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1.
Proc Natl Acad Sci U S A ; 118(3)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33384338

RESUMO

Human adenovirus species D (HAdV-D) types are currently being explored as vaccine vectors for coronavirus disease 2019 (COVID-19) and other severe infectious diseases. The efficacy of such vector-based vaccines depends on functional interactions with receptors on host cells. Adenoviruses of different species are assumed to enter host cells mainly by interactions between the knob domain of the protruding fiber capsid protein and cellular receptors. Using a cell-based receptor-screening assay, we identified CD46 as a receptor for HAdV-D56. The function of CD46 was validated in infection experiments using cells lacking and overexpressing CD46, and by competition infection experiments using soluble CD46. Remarkably, unlike HAdV-B types that engage CD46 through interactions with the knob domain of the fiber protein, HAdV-D types infect host cells through a direct interaction between CD46 and the hexon protein. Soluble hexon proteins (but not fiber knob) inhibited HAdV-D56 infection, and surface plasmon analyses demonstrated that CD46 binds to HAdV-D hexon (but not fiber knob) proteins. Cryoelectron microscopy analysis of the HAdV-D56 virion-CD46 complex confirmed the interaction and showed that CD46 binds to the central cavity of hexon trimers. Finally, soluble CD46 inhibited infection by 16 out of 17 investigated HAdV-D types, suggesting that CD46 is an important receptor for a large group of adenoviruses. In conclusion, this study identifies a noncanonical entry mechanism used by human adenoviruses, which adds to the knowledge of adenovirus biology and can also be useful for development of adenovirus-based vaccine vectors.


Assuntos
Adenovírus Humanos , Proteínas do Capsídeo , Regulação Viral da Expressão Gênica , Internalização do Vírus , Adenovírus Humanos/genética , Adenovírus Humanos/metabolismo , /metabolismo , Proteínas do Capsídeo/biossíntese , Proteínas do Capsídeo/genética , Linhagem Celular , Humanos
2.
J Gen Virol ; 99(1): 135-147, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29154744

RESUMO

The vectorization of rare human adenovirus (HAdV) types will widen our knowledge of this family and their interaction with cells, tissues and organs. In this study we focus on HAdV-56, a member of human Ad species D, and create ease-of-use cloning systems to generate recombinant HAdV-56 vectors carrying foreign genes. We present in vitro transduction profiles for HAdV-56 in direct comparison to the most commonly used HAdV-5-based vector. In vivo characterizations demonstrate that when it is delivered intravenously (i.v.) HAdV-56 mainly targets the spleen and, to a lesser extent, the lungs, whilst largely bypassing liver transduction in mice. HAdV-56 triggered robust inflammatory and cellular immune responses, with higher induction of IFNγ, TNFα, IL5, IL6, IP10, MCP1 and MIG1 compared to HAdV-5 following i.v. administration. We also investigated its potential as a vaccine vector candidate by performing prime immunizations in mice with HAdV-56 encoding luciferase (HAdV-56-Luc). Direct comparisons were made to HAdV-26, a highly potent human vaccine vector currently in phase II clinical trials. HAdV-56-Luc induced luciferase 'antigen'-specific IFNγ-producing cells and anti-HAdV-56 neutralizing antibodies in Balb/c mice, demonstrating a near identical profile to that of HAdV-26. Taken together, the data presented provides further insight into human Ad receptor/co-receptor usage, and the first report on HAdV-56 vectors and their potential for gene therapy and vaccine applications.


