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1.
J Pharm Sci ; 111(4): 1070-1080, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35122832

RESUMO

Most influenza vaccines are administered via intramuscular injection which has several disadvantages that might jeopardize the compliance of vaccinees. Intradermal administration of dissolving-microneedle-arrays (dMNAs) could serve as minimal invasive alternative to needle injections. However, during the production process of dMNAs antigens are subjected to several stresses, which may reduce their potency. Moreover, the needles need to have sufficient mechanical strength to penetrate the skin and subsequently dissolve effectively to release the incorporated antigen. Here, we investigated whether blends of trehalose and pullulan are suitable for the production of stable dMNA fulfilling these criteria. Our results demonstrate that production of trehalose/pullulan-based dMNAs rendered microneedles that were sharp and stiff enough to pierce into ex vivo human skin and subsequently dissolve within 15 min. The mechanical properties of the dMNAs were maintained well even after four weeks of storage at temperatures up to 37°C. In addition, immunization of mice with influenza antigens via both freshly prepared dMNAs and dMNAs after storage (four weeks at 4°C or 37°C) resulted in antibody titers of similar magnitude as found in intramuscularly injected mice and partially protected mice from influenza virus infection. Altogether, our results demonstrate the potential of trehalose/pullulan-based dMNAs as alternative dosage form for influenza vaccination.


Assuntos
Vacinas contra Influenza , Influenza Humana , Administração Cutânea , Animais , Antígenos , Glucanos , Humanos , Influenza Humana/prevenção & controle , Camundongos , Agulhas , Trealose , Vacinação/métodos
2.
Vaccine ; 28(50): 7963-9, 2010 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-20946860

RESUMO

Gram-positive enhancer matrix (GEM) particles, produced from non-genetically modified Lactococcus lactis bacteria have an inherent immunostimulatory activity. It was investigated whether co-administration of GEM particles can reduce the amount of influenza subunit vaccine (HA) necessary to protect mice from viral infection. Decreasing HA amounts of 5, 1, 0.2 and 0.04µg admixed with GEM particles were tested in intramuscular immunizations. Combinations of GEM and seasonal HA (A/Wisconsin/67/2005 [H3N2]) induced significantly higher systemic and better Th1/Th2-type balanced immune responses than HA alone. Addition of GEM to 0.04µg HA resulted in similar HI titers as 1-5µg non-adjuvanted HA. To test the protective efficacy of the adjuvanted combination, mice were immunized with influenza subunit vaccine A/PR/8/34 (H1N1) and then challenged with live virus (A/PR/8/34). Mice immunized with 1µg HA+GEM showed undetectable virus titers in the lungs 5 days after challenge, whereas mice immunized with 1µg HA alone showed detectable levels of virus in the lungs. Interestingly, mice vaccinated with the 0.04µg HA+GEM vaccine demonstrated reduced lung virus titers and a reduction in weight that was similar as that in mice vaccinated with 1µg non-adjuvanted HA. These results indicate that the use of GEM as immunostimulant allows for a strong reduction in the antigen dose as compared to the benchmark vaccine by using GEM particles. Thus, addition of GEM can strongly potentiate immunogenicity of influenza subunit vaccine both quantitatively and qualitatively.


Assuntos
Adjuvantes Imunológicos/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunização , Vacinas contra Influenza/administração & dosagem , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Anticorpos Antivirais/sangue , Relação Dose-Resposta Imunológica , Testes de Hemaglutinação , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/imunologia , Injeções Intramusculares , Lactococcus lactis/química , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Carga Viral
3.
Eur J Pharm Biopharm ; 76(3): 470-4, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20719246

RESUMO

In this study, a liquid formulation of influenza subunit vaccine admixed with Gram-positive enhancer matrix (GEM) particles as adjuvant was delivered to upper and lower parts of intestinal tract. The aim was to determine the most effective immunization site in the intestines. Mice were vaccinated with a liquid formulation of GEM and influenza subunit vaccine orally and rectally. The oral administration of the vaccine with GEM particles induced a better systemic and mucosal immune response than oral (vaccine only) and rectal (with and without adjuvant) immunizations. Rectal administration elicited high IgG1 responses but little IgG2a, indicating a Th2 dominated immune response. In contrast, the oral immunization with GEM particles elicited a balanced IgG1 and IgG2a response. In conclusion, our results demonstrate that GEM-adjuvanted influenza vaccine should be targeted to the upper part of the intestinal tract.


