , Genes Virais , Papillomaviridae/genética , Proteínas Estruturais Virais/genética , Condiloma Acuminado/patologia , Condiloma Acuminado/virologia , Sequência Consenso , Análise Mutacional de DNA , DNA Viral/química , DNA Viral/genética , Humanos , Papillomaviridae/química , Reação em Cadeia da Polimerase , Proteínas Virais/genética
8.
Vaccine ; 15(6-7): 709-19, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9178473

RESUMO

During infection with Varicella-zoster virus (VZV), the envelope proteins are highly immunogenic and glycoprotein E (gE) is one of the most abundant and antigenic. We have previously identified the immunodominant regions of gE and mapped the B-cell epitopes. In this study, we have evaluated the immunogenicity of recombinant hybrid Ty-virus-like particles (VLPs) carrying amino acids (1-134) or (101-161) of gE which contain the immunodominant sequences. VZV-specific antibodies were detected by ELISA in sera from mice and guinea pigs immunized with either gE(1-134)-VLPs or gE (101-161)-VLPs. The dominant B-cell epitopes, mapped by pepscan analysis of the sera, were found in peptides spanning amino acids 41-60, 56-75, 101-120, 116-135, 131-150 and 141-161. These sera also showed neutralizing activity against VZV in vitro. Epitopes recognized by neutralizing MAbs were mapped to both gE sequences (3B3 MAb recognizing amino acids 141-161 and IFB9 MAb recognizing amino acids 71-90). Lymphocyte proliferative responses to VZV were detected in four different mouse strains immunized with either gE(1-134)-VLPs or gE(101-134)-VLPs in alum. All mouse strains immunized with gE(1-134)-VLPs recognized epitopes in amino acids 11-30 and 71-90 and all those immunized with gE(101-161)-VLPs recognized epitopes in amino acids 91-110 and 106-125. These results indicate that VLPs carrying these gE sequences can prime potent humoral and cellular anti-VZV responses in small animals and warrant further investigation as potential vaccine candidates against varicella-zoster infections.


Assuntos
Anticorpos Antivirais/imunologia , Herpesvirus Humano 3/imunologia , Retroelementos , Linfócitos T/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Mapeamento de Epitopos , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Cobaias , Epitopos Imunodominantes/imunologia , Masculino , Camundongos , Camundongos Endogâmicos , Dados de Sequência Molecular , Testes de Neutralização , Peptídeos/genética , Peptídeos/imunologia , Linfócitos T/citologia , Proteínas do Envelope Viral/genética
9.
Int Immunol ; 9(2): 273-80, 1997 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9040009

RESUMO

The group I (Der p 1) allergen of Dermatophagoides pteronyssinus (house dust mite, HDM) contains several T helper (Th) epitopes recognized by C57BL/6 mice, with the peptide (111-139) containing a dominant MHC class II-restricted epitope (113-127). Since CD8+ T cells are thought to play a role in the regulation of allergic disease, we examined the Der p 1 sequence for potential MHC class I-binding motifs and observed that residues 111-119 (FGISNYCQI) contain motifs for H-2Db and Kb. Furthermore, immunization of C57BL/6 mice with unadjuvanted Ty virus-like particles (VLP) carrying Der p 1 (111-139), a method known to induce murine cytotoxic T lymphocyte (CTL) responses, primed Der p 1 (111-119)-specific Db-restricted CTL which produce high levels of IFN-gamma and low levels of IL-5 and IL-6 in vitro (T1-type CTL). VLP carrying the minimal epitope (FGISNYCQI) also induced a CTL response following immunization without adjuvant by various routes. Der p 1 (111-139)-VLP adjuvanted with alum did not prime CTL in C57BL/6 mice but were found to prime Th1-type CD4+ T cells that recognize the overlapping peptide (113-127) and native protein. The ability to successfully predict allergen-specific CD8+ T cell epitopes and prime CD8+ and/or CD4+ T cell responses provides an opportunity to dissect the relative roles of these T cells in the regulation of allergic responses and may offer therapeutic potential for reprogramming Th2-type allergic responses.