Assuntos
Adenovírus Humanos/imunologia , Expressão Gênica/efeitos dos fármacos , Terapia Genética/métodos , Vetores Genéticos/imunologia , Vacinação , Vacinas Virais/biossíntese , Adenovírus Humanos/genética , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/sangue , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Quimiocina CXCL10/genética , Quimiocina CXCL10/imunologia , Quimiocina CXCL9/genética , Quimiocina CXCL9/imunologia , Feminino , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Injeções Intravenosas , Interferon gama/genética , Interferon gama/imunologia , Interleucina-5/genética , Interleucina-5/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/efeitos dos fármacos , Baço/imunologia , Transgenes , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Vacinas Virais/administração & dosagem
3.
Mol Ther ; 24(1): 6-16, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26478249

RESUMO

Recombinant vectors based on human adenovirus serotype 5 (HAdV-5) have been extensively studied in preclinical models and clinical trials over the past two decades. However, the thorough understanding of the HAdV-5 interaction with human subjects has uncovered major concerns about its product applicability. High vector-associated toxicity and widespread preexisting immunity have been shown to significantly impede the effectiveness of HAdV-5-mediated gene transfer. It is therefore that the in-depth knowledge attained working on HAdV-5 is currently being used to develop alternative vectors. Here, we provide a comprehensive overview of data obtained in recent years disqualifying the HAdV-5 vector for systemic gene delivery as well as novel strategies being pursued to overcome the limitations observed with particular emphasis on the ongoing vectorization efforts to obtain vectors based on alternative serotypes.


Assuntos
Adenovírus Humanos/imunologia , Vetores Genéticos/toxicidade , Adenovírus Humanos/genética , Técnicas de Transferência de Genes , Vetores Genéticos/imunologia , Humanos , Imunidade Inata
4.
Hum Gene Ther ; 25(4): 318-27, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24593243

RESUMO

Abstract Once adenovirus vector-based vaccines are licensed for the prevention of important infectious diseases, manufacturing processes capable of reliably delivering large numbers of vaccine doses will be required. The highest burden of disease for many infectious pathogens under investigation occurs in resource-poor settings. Therefore, the price per dose will be an important determinant of success. This review describes common practices for manufacturing replication-incompetent adenovirus vectors at clinical scale. Recent innovations and strategies aimed at improving the cost-effectiveness of manufacturing and ensuring high-volume vaccine production and purification are described. Hereto, technologies to increase bioreactor yields are reviewed. In addition, the use of single-use perfusion bioreactors, modification of some purification steps to avoid the use of expensive endonucleases, and use of charged filters during anion exchange all have the potential to bring down the cost of goods and are thus described. Finally, processes for ensuring quality throughout the manufacturing process, methods for testing viral identity, and safety of master seeds through to the end vaccine product are described.


Assuntos
Adenoviridae/genética , Reatores Biológicos , Biotecnologia , Vetores Genéticos/genética , Vacinas Sintéticas/genética , Adenoviridae/imunologia , Animais , Biotecnologia/métodos , Biotecnologia/normas , Vetores Genéticos/imunologia , Humanos , Vacinas Sintéticas/imunologia
5.
Clin Vaccine Immunol ; 17(11): 1687-94, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20826614

RESUMO

The most advanced malaria vaccine, RTS,S, is comprised of a portion of the Plasmodium falciparum circumsporozoite (CS) protein, fused to and admixed with the hepatitis B virus surface antigen, and an adjuvant [corrected].This vaccine confers short-term protection against malaria infection, with an efficacy of about 50%, and induces particularly B-cell and CD4(+) T-cell responses.In the present study, we tested the hypothesis that the Th1 immune response to CS protein,in particular the CD8(+) T-cell response, which is needed for strong and lasting malaria immunity, is boosted to sustainable levels by adenovirus vectors 35 and 26 with a homologous insert (Ad35.CS/Ad26.CS) [corrected]. In this study, we evaluated immune responses induced with vaccination regimens based on an adjuvant-containing, yeast-produced complete CS protein followed by two recombinant low-seroprevalence adenoviruses expressing P. falciparum CS antigen, Ad35.CS (subgroup B) and Ad26.CS (subgroup D). Our results show that (i) the yeast (Hansenula polymorpha)produced, adjuvanted full-length CS protein is highly potent in inducing high CS-specific humoral responses in mice but produces poor T-cell responses, (ii) the Ad35.CS vector boosts the gamma interferon-positive (IFN-γ(+)) CD8(+) T-cell response induced by the CS protein immunization and shifts the immune response toward the Th1 type, and (iii) a three-component heterologous vaccination comprised of a CS protein prime followed by boosts with Ad35.CS and Ad26.CS elicits an even more robust and sustainable IFN-γ(+) CD8(+) T-cell response than one- or two-component regimens. The Ad35.CS/Ad26.CS combination boosted particularly the IFN-γ(+) and tumor necrosis factor alpha-positive (TNF-α(+)) T cells, confirming the shift of the immune response from the Th2 type to the Th1 type. These results support the notion of first immunizations of infants with an adjuvanted CS protein vaccine, followed by a booster Ad35.CS/Ad26.CS vaccine at a later age, to induce lasting protection against malaria for which the Th1 response and immune memory is required.