Assuntos
Adjuvantes Imunológicos , Anticorpos Antivirais/sangue , Trato Gastrointestinal/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Lactococcus lactis/imunologia , Administração Oral , Administração Retal , Animais , Imunidade nas Mucosas , Imunização , Imunoglobulina G/sangue , Camundongos , Camundongos Endogâmicos BALB C , Vacinação , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia
4.
J Control Release ; 136(1): 71-8, 2009 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-19331846

RESUMO

Transcutaneous immunization (TCI) is limited by poor permeation of macromolecules across the skin. Microneedle arrays form transient conduits and enhance the transport of vaccine molecules across the skin barrier without pain sensation. Here we investigated in mouse the immune responses after TCI using two model antigens, diphtheria toxoid (DT) and influenza subunit vaccine. The electric applicator enabled shorter microneedle (300 microm) to pierce mouse skin effectively, as shown by Trypan blue staining and trans-epidermal water loss measurement. The vaccines were topically applied with and without cholera toxin (CT) on microneedle-treated skin. In DT TCI, microneedle array pretreatment of the skin was essential to achieve substantial IgG and toxin-neutralizing antibody titers. Addition of CT further boosted the immune response to similar levels as observed after subcutaneous injection of AlPO4-adsorbed DT (DT-alum). In contrast, microneedle array pretreatment showed no effect on the immune response to plain influenza vaccine. This response was strongly improved by inclusion of CT, independent of microneedle treatment. These results indicate that TCI of DT and CT with microneedle treatment results in comparable protection as injection of DT-alum, and TCI of influenza vaccine adjuvanted with CT is superior to the injection of plain vaccine.


Assuntos
Toxoide Diftérico/administração & dosagem , Imunização/instrumentação , Vírus da Influenza A Subtipo H3N2/imunologia , Vacinas contra Influenza/administração & dosagem , Administração Cutânea , Animais , Toxina da Cólera/administração & dosagem , Toxina da Cólera/imunologia , Difteria/prevenção & controle , Toxoide Diftérico/imunologia , Feminino , Humanos , Imunoglobulina G/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C
5.
Ann Rheum Dis ; 68(6): 873-8, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18625625

RESUMO

OBJECTIVES: Wegener's granulomatosis (WG) is a systemic vasculitis characterised by relapsing and remitting disease activity. Immunosuppressive drugs are used to control disease, but increase susceptibility to infection. Therefore, influenza vaccination should be considered in WG patients. This study was performed to assess the immunogenicity of influenza vaccination in WG patients. METHODS: A randomised, controlled trial was performed in WG patients with quiescent disease, defined as a Birmingham vasculitis activity score (BVAS) less than 2. Patients were randomly assigned to receive influenza vaccination (n = 49) or to participate as controls (n = 23). In addition, healthy controls (n = 49) were vaccinated. At entry and at 1 and 3-4 months after entry, antibody responses to vaccination were determined. Furthermore, disease activity was measured (BVAS), adverse effects were recorded and antineutrophil cytoplasmic autoantibody (ANCA) titres were determined. RESULTS: WG patients achieved high seroprotection rates to all three influenza strains, comparable with healthy controls. Only the A/H1N1 strain patients had a lower seroconversion rate (p = 0.002) and geometric mean titre (p = 0.037) than controls. After 1 month, one control and one vaccinated WG patient had developed active disease. At 3-4 months, two additional control patients had developed active disease compared with none of the vaccinated patients (p = 0.099). Vaccination did not influence ANCA titres. Adverse effects did not differ between patients and healthy controls. CONCLUSIONS: Influenza vaccination in WG patients with quiescent disease induced a sufficient antibody response. TRIAL REGISTRATION NUMBER: NTR1130.