Assuntos
Alérgenos/imunologia , Glicoproteínas/imunologia , Epitopos Imunodominantes/imunologia , Ativação Linfocitária , Ácaros/imunologia , Linfócitos T Citotóxicos/imunologia , Células Th1/imunologia , Administração Intranasal , Animais , Antígenos de Dermatophagoides , Antígenos CD5/imunologia , Antígenos CD8/imunologia , Citocinas/biossíntese , Testes Imunológicos de Citotoxicidade , Feminino , Genes MHC Classe I/imunologia , Epitopos Imunodominantes/classificação , Injeções Intramusculares , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos C57BL , Baço/citologia , Linfócitos T Citotóxicos/fisiologia , Células Th1/metabolismo
10.
J Med Virol ; 53(4): 332-9, 1997 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-9407380

RESUMO

Herpesviruses produce assembly proteins (AP) that act as scaffolding proteins for the assembly of the viral capsids. The products of the assemblin gene, which encodes both maturational protease and AP, have been established for herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (CMV). We cloned an inframe ORF (encoding amino acids 304-605), found within the ORF 33 assemblin gene of VZV, into a yeast expression vector. The 34-kDa AP was expressed as a fusion protein with the particle-forming Ty p1 protein, resulting in high-level production of hybrid AP-virus-like particles (AP-VLPs). When AP-VLPs were injected into mice and rabbits, antibodies were produced that reacted with, but that did not neutralise, native VZV. Three of four inbred strains of mice immunised with AP-VLPs produced a VZV-specific T-cell response. The mouse and rabbit sera reacted with six bands on native VZV by Western blot analysis. The dominant bands were found at 34 and 38 kDa. Bands were also seen at 66, 63, 41, and 31 kDa. The 38-kDa protein may represent the mature AP derived from the 41-kDa precursor AP, itself the release product from the full-length 66-kDa assemblin. The 34-kDa protein probably represents the product of the inframe co-translational gene within ORF 33 encoding amino acids 304-605. The genetic organisation and proteolytic maturation of VZV assemblin are, therefore, analogous to those of other herpesviruses.


Assuntos
Endopeptidases/genética , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/fisiologia , Serina Endopeptidases , Proteínas Virais/genética , Proteínas Virais/imunologia , Animais , Western Blotting , Linhagem Celular , Clonagem Molecular , Endopeptidases/análise , Feminino , Herpesvirus Humano 3/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fases de Leitura Aberta , Coelhos , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/imunologia , Montagem de Vírus
11.
Eur J Immunol ; 26(11): 2595-600, 1996 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-8921944

RESUMO

Previous reports have indicated that both dendritic cells and macrophages have the ability to induce cytotoxic T lymphocyte (CTL) and T helper (Th) cell responses in vivo. Dendritic cells process exogenous antigens conventionally for presentation on major histocompatibility complex (MHC) class II molecules. However, unconventional processing of exogenous antigens in vitro for presentation on MHC class I molecules is still an open question. In this study, we report that a cloned dendritic cell line (D2SC/1) is able to present cell debris-associated exogenous viral proteins to MHC class I-restricted CTL in vitro. The dendritic cell line was very efficient in processing recombinant lymphocytic choriomeningitis virus nucleoprotein (LCMV NP) and presenting the class I-restricted epitope to CTL primed in vivo. Peritoneal macrophages could also process the recombinant LCMV NP for subsequent MHC class I presentation, but were less efficient compared to the dendritic cells. Furthermore, recombinant yeast-derived virus-like particles carrying the HIV-1 V3 loop (V3-VLP), which are protenaceous and do not contain any lipid, were also found to be efficiently processed by the dendritic cell line for presentation of the class I-restricted epitope. These results clearly indicate that viral proteins, in particulate form or associated with cell debris, are processed by dendritic cells for CTL induction.