Assuntos
Adenoviridae/genética , Vetores Genéticos/genética , Vacinas Antimaláricas/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Células Th1/imunologia , Animais , Anticorpos Antiprotozoários/sangue , Linfócitos T CD8-Positivos/imunologia , Feminino , Humanos , Imunização Secundária/métodos , Interferon gama/metabolismo , Vacinas Antimaláricas/administração & dosagem , Vacinas Antimaláricas/genética , Camundongos , Camundongos Endogâmicos BALB C , Pichia/genética , Proteínas de Protozoários/genética , Vacinação/métodos , Vacinas de Subunidades/administração & dosagem , Vacinas de Subunidades/genética , Vacinas de Subunidades/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
6.
Expert Rev Vaccines ; 8(5): 577-92, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19397415

RESUMO

Classical vaccination approaches, based on a single vaccine administered in a homologous prime-boost schedule and optimized to induce primarily neutralizing antibodies, are unlikely to be sufficiently efficacious to prevent TB, malaria or HIV infections. Novel vaccines, capable of inducing a more powerful immune response, in particular T-cell immunity, are desperately needed. Combining different vaccine modalities that are able to complement each other and induce broad and sustainable immunity is a promising approach. This review provides an overview of heterologous prime-boost vaccination modalities currently in development for the 'big three' poverty-related diseases and emphasizes the need for innovative vaccination approaches.


Assuntos
Imunização Secundária/métodos , Vacinação/métodos , Vacinas/administração & dosagem , Vacinas/imunologia , Infecções por HIV/prevenção & controle , Humanos , Malária/prevenção & controle , Tuberculose/prevenção & controle , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Vacinas de Subunidades/administração & dosagem , Vacinas de Subunidades/imunologia
7.
J Virol ; 82(10): 4844-52, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18337575

RESUMO

Recombinant adenovirus serotype 5 (rAd5) vaccine vectors for human immunodeficiency virus type 1 (HIV-1) and other pathogens have been shown to elicit antigen-specific cellular immune responses. Rare serotype rAd vectors have also been constructed to circumvent preexisting anti-Ad5 immunity and to facilitate the development of novel heterologous rAd prime-boost regimens. Here we show that rAd5, rAd26, and rAd48 vectors elicit qualitatively distinct phenotypes of cellular immune responses in rhesus monkeys and can be combined as potent heterologous prime-boost vaccine regimens. While rAd5-Gag induced primarily gamma interferon-positive (IFN-gamma(+)) and IFN-gamma(+)/tumor necrosis factor alpha(+) (TNF-alpha(+)) T-lymphocyte responses, rAd26-Gag and rAd48-Gag induced higher proportions of interleukin-2(+) (IL-2(+)) and polyfunctional IFN-gamma(+)/TNF-alpha(+)/IL-2(+) T-lymphocyte responses. Priming with the rare serotype rAd vectors proved remarkably effective for subsequent boosting with rAd5 vectors. These data demonstrate that the rare serotype rAd vectors elicited T-lymphocyte responses that were phenotypically distinct from those elicited by rAd5 vectors and suggest the functional relevance of polyfunctional CD8(+) and CD4(+) T-lymphocyte responses. Moreover, qualitative differences in cellular immune responses may prove critical in determining the overall potency of heterologous rAd prime-boost regimens.