Assuntos
Anticorpos Antivirais/sangue , Granulomatose com Poliangiite/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Orthomyxoviridae/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Anticitoplasma de Neutrófilos/sangue , Estudos de Casos e Controles , Distribuição de Qui-Quadrado , Feminino , Granulomatose com Poliangiite/tratamento farmacológico , Humanos , Imunossupressores/uso terapêutico , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/efeitos adversos , Influenza Humana/prevenção & controle , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Resultado do Tratamento , Vasculite/imunologia
6.
Vaccine ; 27(6): 947-55, 2009 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-19059296

RESUMO

The increasing number of human influenza H5N1 infections accentuates the need for the development of H5N1 vaccine candidates to prevent a potential influenza pandemic. The use of adjuvants in such vaccines can contribute significantly to antigen dose-sparing. In this study, we evaluated the capacity of the non-toxic Neisseria meningitidis lipopolysaccharide analog LpxL1 to function as an adjuvant for an influenza H5N1 virosomal vaccine. Inactivated influenza H5N1 virus (NIBRG-14) was used to construct virosomes (reconstituted virus envelopes) with LpxL1 incorporated in the virosomal membrane thus combining the influenza hemagglutinin (HA) antigen and the adjuvant in the same particle. Mice were immunized in a one- or two-dose immunization regimen with H5N1 virosomes with or without incorporated LpxL1. After a single immunization, H5N1 virosomes with incorporated LpxL1 induced significantly enhanced H5N1-specific total IgG titers as compared to non-adjuvanted virosomes but hemagglutination inhibition (HI) titers remained low. In the two-dose immunization regimen, LpxL1-modified H5N1 virosomes induced HI titers above 40 which were significantly higher than those obtained with non-adjuvanted virosomes. Incorporation of LpxL1 had little effect on virosome-induced IgG1 levels, but significantly increased IgG2a levels in both the one- and two-dose immunization regimen. Compared to non-adjuvanted virosomes, LpxL1-modified virosomes induced similar numbers of IFNgamma-producing T cells but decreased numbers of IL-4-producing T cells irrespective of the number of immunizations. We conclude that LpxL1 incorporated in H5N1 influenza virosomes has the capacity to function as a potent adjuvant particularly stimulating Th1-type immune reactions.


Assuntos
Adjuvantes Imunológicos/farmacologia , Virus da Influenza A Subtipo H5N1/imunologia , Vacinas contra Influenza/imunologia , Lipopolissacarídeos/farmacologia , Animais , Anticorpos Antivirais/sangue , Feminino , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunização Secundária , Imunoglobulina G/sangue , Interferon gama/metabolismo , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Neisseria meningitidis/química , Linfócitos T/imunologia , Vacinas Virossomais/imunologia
7.
Pharm Res ; 25(6): 1256-73, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18338241

RESUMO

Influenza vaccination represents the cornerstone of influenza prevention. However, today all influenza vaccines are formulated as liquids that are unstable at ambient temperatures and have to be stored and distributed under refrigeration. In order to stabilize influenza vaccines, they can be brought into the dry state using suitable excipients, stabilizers and drying processes. The resulting stable influenza vaccine powder is independent of cold-chain facilities. This can be attractive for the integration of the vaccine logistics with general drug distribution in Western as well as developing countries. In addition, a stockpile of stable vaccine formulations of potential vaccines against pandemic viruses can provide an immediate availability and simple distribution of vaccine in a pandemic outbreak. Finally, in the development of new needle-free dosage forms, dry and stable influenza vaccine powder formulations can facilitate new or improved targeting strategies for the vaccine compound. This review represents the current status of dry stable inactivated influenza vaccine development. Attention is given to the different influenza vaccine types (i.e. whole inactivated virus, split, subunit or virosomal vaccine), the rationale and need for stabilized influenza vaccines, drying methods by which influenza vaccines can be stabilized (i.e. lyophilization, spray drying, spray-freeze drying, vacuum drying or supercritical fluid drying), the current status of dry influenza vaccine development and the challenges for ultimate market introduction of a stable and effective dry-powder influenza vaccine.


Assuntos
Vacinas contra Influenza/administração & dosagem , Estabilidade de Medicamentos , Liofilização , Humanos , Vacinas contra Influenza/química , Vacinas contra Influenza/imunologia , Pós , Vacinas Atenuadas/imunologia
8.
Vaccine ; 26(1): 67-76, 2007 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-18061315

RESUMO

In this paper we investigated to which part of the gastro-intestinal (GI) tract, the upper or lower part, an oral influenza vaccine should be targeted to result in an effective immune response in mice. Our study demonstrates that without adjuvant substantial systemic but low respiratory mucosal immune responses were induced in mice after delivery of influenza subunit vaccine to the upper GI-tract (intragastric) as well as the lower GI-tract (intracolonically). When the vaccine was adjuvanted with Escherichia coli heat-labile enterotoxin (LT) these responses were significantly enhanced. Interestingly, intracolonic administration of vaccine with adjuvant also resulted in enhanced cellular immune responses and the desired Th1-skewing of these responses. Intragastric administration of the adjuvanted vaccine also increased T-helper responses. However, Th1-skewing was absent. In conclusion, the right combination of strong mucosal adjuvant (e.g. LT) and antigen delivery site (e.g. the lower part of the gastro-intestinal tract) might result in effective vaccination via the oral route.