Assuntos
Apresentação de Antígeno/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Antígenos H-2/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Células Cultivadas , Células Dendríticas/virologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular
12.
Vaccine ; 14(10): 971-6, 1996 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-8873390

RESUMO

We have previously described the generation of HIV-1 V3-specific cytotoxic T-lymphocytes (CTL) responses in BALB/c (H-2d) mice following immunization with Ty-virus-like particles carrying the V3 loop of gp120 (V3-VLPs) without adjuvant. In this study the effects of various adjuvants on CTL induction by V3-VLPs was examined. Mice immunized with V3-VLPs formulated in aqueous-based adjuvants, Detox, gamma-inulin, galactosaminylmuramyl dipeptide and Chemivax generated V3-specific CTL responses, although at reduced levels when compared to the no adjuvant group. V3-VLPs prepared in Alhydrogel, algamulin or as an oil emulsion in SAF-MF failed to generate V3-specific CTL responses. The mechanism whereby alum prevented the induction of a CTL response was investigated further. Immunization with V3-VLPs prepared in non-saturating doses of alum or alum plus EDTA primed for strong CTL responses, indicating that free VLPs do, but alum-bound VLPs do not enter the MHC class I processing pathway of antigen-presenting cells (APCs). Furthermore, V3-VLPs with very low doses of alum led to an enhancement of the CTL response. The formulation of hybrid Ty-VLPs in oil based or precipitating adjuvants, therefore, inhibits access to the MHC class I processing pathway of APCs. The intact particulate structure of hybrid VLPs is therefore strictly necessary for CTL induction.


Assuntos
Adjuvantes Imunológicos/farmacologia , Compostos de Alúmen/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Vírion/imunologia , Sequência de Aminoácidos , Animais , Linfonodos/citologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Linfócitos T Citotóxicos/imunologia
13.
Immunology ; 87(2): 171-8, 1996 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-8698376

RESUMO

The induction of cytotoxic T-lymphocyte (CTL) responses to viral proteins is thought to be an essential component of protective immunity against viral infections. Methods for generating such responses in a reproducible manner would be of great value in vaccine development. We demonstrate here that the recombinant antigen-presentation system based on the yeast transposon (Ty) particle-forming p1 protein is a potent means of inducing CTL responses to a variety of viral CTL epitopes, including influenza virus nucleoprotein (two epitopes), Sendai virus and vesicular stomatitis virus nucleoproteins, and the V3 loop of human immunodeficiency virus type-1 (HIV-1) gp120. CTL were primed by hybrid Ty-virus-like particles (VLP) carrying the minimal epitope or as much as 19,000 MW of protein. Ty-VLP carrying two different epitopes (dual-epitope Ty-VLP) were capable of priming CTL responses in two different strains of mice or against two epitopes in the same individual. Furthermore, co-administration of a mixture of two different Ty-VLP carrying single epitopes could induce responses to both epitopes in the same individual. Ty-VLP appear to represent a reproducible and flexible system for inducing CTL responses in mice, and warrant further evaluation in primates.


Assuntos
Antígenos Virais/imunologia , Elementos de DNA Transponíveis/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Nucleoproteínas/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas de Ligação a RNA , Linfócitos T Citotóxicos/imunologia , Proteínas do Core Viral/imunologia , Sequência de Aminoácidos , Animais , Citotoxicidade Imunológica , Epitopos/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo , Proteínas Recombinantes/imunologia
14.
Virology ; 214(2): 531-40, 1995 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-8553555