Assuntos
Adenoviridae/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Vetores Genéticos/imunologia , Vacinas contra a SAIDS/imunologia , Animais , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Imunização/métodos , Imunização Secundária/métodos , Interferon gama/biossíntese , Interleucina-2/biossíntese , Macaca mulatta , Fator de Necrose Tumoral alfa/biossíntese
8.
J Virol ; 81(9): 4654-63, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17329340

RESUMO

Recombinant adenovirus serotype 5 (rAd5) vector-based vaccines are currently being developed for both human immunodeficiency virus type 1 and other pathogens. The potential limitations associated with rAd5 vectors, however, have led to the construction of novel rAd vectors derived from rare Ad serotypes. Several rare serotype rAd vectors have already been described, but a detailed comparison of multiple rAd vectors from subgroups B and D has not previously been reported. Such a comparison is critical for selecting optimal rAd vectors for advancement into clinical trials. Here we describe the construction of three novel rAd vector systems from Ad26, Ad48, and Ad50. We report comparative seroprevalence and immunogenicity studies involving rAd11, rAd35, and rAd50 vectors from subgroup B; rAd26, rAd48, and rAd49 vectors from subgroup D; and rAd5 vectors from subgroup C. All six rAd vectors from subgroups B and D exhibited low seroprevalence in a cohort of 200 individuals from sub-Saharan Africa, and they elicited Gag-specific cellular immune responses in mice both with and without preexisting anti-Ad5 immunity. The rAd vectors from subgroup D were also evaluated using rhesus monkeys and were shown to be immunogenic after a single injection. The rAd26 vectors proved the most immunogenic among the rare serotype rAd vectors studied, although all rare serotype rAd vectors were still less potent than rAd5 vectors in the absence of anti-Ad5 immunity. These studies substantially expand the portfolio of rare serotype rAd vectors that may prove useful as vaccine vectors for the developing world.


Assuntos
Infecções por Adenoviridae/epidemiologia , Adenoviridae/genética , Vetores Genéticos/genética , Vacinas Sintéticas/genética , Vacinas Virais/genética , Infecções por Adenoviridae/sangue , África ao Sul do Saara/epidemiologia , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos/imunologia , Humanos , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Testes de Neutralização , Análise de Sequência de DNA , Estudos Soroepidemiológicos , Sorotipagem
9.
J Virol ; 80(24): 12009-16, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17035318

RESUMO

The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations has led to the development of recombinant adenovirus (rAd) vectors derived from rare Ad serotypes as vaccine candidates for human immunodeficiency virus type 1 and other pathogens. Vaccine vectors have been constructed from Ad subgroup B, including rAd11 and rAd35, as well as from Ad subgroup D, including rAd49. However, the optimal combination of vectors for heterologous rAd prime-boost vaccine regimens and the extent of cross-reactive vector-specific neutralizing antibodies (NAbs) remain poorly defined. We have shown previously that the closely related vectors rAd11 and rAd35 elicited low levels of cross-reactive NAbs. Here we show that these cross-reactive NAbs correlated with substantial sequence homology in the hexon hypervariable regions (HVRs) and suppressed the immunogenicity of heterologous rAd prime-boost regimens. In contrast, vectors with lower hexon HVR homology, such as rAd35 and rAd49, did not elicit detectable cross-reactive vector-specific NAbs. Consistent with these findings, rAd35-rAd49 vaccine regimens proved more immunogenic than both rAd35-rAd5 and rAd35-rAd11 regimens in mice with anti-Ad5 immunity. These data suggest that optimal heterologous rAd prime-boost regimens should include two vectors that are both rare in human populations to circumvent preexisting antivector immunity as well as sufficiently immunologically distinct to avoid cross-reactive antivector immunity.