Assuntos
Vacinas contra Influenza/administração & dosagem , Administração Oral , Animais , Anticorpos Antivirais/sangue , Toxinas Bacterianas/administração & dosagem , Colo/imunologia , Enterotoxinas/administração & dosagem , Proteínas de Escherichia coli/administração & dosagem , Feminino , Imunização , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Vacinas contra Influenza/imunologia , Interferon gama/biossíntese , Interleucina-2/biossíntese , Interleucina-4/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Estômago/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem
9.
Vaccine ; 25(52): 8707-17, 2007 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-17996993

RESUMO

In this study pulmonary vaccination with a new influenza subunit vaccine powder was evaluated. Vaccine powder was produced by spray-freeze drying (SFD) using the oligosaccharide inulin as stabilizer. Immune responses after pulmonary vaccination of BALB/c mice with vaccine powder were determined and compared to those induced by intramuscular and pulmonary vaccination with a conventional liquid subunit vaccine. All vaccinations were performed without adjuvant. Pulmonary vaccination with liquid subunit vaccine resulted in systemic humoral (IgG) immune responses similar to intramuscular immunization. In contrast, the vaccine powder delivered by the pulmonary route, induced not only systemic humoral (IgG) responses, but also cell-mediated (Il-4, IFN-gamma) and mucosal immune responses (IgA, IgG). This study demonstrates that the combination of pulmonary antigen delivery and antigen powder production by SFD improves the immunogenic potential of (influenza subunit) antigen. In conclusion, vaccination with a non-adjuvanted SFD subunit vaccine powder by inhalation might be feasible and could be an alternative to conventional parenteral vaccine administration.


Assuntos
Administração por Inalação , Aerossóis/administração & dosagem , Crioprotetores/administração & dosagem , Vacinas contra Influenza/imunologia , Inulina/administração & dosagem , Animais , Anticorpos Antivirais/sangue , Líquido da Lavagem Broncoalveolar/química , Feminino , Liofilização , Testes de Inibição da Hemaglutinação , Imunoglobulina A/análise , Imunoglobulina A/sangue , Imunoglobulina G/análise , Imunoglobulina G/sangue , Vacinas contra Influenza/administração & dosagem , Interferon gama/biossíntese , Interleucina-4/biossíntese , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Líquido da Lavagem Nasal/química , Baço/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia
10.
Vaccine ; 25(35): 6447-57, 2007 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-17673338

RESUMO

The development of a stable influenza subunit vaccine in the dry state was investigated. The influence of various carbohydrates, buffer types and freezing rates on the integrity of haemagglutinin after freeze-thawing or freeze-drying was investigated with a range of analytical and immunological methods. The use of fast freezing, Hepes buffer and carbohydrates (trehalose, inulin or dextran) as cryo- and lyoprotectants resulted in a significant reduction or even absence of conformational changes of HA as revealed by the used methods. The subunit vaccine in the powder was shown to remain immunogenic in an in vivo study in mice, using reconstituted powder. Moreover, the HA potency of the influenza subunit vaccine powder was stable for at least 26 weeks at room temperature.


Assuntos
Hemaglutininas/química , Hemaglutininas/imunologia , Vacinas contra Influenza/imunologia , Animais , Antígenos/imunologia , Carboidratos/química , Dicroísmo Circular , Diálise , Desenho de Fármacos , Ensaio de Imunoadsorção Enzimática , Liofilização , Congelamento , Testes de Inibição da Hemaglutinação , Concentração de Íons de Hidrogênio , Hidrólise , Imunodifusão , Vírus da Influenza A Subtipo H3N2/imunologia , Camundongos , Pós , Conformação Proteica , Espectrometria de Fluorescência , Tripsina/química
11.
Ann Rheum Dis ; 65(7): 913-8, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16322083

RESUMO

OBJECTIVE: to assess the safety and efficacy of influenza vaccination in patients with systemic lupus erythematosus (SLE), and to evaluate the influence of immunosuppressive drugs on the immune response. METHODS: SLE patients (n=56) and healthy controls (n=18) were studied. All patients had quiescent disease (SLE disease activity indexor=40 against A/H3N2 (p=0.030) compared with the other patient groups. CONCLUSIONS: Influenza vaccination in SLE patients with quiescent disease is safe but is less effective than in controls. Use of azathioprine was associated with a trend to decreased vaccination efficacy.