RESUMO

The envelope proteins of varicella-zoster virus (VZV) are highly immunogenic and one of the most abundant is glycoprotein E (gE). However, its immunodominant regions and epitopes have not been identified. In this study, using human sera from individuals with recent varicella or zoster infections, we have localized antigenic sequences of gE using recombinant hybrid Ty-virus-like particles (VLPs) carrying overlapping fragments of the gE protein. gE(1-134)-VLPs (particles carrying amino acids 1-134 of gE) and, to a lesser extent, gE(101-161)-VLPs were found to be the most antigenic when tested by Western blotting and ELISA. Other fragments of gE (spanning residues 161-623) showed weak or no antigenicity. Pepscan analysis of human sera on overlapping synthetic peptides representing residues 1-135 of gE revealed that the most antigenic region was between residues 50 and 135. Three immunodominant sequences (residues 86-105, 116-135, and, to a lesser extent, 56-75) were detected using sera from both varicella and zoster patients. All sera from varicella, but not zoster, patients reacted strongly with an epitope in peptide 66-85. Other epitopes were recognized weakly by some varicella or zoster sera. More sera need to be tested to assess the potential disease specificity of these epitopes. The neutralizing monoclonal antibody (MAb) IF-B9 reacted with residues 71-90; however, another neutralizing MAb, SG1A, which bound to both gE(1-134)-VLPs and gE(101-161)-VLPs did not bind to any peptide. The identification of immunodominant sequences of gE will help toward the development of a subunit VZV vaccine.


Assuntos
Epitopos de Linfócito B/imunologia , Herpesvirus Humano 3/isolamento & purificação , Epitopos Imunodominantes/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Sítios de Ligação , Varicela/sangue , Varicela/imunologia , Mapeamento de Epitopos , Herpes Zoster/sangue , Herpes Zoster/imunologia , Herpesvirus Humano 3/imunologia , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/imunologia , Relação Estrutura-Atividade , Proteínas do Envelope Viral/genética
15.
Ann N Y Acad Sci ; 754: 202-13, 1995 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-7625653

RESUMO

The development of technologies to produce recombinant proteins for use in the pharmaceutical industry has made substantial advances, in particular in the area of generating antigens containing multiple copies of important immunological regions. One such antigen-carrier system is based on the ability of a protein encoded by the yeast retrotransposon, Ty, to self-assemble into virus-like particles. Ty-fusion proteins retain this ability to form particles, and a range of hybrid VLPs carrying a variety of heterologous antigens have been produced and shown to induce potent immune responses. In particular, hybrid VLPs carrying the core protein p24 of HIV (p24-VLPs) have been shown to induce antibody and T-cell proliferative responses in both experimental animals and human volunteers, and immunization of rabbits with VLPs carrying the principal neutralizing determinant of HIV (V3-VLPs) resulted in the induction of neutralizing antibody responses and T-cell proliferation. Further studies with V3-VLPs have shown that this particulate antigen stimulates enhanced V3-specific lymphoproliferative responses as compared to whole recombinant gp120 or to V3 peptide conjugated to albumin. The V3-VLPs also induce potent CTL responses following immunization of mice in the absence of adjuvant. These responses are MHC class I restricted and are mediated by CD8-positive cells. These observations therefore demonstrate that hybrid Ty-VLPs induce both humoral and cellular immune responses against HIV and suggest that these immunogens may be important in combatting AIDS and other infections.


Assuntos
Vacinas contra a AIDS/química , Proteínas Fúngicas/imunologia , Retroelementos/imunologia , Saccharomyces cerevisiae/imunologia , Vacinas Sintéticas/química , Vacinas contra a AIDS/imunologia , Animais , Citotoxicidade Imunológica , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T Citotóxicos/imunologia , Vacinas Sintéticas/imunologia
16.
Int Rev Immunol ; 11(2): 133-41, 1994.
Artigo em Inglês | MEDLINE | ID: mdl-8046274