Assuntos
Adenoviridae/imunologia , Reações Cruzadas/imunologia , Vetores Genéticos/imunologia , Vacinas Sintéticas/imunologia , Adenoviridae/genética , Animais , Anticorpos/imunologia , Antígenos Virais/genética , Proteínas do Capsídeo/genética , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos C57BL , Testes de Neutralização
10.
J Clin Microbiol ; 44(10): 3781-3, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17021110

RESUMO

We assessed neutralizing antibody titers to adenovirus serotype 5 (Ad5) and six rare adenovirus serotypes, serotypes 11, 35, 50, 26, 48, and 49, in pediatric populations in sub-Saharan Africa. We observed a clear age dependence of Ad5-specific neutralizing antibody titers. These data will help to guide the development of Ad vector-based vaccines for human immunodeficiency virus type 1 and other pathogens.


Assuntos
Infecções por Adenovirus Humanos/imunologia , Adenovírus Humanos/imunologia , Envelhecimento , Anticorpos Antivirais/sangue , Infecções por Adenovirus Humanos/sangue , Infecções por Adenovirus Humanos/epidemiologia , Adolescente , África ao Sul do Saara/epidemiologia , Criança , Pré-Escolar , Humanos , Lactente , Transmissão Vertical de Doença Infecciosa , Estudos Soroepidemiológicos
11.
J Gen Virol ; 87(Pt 10): 2891-2899, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16963747

RESUMO

Recombinant adenoviral vectors based on type 5 (rAd5) show great promise as a vaccine carrier. However, neutralizing activity against Ad5 is prevalent and high-titred among human populations, and significantly dampens Ad5-based vaccine modalities. The generation of alternative adenoviral vectors with low seroprevalence thus receives much research attention. Here, it is shown that a member from human adenovirus subgroup D, i.e. Ad49, does not cross-react with Ad5 neutralizing activity, making it a candidate serotype for vector development. Therefore, a plasmid system that allows formation of replication-incompetent adenovirus serotype 49 vaccine vectors (rAd49) was constructed and it was demonstrated that rAd49 can be successfully propagated to high titres on existing Ad5.E1-complementing cell lines such as PER.C6. Using an rAd49 vector carrying the luciferase marker gene, detailed seroprevalence studies were performed, demonstrating that rAd49 has low seroprevalence and neutralizing antibody titres worldwide. Also, we have initiated rAd49 vector receptor usage suggesting that rAd49 utilizes hCD46 as a cellular receptor. Finally, the immunogenicity of the rAd49 vector was assessed and it was shown that an rAd49.SIVGag vaccine induces strong anti-SIVGag CD8+ T-lymphocytes in naïve mice, albeit less than an rAd5.SIVGag vaccine. However, in mice with high anti-Ad5 immunity the rAd5.SIVGag vaccine was severely blunted, whereas the anti-SIVGag response was not significantly suppressed using the rAd49.SIVGag vaccine. These data demonstrate the potential of a replication deficient human group D adenoviral vector for vaccination purposes.


Assuntos
Adenovírus Humanos/crescimento & desenvolvimento , Adenovírus Humanos/imunologia , Replicação Viral , Adenovírus Humanos/genética , Adenovírus Humanos/fisiologia , Animais , Anticorpos Antivirais , Linhagem Celular , Engenharia Genética , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Humanos , Proteína Cofatora de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Especificidade de Órgãos , Vacinas Sintéticas , Vacinas Virais/imunologia
12.
Nature ; 441(7090): 239-43, 2006 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-16625206