Assuntos
Vacinas contra Influenza/administração & dosagem , Lúpus Eritematoso Sistêmico/imunologia , Adulto , Idoso , Anticorpos Antivirais/sangue , Azatioprina/uso terapêutico , Estudos de Casos e Controles , Feminino , Testes de Inibição da Hemaglutinação , Humanos , Hidroxicloroquina/uso terapêutico , Imunossupressores/uso terapêutico , Vacinas contra Influenza/efeitos adversos , Vacinas contra Influenza/imunologia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Masculino , Pessoa de Meia-Idade , Orthomyxoviridae/imunologia , Prednisona/uso terapêutico , Estatísticas não Paramétricas , Vacinação/efeitos adversos
12.
Gene Ther ; 13(5): 400-11, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16267567

RESUMO

Application of RNA interference for in vivo evaluation of gene function or for therapeutic interventions has been hampered by a lack of suitable delivery methods for small interfering RNA (siRNA). Here, we present reconstituted viral envelopes (virosomes) derived from influenza virus as suitable vehicles for in vitro as well as in vivo delivery of siRNAs. Virosomes are vesicles that bear in their membrane the influenza virus spike protein hemagglutinin (HA). This protein mediates binding of native virus to and fusion with cellular target membranes. Accordingly, virosomes with membrane-incorporated HA bind to cells, are taken up by receptor-mediated endocytosis, and fuse with the endosomal membrane to release their contents into the cytoplasm. When complexed to cationic lipids, siRNA was successfully encapsulated in virosomes. Virosomes with encapsulated siRNA fused with target membranes in a pH-dependent manner and delivered the encapsulated siRNA to several cell lines in vitro. Virosome-delivered siRNA markedly downregulated the synthesis of newly induced and constitutively expressed green fluorescent protein. Moreover, intraperitoneal injection of siRNA-loaded virosomes resulted in delivery of the nucleotides to cells in the peritoneal cavity. Our results indicate that virosomes are a promising delivery device for in vivo application, especially where topical administration of siRNA, for example, to the respiratory tract is envisaged.


Assuntos
Terapia Genética/métodos , Orthomyxoviridae , RNA Interferente Pequeno/genética , Virossomos/administração & dosagem , Animais , Linhagem Celular , Cricetinae , Citometria de Fluxo , Expressão Gênica , Engenharia Genética , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Injeções Intraperitoneais , Camundongos , Microscopia Eletrônica , Virologia/métodos , Virossomos/ultraestrutura
13.
Br J Cancer ; 82(8): 1474-9, 2000 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-10780529

RESUMO

Immunotherapy of tumours by induction of tumour-specific cytotoxic T-lymphocytes (CTLs) will only be effective for tumours with a functional antigen processing and presentation machinery. However, many tumours are known to down-regulate expression of major histocompatibility complex (MHC) class I molecules and/or to impair antigen processing. It is therefore desirable to evaluate the ability of a given tumour to present antigenic epitopes before developing an immunotherapy protocol. In this study we have used influenza virus as a tool to determine the antigen-presenting capacities of the murine neuroblastoma C1300 cell line NB41A3, a frequently used model for human neuroblastoma. Immunofluorescence analyses revealed low and moderate expression of MHC class I molecules Dd and Kk respectively. Nevertheless, infected NB41 A3 cells were lysed efficiently by influenza-specific CTLs. These results demonstrate that all steps of the antigen-processing pathway function properly in the NB tumour cells, and that the limited MHC class I expression suffices for efficient recognition by CTLs. In addition, lysis of the NB tumour cells shows that the cells are susceptible to CTL-induced apoptosis, a pathway that is often impaired in tumour cells. These characteristics make neuroblastoma a suitable target for immunotherapy. The presented assay allows evaluation of various immunological properties of tumour cells and, thus, represents a valuable tool to assess whether a given tumour will be susceptible to immunotherapy or not.