RESUMO

Vaccines need to activate antigen presenting cells, overcome genetic restriction in T-cell responses and elicit both T and B memory cells. In order to produce recombinant vaccines which can do this, considerable effort has been put into developing particulate antigen presentation systems to generate polyvalent, high molecular weight antigens which should maximally stimulate the immune system. One such antigen-carrier system is based on the ability of a protein encoded by the yeast retrotransposon, Ty, to self-assemble into virus-like particles (VLPs). Ty-fusion proteins retain this ability to form particles and a range of hybrid VLPs carrying a variety of heterologous antigens have been produced and shown to elicit potent immune responses. Hybrid VLPs carrying human immunodeficiency virus (HIV) antigens stimulate the three main components of the immune system, namely antibody synthesis, T-cell proliferative responses and cytotoxic T-lymphocyte (CTL) responses.


Assuntos
Elementos de DNA Transponíveis , Proteínas Fúngicas/imunologia , Vetores Genéticos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Saccharomyces cerevisiae/genética , Proteínas Virais/imunologia , Animais , Apresentação de Antígeno/imunologia , Linfócitos B/imunologia , Proteínas Recombinantes de Fusão/fisiologia , Linfócitos T Auxiliares-Indutores/imunologia
17.
J Immunol ; 151(2): 1097-107, 1993 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-8335892

RESUMO

In general, it has proven difficult to induce CTL responses using simple proteins or peptides without resorting to specialized adjuvants. In this study we show that particulate polymeric Ag in the form of hybrid Ty virus-like particles carrying the V3 region of HIV-1 gp120/160 envelope protein (V3:Ty-VLP) induce V3-specific CTL in BALB/c mice in the absence of adjuvant or lipid vehicle. In vitro restimulation of splenocytes with V3 peptide was necessary in order to generate effector CTL. Th cell activation was not required for this in vitro restimulation phase. The CTL induced by the V3:Ty-VLP were CD8+ve, H-2d-restricted, and HIV-1 isolate-specific (IIIB or MN). Co-administration of IIIB V3:Ty-VLP and MN V3:Ty-VLP primed both IIIB and MN V3-specific CTL. However, only IIIB V3-specific CTL were primed by hybrid Ty-VLP carrying IIIB, MN, and RF V3 loop sequences on the same particle indicating that there is intra- but not intermolecular competition between CTL epitopes. In direct comparisons, V3:Ty-VLP were substantially more potent than rgp120. Rgp160 and a 40mer IIIB V3 peptide both failed to prime V3-specific CTL. These data suggest that the particulate nature of hybrid Ty-VLP facilitates uptake into APC with subsequent access to the MHC class I processing pathway and that they may be useful vaccine vehicles for inducing cytolytic immunity against HIV-1 and other intracellular pathogens.


Assuntos
Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Citotóxicos/imunologia , Sequência de Aminoácidos , Animais , Antígenos CD8/análise , Feminino , Antígenos H-2/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular
18.
J Virol ; 67(6): 3191-8, 1993 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8497047

RESUMO

In attempts to increase the immunogenicity of recombinant antigens, a number of particulate antigen presentation systems have been developed. In this study, we used human immunodeficiency virus Gag particles as carriers for the human immunodeficiency virus envelope V3 region. Gag:V3 fusion proteins were expressed from baculovirus expression vectors; they migrated to the insect cell membrane and budded from the cells as hybrid particles. An immunization study carried out with rats showed that the particles elicited a strong anti-Gag antibody response and a weak antibody response to the V3 region. A strong anti-V3 cytolytic T-cell response was elicited in immunized mice. These data show that retroviral Gag particles can be used as antigen presentation vehicles.