RESUMO

A common viral immune evasion strategy involves mutating viral surface proteins in order to evade host neutralizing antibodies. Such immune evasion tactics have not previously been intentionally applied to the development of novel viral gene delivery vectors that overcome the critical problem of anti-vector immunity. Recombinant, replication-incompetent adenovirus serotype 5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 and other pathogens have proved highly immunogenic in preclinical studies but will probably be limited by the high prevalence of pre-existing anti-Ad5 immunity in human populations, particularly in the developing world. Here we show that rAd5 vectors can be engineered to circumvent anti-Ad5 immunity. We constructed novel chimaeric rAd5 vectors in which the seven short hypervariable regions (HVRs) on the surface of the Ad5 hexon protein were replaced with the corresponding HVRs from the rare adenovirus serotype Ad48. These HVR-chimaeric rAd5 vectors were produced at high titres and were stable through serial passages in vitro. HVR-chimaeric rAd5 vectors expressing simian immunodeficiency virus Gag proved comparably immunogenic to parental rAd5 vectors in naive mice and rhesus monkeys. In the presence of high levels of pre-existing anti-Ad5 immunity, the immunogenicity of HVR-chimaeric rAd5 vectors was not detectably suppressed, whereas the immunogenicity of parental rAd5 vectors was abrogated. These data demonstrate that functionally relevant Ad5-specific neutralizing antibodies are focused on epitopes located within the hexon HVRs. Moreover, these studies show that recombinant viral vectors can be engineered to circumvent pre-existing anti-vector immunity by removing key neutralizing epitopes on the surface of viral capsid proteins. Such chimaeric viral vectors may have important practical implications for vaccination and gene therapy.


Assuntos
Adenoviridae/genética , Adenoviridae/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Engenharia Genética , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Adenoviridae/classificação , Adenoviridae/fisiologia , Animais , Linfócitos T CD8-Positivos/imunologia , DNA Recombinante/genética , Terapia Genética , Macaca mulatta/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Testes de Neutralização , Vacinas
13.
J Virol ; 79(22): 14161-8, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16254351

RESUMO

Preexisting immunity to adenovirus serotype 5 (Ad5) has been shown to suppress the immunogenicity of recombinant Ad5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 (HIV-1) in both preclinical studies and clinical trials. A potential solution to this problem is to utilize rAd vectors derived from rare Ad serotypes, such as Ad35. However, rAd35 vectors have appeared less immunogenic than rAd5 vectors in preclinical studies to date. In this study, we explore the hypothesis that the differences in immunogenicity between rAd5 and rAd35 vectors may be due in part to differences between the fiber proteins of these viruses. We constructed capsid chimeric rAd35 vectors containing the Ad5 fiber knob (rAd35k5) and compared the immunogenicities of rAd5, rAd35k5, and rAd35 vectors expressing simian immunodeficiency virus Gag and HIV-1 Env in mice and rhesus monkeys. In vitro studies demonstrated that rAd35k5 vectors utilized the Ad5 receptor CAR rather than the Ad35 receptor CD46. In vivo studies showed that rAd35k5 vectors were more immunogenic than rAd35 vectors in both mice and rhesus monkeys. These data suggest that the Ad5 fiber knob contributes substantially to the immunogenicity of rAd vectors. Moreover, these studies demonstrate that capsid chimeric rAd vectors can be constructed to combine beneficial immunologic and serologic properties of different Ad serotypes.


Assuntos
Infecções por Adenoviridae/imunologia , Adenoviridae/imunologia , Proteínas do Capsídeo/genética , Vacinas Virais , Adenoviridae/classificação , Adenoviridae/genética , Animais , Epitopos/química , Epitopos/imunologia , Imunização , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Sorotipagem , Replicação Viral
14.
J Virol ; 79(15): 9694-701, 2005 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16014931

RESUMO

The high prevalence of preexisting immunity to adenovirus serotype 5 (Ad5) in human populations will likely limit the immunogenicity and clinical utility of recombinant Ad5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 and other pathogens. A potential solution to this problem is to utilize rAd vaccine vectors derived from rare Ad serotypes such as Ad35 and Ad11. We have previously reported that rAd35 vectors were immunogenic in the presence of anti-Ad5 immunity, but the immunogenicity of heterologous rAd prime-boost regimens and the extent that cross-reactive anti-vector immunity may limit this approach have not been fully explored. Here we assess the immunogenicity of heterologous vaccine regimens involving rAd5, rAd35, and novel rAd11 vectors expressing simian immunodeficiency virus Gag in mice both with and without anti-Ad5 immunity. Heterologous rAd prime-boost regimens proved significantly more immunogenic than homologous regimens, as expected. Importantly, all regimens that included rAd5 were markedly suppressed by anti-Ad5 immunity. In contrast, rAd35-rAd11 and rAd11-rAd35 regimens elicited high-frequency immune responses both in the presence and in the absence of anti-Ad5 immunity, although we also detected clear cross-reactive Ad35/Ad11-specific humoral and cellular immune responses. Nevertheless, these data suggest the potential utility of heterologous rAd prime-boost vaccine regimens using vectors derived from rare human Ad serotypes.