Assuntos
Vírus da Influenza A/imunologia , Neuroblastoma/imunologia , Neuroblastoma/terapia , Linfócitos T Citotóxicos/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Feminino , Genes MHC Classe I , Antígenos de Histocompatibilidade Classe I/análise , Humanos , Interferon gama/farmacologia , Células L , Camundongos , Camundongos Endogâmicos A , Proteínas Recombinantes , Linfócitos T Citotóxicos/virologia , Células Tumorais Cultivadas
14.
Vaccine ; 18(14): 1327-33, 2000 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-10618529

RESUMO

Priming of cytotoxic T lymphocyte (CTL) activity with exogenous antigen requires introduction of the antigen into the MHC class I presentation pathway of antigen-presenting cells. In the present study, we used fusogenic reconstituted envelopes (virosomes), derived from influenza virus, as a carrier system for delivery of a synthetic soluble peptide corresponding to a major murine CTL epitope of the influenza virus nucleoprotein (NP). Virosomes containing encapsulated NP-peptide efficiently sensitized target cells for recognition by influenza-specific CTLs generated through priming of mice with infectious virus. Intramuscular immunization of mice with peptide-containing virosomes induced a potent class I MHC-restricted CTL response against influenza-infected target cells. By contrast, an equal dose of NP-peptide encapsulated in fusion-inactivated virosomes did not induce CTL activity, indicating an essential role of the membrane fusion activity of the virosomes in the induction of the response. Likewise, NP-peptide encapsulated in liposomes, NP-peptide mixed with empty virosomes and NP-peptide in IFA failed to induce a CTL response. These results demonstrate that fusion-active virosomes represent a promising delivery system for induction of class I MHC-restricted CTL activity with non-replicating viral antigens.


Assuntos
Nucleoproteínas/imunologia , Orthomyxoviridae/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Feminino , Antígenos de Histocompatibilidade Classe I/imunologia , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Peptídeos/imunologia , Fenótipo
15.
J Inherit Metab Dis ; 20(4): 569-77, 1997 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9266394

RESUMO

Heat shock protein 60 (hsp60) is a mitochondrial matrix protein involved in the folding and correct assembly of polypeptides into complex mitochondrial enzymes. Its deficiency has recently been described as the most likely primary cause of congenital lactic acidaemia with multiple mitochondrial enzyme deficiencies in a female patient. We describe a new case of a girl with a substantially decreased amount of hsp60 in cultured fibroblasts. She presented from birth with hypotonia, unusual facial features, feeding difficulties and failure to thrive. Death occurred at age 4.5 years. Biochemical findings included metabolic acidosis with lactic acidaemia, hyperammonaemia and intermittent ketosis. In contrast to the previously reported case, organic acid analysis showed an altered profile throughout her life. In agreement with this profile, various mitochondrial enzyme activities were deficient in cultured fibroblasts, including enzymes of the respiratory chain and the Krebs cycle, the pyruvate dehydrogenase complex and the mitochondrial biotindependent carboxylases. Fibroblast mitochondria showed ultrastructural abnormalities, were swollen, and were mainly localized around the nucleus. The description of a second case of multiple mitochondrial enzyme deficiencies with reduced amount of hsp60 supports the idea that hsp60 deficiency might be a more common cause of mitochondrial disease. This opens new possibilities for the diagnosis and understanding of congenital lactic acidaemia.


Assuntos
Enzimas/deficiência , Proteínas de Choque Térmico/deficiência , Erros Inatos do Metabolismo/metabolismo , Mitocôndrias Hepáticas/enzimologia , Encefalomiopatias Mitocondriais/metabolismo , Aminoácidos/metabolismo , Western Blotting , Cromatografia por Troca Iônica , Coenzimas/metabolismo , Enzimas/sangue , Enzimas/urina , Evolução Fatal , Feminino , Fibroblastos/enzimologia , Humanos , Lactente , Erros Inatos do Metabolismo/enzimologia , Mitocôndrias Hepáticas/ultraestrutura , Encefalomiopatias Mitocondriais/enzimologia
16.
Immunol Lett ; 57(1-3): 69-74, 1997 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-9232428