Assuntos
Síndrome da Imunodeficiência Adquirida/imunologia , Produtos do Gene env/imunologia , Produtos do Gene gag/imunologia , Anticorpos Anti-HIV/biossíntese , HIV/imunologia , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Sequência de Bases , Citotoxicidade Imunológica , Portadores de Fármacos , Produtos do Gene env/genética , Produtos do Gene gag/genética , HIV/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mariposas , Proteínas Recombinantes de Fusão/imunologia
19.
Immunology ; 77(3): 315-21, 1992 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-1362183

RESUMO

The induction of CD4+ T-helper (Th) cell responses is likely to be an important requirement of vaccine candidates designed to prevent or moderate human immunodeficiency virus-1 (HIV-1) infection. We have investigated the ability of hybrid Ty virus-like particles carrying the V3 loop region of the HIV-1 IIIB envelope gp120 (V3:Ty-VLP) to elicit V3-specific proliferative responses. Significant proliferation in response to stimulation in vitro with homologous IIIB V3 peptide was observed following immunization of mice with V3:Ty-VLP either as an aluminium hydroxide precipitate or without adjuvant. Responses to MN V3 peptide were also observed in certain mouse haplotypes. To assess the effect of presenting the V3 loop in this particulate form, we compared the responses induced by V3:Ty-VLP with those obtained with two non-particulate immunogens, recombinant gp120 (rgp120) and V3 peptide conjugated to albumin. V3-specific responses to V3 peptide in vitro were reproducibly higher following immunization with V3:Ty-VLP than with either rgp120 or V3-albumin coagulate (V3-alb). The data indicate that immunization with the V3 loop as a hybrid Ty-VLP results in enhanced proliferative responses to V3 peptide and recognition of rgp120 in vitro. Some cross-reactivity of Th cells for V3 sequences from different isolates was also observed.


Assuntos
Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Imunização , Fragmentos de Peptídeos/imunologia , Adjuvantes Imunológicos , Animais , Linfócitos T CD4-Positivos/imunologia , Divisão Celular/imunologia , Células Cultivadas , Relação Dose-Resposta Imunológica , Feminino , Interleucina-2/biossíntese , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos
20.
J Natl Cancer Inst ; 84(9): 699-703, 1992 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-1569603

RESUMO

BACKGROUND: Antitumor antibodies have traditionally been made to whole tumors or tumor extract. The use of defined synthetic antigens would be desirable for producing monoclonal antibodies. PURPOSE: Our purpose was to determine if antipeptide antibody to MUC2 had antitumor activity and specificity. METHODS: A 29-amino-acid peptide to MUC2 was synthesized and monoclonal antibodies were produced after immunizing BALB/c mice with peptide-keyhole-limpet hemocyanin in complete Freund's adjuvant, and the monoclonal antibodies were tested on peptides and human tissues. RESULTS: CCP31, CCP37, and CCP58 monoclonal antibodies were produced using MUC2 MI-29 (KYPTTTPISTTTMVTPTPTPTGTQTPTTT) containing one repeat unit of 23 amino acids and part of the next repeat of four amino acids. These antibodies reacted with the MUC2-derived peptide but not with MUC1- or MUC3-derived peptides. One of the monoclonal antibodies, CCP58, reacted strongly with human colon cancer and normal intestine in both fresh and formalin-fixed tissues; two other antibodies, CCP37 and CCP31, reacted only with fresh human tissues of normal colon and malignant colon tumors by immunoperoxidase staining. In addition, CCP37 and CCP58 reacted strongly with human gastric cancer; all antibodies reacted weakly with human salivary gland, and none reacted with tissues from normal human lung, kidney, stomach, pancreas, or endometrium. By analysis of mucin molecules by Western blotting, the antigen detected by monoclonal antibodies CCP37 and CCP58 was found to be of a high relative molecular mass (520 kd). CONCLUSIONS: Anti-MUC2 peptide antibodies appear to be relatively tissue specific and represent a new method of producing antitumor antibodies.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Neoplasias do Colo/imunologia , Mucinas/imunologia , Sequência de Aminoácidos , Especificidade de Anticorpos , Humanos , Técnicas Imunoenzimáticas , Dados de Sequência Molecular , Peso Molecular , Mucinas/química , Peptídeos/química , Peptídeos/imunologia , Neoplasias Gástricas/imunologia
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