Assuntos
Adenovírus Humanos/imunologia , Vetores Genéticos/imunologia , Vírus Reordenados/imunologia , Vacinas Virais/imunologia , Animais , Formação de Anticorpos , Reações Cruzadas , Avaliação Pré-Clínica de Medicamentos , Produtos do Gene gag/genética , Terapia Genética , Imunidade Celular , Imunização Secundária , Injeções Intramusculares , Camundongos , Camundongos Endogâmicos C57BL , Vírus da Imunodeficiência Símia/genética , Vacinas Sintéticas/imunologia , Vacinas Virais/administração & dosagem
15.
J Immunol ; 174(11): 7179-85, 2005 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-15905562

RESUMO

The utility of recombinant adenovirus serotype 5 (rAd5) vector-based vaccines for HIV-1 and other pathogens will likely be limited by the high prevalence of pre-existing Ad5-specific neutralizing Abs (NAbs) in human populations. However, the immunodominant targets of Ad5-specific NAbs in humans remain poorly characterized. In this study, we assess the titers and primary determinants of Ad5-specific NAbs in individuals from both the United States and the developing world. Importantly, median Ad5-specific NAb titers were >10-fold higher in sub-Saharan Africa compared with the United States. Moreover, hexon-specific NAb titers were 4- to 10-fold higher than fiber-specific NAb titers in these cohorts by virus neutralization assays using capsid chimeric viruses. We next performed adoptive transfer studies in mice to evaluate the functional capacity of hexon- and fiber-specific NAbs to suppress the immunogenicity of a prototype rAd5-Env vaccine. Hexon-specific NAbs were remarkably efficient at suppressing Env-specific immune responses elicited by the rAd5 vaccine. In contrast, fiber-specific NAbs exerted only minimal suppressive effects on rAd5 vaccine immunogenicity. These data demonstrate that functionally significant Ad5-specific NAbs are directed primarily against the Ad5 hexon protein in both humans and mice. These studies suggest a potential strategy for engineering novel Ad5 vectors to evade dominant Ad5-specific NAbs.


Assuntos
Adenovírus Humanos/imunologia , Anticorpos Antivirais/fisiologia , Proteínas do Capsídeo/imunologia , Vetores Genéticos/imunologia , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Adenovírus Humanos/genética , Adulto , Animais , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/administração & dosagem , Proteínas do Capsídeo/genética , Relação Dose-Resposta Imunológica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/metabolismo , Humanos , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Epitopos Imunodominantes/metabolismo , Imunossupressores/metabolismo , Imunossupressores/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Testes de Neutralização , Estudos Soroepidemiológicos , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genética
16.
J Leukoc Biol ; 77(3): 337-43, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15601667

RESUMO

Recently, we described the molecular identification of dendritic cell-specific TrAnsMembrane protein (DC-STAMP), a multimembrane-spanning protein preferentially expressed by human DC (hDC). In this report, we describe the identification and expression profile of the murine homologue of DC-STAMP (mDC-STAMP) as well as the characterization of the DC-STAMP protein. The results demonstrate that mDC-STAMP is over 90% homologous to hDC-STAMP and is also preferentially expressed by DC in vitro and ex vivo. mDC-STAMP expression is enhanced by interleukin-4 and down-regulated upon DC maturation. Analysis of differently tagged DC-STAMP proteins further demonstrates that hDC-STAMP and mDC-STAMP are glycosylated and primarily localize to an intracellular compartment. Applying confocal microscopy and electron microscopy, we demonstrate that hDC-STAMP localizes to the endoplasmic reticulum (ER) in human embryonic kidney 293 cells as well as hDC transduced with an adenovirus encoding hDC-STAMP-green fluorescent protein fusion protein. These data imply that DC-STAMP may exert its effect in the ER.