RESUMO

Several studies have shown that immunization with heat shock proteins (HSPs) purified from tumors of virus-infected cells induces specific cytotoxic T-cell (CTL) activity. This immune response is directed against peptides bound to the HSPs rather than against the HSPs themselves. The peptides are derived from tumor- or virus-specific proteins which are degraded in the course of normal protein turnover and processing for presentation by MHC class I molecules. The HSPs appear to function as carriers for the antigenic peptides. Upon immunization they ensure their uptake by specialized macrophages and their introduction into the MHC class I presentation route which is otherwise accessible only for intracellular proteins. Using influenza virus nucleoprotein (NP) as a model antigen, we have tested whether an HSP-based vaccine can be preduced by overexpressing an antigen in cultured cells prior to purification of the HSP's. The transfection system based on the Semliki Forest virus (SFV) replicon was employed to achieve high expression of NP. Since SFV-mediated transfection of murine cells was inefficient we used the hamster-derived cell line BHK21, which can be transfected with 100% efficiency, as a source for NP peptide-loaded gp96. The protein was purified from transfected cells and used for first vaccination studies. The hamster gp96 preparation was well tolerated in mice, an antibody response against the foreign protein was not observed. Preliminary results suggest that a cellular immune response against NP was indeed induced. SFV transfection is applicable for any known antigen and is therefore considered to be an elegant means for the production of HSP-based vaccines capable of inducing a cellular immune response.


Assuntos
Antígenos de Neoplasias/imunologia , Antígenos Virais/imunologia , Proteínas de Choque Térmico/imunologia , Vacinas contra Influenza/imunologia , Nucleoproteínas/imunologia , Proteínas de Ligação a RNA , Proteínas do Core Viral/imunologia , Animais , Antígenos de Neoplasias/isolamento & purificação , Linhagem Celular , Cricetinae , Vetores Genéticos , Proteínas de Choque Térmico/isolamento & purificação , Imunização , Camundongos , Proteínas do Nucleocapsídeo , Nucleoproteínas/biossíntese , Nucleoproteínas/genética , Peptídeos/imunologia , Vírus da Floresta de Semliki/genética , Transfecção , Células Tumorais Cultivadas , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/genética , Vírion
17.
Pediatr Neurol ; 16(2): 160-2, 1997 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-9090694

RESUMO

A boy with delayed psychomotor development, attention deficit disorder, and therapy-resistant epilepsy was treated with valproate. The patient died of liver failure after 4 months of valproate treatment. Postmortem investigation of cultured fibroblasts suggested medium chain acyl-CoA dehydrogenase deficiency, an unexpected finding since the boy had not presented typical manifestations of this disease. Because medium chain acyl-CoA dehydrogenase is an important enzyme in the beta-oxidation of fatty acids, our patient probably had a genetically reduced tolerance to valproate. This drug should be omitted in the treatment of seizures in patients with possible medium chain acyl-CoA dehydrogenase deficiency.


Assuntos
Acil-CoA Desidrogenases/deficiência , Anticonvulsivantes/efeitos adversos , Epilepsia Tônico-Clônica/tratamento farmacológico , Falência Hepática/induzido quimicamente , Ácido Valproico/efeitos adversos , Acil-CoA Desidrogenase , Células Cultivadas , Criança , Epilepsia Tônico-Clônica/enzimologia , Evolução Fatal , Fibroblastos/enzimologia , Humanos , Falência Hepática/enzimologia , Masculino , Pele/citologia , Pele/enzimologia
18.
Eur J Biochem ; 237(1): 288-94, 1996 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-8620886

RESUMO

In order to study mitochondrial protein import in the context of whole cell metabolism, we have used the transfection technique based on Semliki Forest virus (SFV) to express a mitochondrial precursor protein within BHK21 cells and human fibroblasts. Recombinant SFV particles mediate a highly efficient, transient transfection of higher eukaryotic cells. The mitochondrial precursor protein used is a fusion protein consisting of the mitochondrial targeting sequence of Neurospora crassa ATPase subunit 9 and mouse dihydrofolate (H2folate) reductase. Transfected BHK21 cells synthesized substantial amounts of subunit-9-H2folate-reductase. Immunofluorescence staining revealed that the protein colocalized with the mitochondria. The precursor protein was processed to the intermediate and mature form, implying that is was successfully imported into the mitochondrial matrix. Import was dependent on a proton gradient across the mitochondrial membranes since uncoupling of oxidative phosphorylation inhibited the process. The mature-sized protein was folded into a protease-resistant conformation. These results indicate that, in mammalian cells, transport of the precursor subunit-9-H2folate-reductase into mitochondria and its subsequent maturation occurs in a similar way as in lower eukaryotes. Import and processing of the fusion protein proceeded very rapidly in BHK21 cells but were substantially slower in human fibroblasts. SFV-mediated transfection proved to be excellently suited to study protein import into mitochondria of living cells and is probably applicable to transport studies with other organelles as well. The approach could also be helpful in the diagnosis of hereditary disorders or organelle protein import.


Assuntos
Mitocôndrias/metabolismo , Precursores de Proteínas/genética , Adenosina Trifosfatases/genética , Animais , Transporte Biológico , Linhagem Celular , Cricetinae , Eletroporação , Humanos , Camundongos , Precursores de Proteínas/metabolismo , RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vírus da Floresta de Semliki/genética , Frações Subcelulares/enzimologia , Tetra-Hidrofolato Desidrogenase/biossíntese , Transfecção
19.
Virchows Arch ; 427(2): 159-65, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-7582246

RESUMO

We have described two mitochondrial (mt) myopathy patients with reduced activities of various mt enzymes associated with significantly decreased amounts of heat shock protein 60 (hsp60). Experimental evidence suggested that the lack of hsp60 was the primary defect. Since hsp60 is essential for the proper folding of enzyme subunits in the mt matrix a partial deficiency of this protein can explain the observed defects of the mitochondria. Here we report on morphological studies aimed at obtaining more insight into the relation between lack of hsp60 and pathological changes of the mitochondria. Under standard culture conditions mitochondria in the partially hsp60 deficient fibroblasts showed profound morphological aberrations. In contrast, the mitochondria in fibroblasts from a MELAS patient and a cytochrome c oxidase-deficient patient appeared normal. Under stress conditions the integrity of the hsp60 deficient mitochondria declined even further: heat shock induced a temporary collapse of the electrochemical potential across the inner mt membrane, but did not affect the ultrastructure of the mitochondria; prolonged growth in confluent cultures resulted in decrease in mt number. The altered mt morphology in the hsp60 deficient cells is probably indicative of the severely impaired mt metabolism whereas the decreased stress tolerance is likely to be a direct result of paucity of the heat shock protein. Both variables are potentially useful in the diagnosis and molecular characterization of mt disorders with systemic manifestation and multiple enzyme deficiency.


Assuntos
Chaperonina 60/análise , Fibroblastos/patologia , Temperatura Alta/efeitos adversos , Mitocôndrias/patologia , Miopatias Mitocondriais/patologia , Células Cultivadas , Fibroblastos/química , Fibroblastos/ultraestrutura , Humanos , Microscopia Eletrônica
20.
Biochim Biophys Acta ; 1227(3): 200-6, 1994 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-7986829

RESUMO

In a recent paper (Agsteribbe et al. (1993) Biochem. Biophys. Res. Commun. 193, 146-154) we suggested deficiency of heat shock protein 60 (hsp60) as the possible cause of a systemic mitochondrial encephalomyopathy with multiple deficiency of mitochondrial enzymes. In this paper we present new data which strongly support this hypothesis. Hsp60 deficiency appeared to be not a common side effect of impaired mitochondrial metabolism as eight out of ten fibroblast cultures from patients with systemic mitochondrial myopathy contained normal quantities of the protein. The low steady state amount of hsp60 in the fibroblasts of our patient is caused by decreased synthesis of the protein and not by its enhanced degradation indicating that the hsp60 deficiency is indeed a primary defect. Processing of hsp60 but not of other mitochondrial proteins is markedly retarded in the patient cells. Other functional properties of the patient hsp60 like the assembly of hsp60 monomers to the native 14mer complex and the affinity of this complex to denatured protein are not impaired. Our results underline that a primary defect in hsp60 synthesis and/or processing causing a low steady state amount of hsp60 is the molecular basis of this mitochondrial disorder. The presented data provide for the first time substantial evidence that deficiency of a heat shock protein can give rise to pathological conditions in man.


Assuntos
Chaperonina 60/biossíntese , Mitocôndrias/enzimologia , Encefalomiopatias Mitocondriais/etiologia , Chaperonina 60/análise , Feminino , Fibroblastos/metabolismo , Humanos , Recém-Nascido
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