Assuntos
Células Dendríticas/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Clonagem Molecular , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Homologia de Sequência de Aminoácidos
17.
J Virol ; 78(23): 13207-15, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15542673

RESUMO

A novel plasmid-based adenovirus vector system that enables manufacturing of replication-incompetent (DeltaE1) adenovirus type 11 (Ad11)-based vectors is described. Ad11 vectors are produced on PER.C6/55K cells yielding high-titer vector batches after purification. Ad11 seroprevalence proves to be significantly lower than that of Ad5, and neutralizing antibody titers against Ad11 are low. Ad11 seroprevalence among human immunodeficiency virus-positive (HIV(+)) individuals is as low as that among HIV(-) individuals, independent of the level of immune suppression. The low level of coinciding seroprevalence between Ad11 and Ad35 in addition to a lack of correlation between high neutralizing antibody titers towards either adenovirus strongly suggest that the limited humoral cross-reactive immunity between these two highly related B viruses appears not to preclude the use of both vectors in the same individual. Ad11 transduces primary cells including smooth muscle cells, synoviocytes, and dendritic cells and cardiovascular tissues with higher efficiency than Ad5. Ad11 and Ad35 appear to have a similar tropism as judged by green fluorescent protein expression levels determined by using a panel of cancer cell lines. In addition, Ad5 preimmunization did not significantly affect Ad11-mediated transduction in C57BL/6 mice. We therefore conclude that the Ad11-based vector represents a novel and useful candidate gene transfer vehicle for vaccination and gene therapy.


Assuntos
Adenovírus Humanos/genética , Terapia Genética , Vetores Genéticos/genética , Replicação Viral , Adenovírus Humanos/imunologia , Adulto , Idoso , Animais , Anticorpos Antivirais/sangue , Antígenos CD/análise , Reações Cruzadas , Vetores Genéticos/imunologia , Humanos , Proteína Cofatora de Membrana , Glicoproteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Estudos Soroepidemiológicos , Tropismo , Vacinação
18.
Eur J Epidemiol ; 19(6): 513-6, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15330122

RESUMO

Considerable progress has been made over the past several years in the development of an HIV vaccine. As a result, a growing number of vaccine modalities are being investigated in pre-clinical and phase I/II clinical trials. However, a number of major scientific challenges still remain. It is widely believed that the ideal vaccine should elicit both neutralizing antibodies and cytotoxic T lymphocytes (CTL) against diverse isolates of HIV, but the precise correlates of immunity have not been defined. Recombinant live vector-based vaccines and plasmid DNA vaccines have been shown to induce CTL, either alone or in combination, and these CTL-based vaccines have shown partial protective efficacy in nonhuman primates challenge studies. An immunogen that elicits broadly reactive neutralizing antibodies, however, has yet to be developed.


Assuntos
Vacinas contra a AIDS , Síndrome de Imunodeficiência Adquirida/prevenção & controle , HIV/imunologia , Anticorpos Anti-HIV/biossíntese , Anticorpos Anti-HIV/imunologia , Humanos , Linfócitos T Citotóxicos/imunologia
19.
J Virol ; 77(15): 8263-71, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12857895

RESUMO

Replication-deficient human adenovirus type 5 (Ad5) can be produced to high titers in complementing cell lines, such as PER.C6, and is widely used as a vaccine and gene therapy vector. However, preexisting immunity against Ad5 hampers consistency of gene transfer, immunological responses, and vector-mediated toxicities. We report the identification of human Ad35 as a virus with low global prevalence and the generation of an Ad35 vector plasmid system for easy insertion of heterologous genes. In addition, we have identified the minimal sequence of the Ad35-E1B region (molecular weight, 55,000 [55K]), pivotal for complementation of fully E1-lacking Ad35 vector on PER.C6 cells. After stable insertion of the 55K sequence into PER.C6 cells a cell line was obtained (PER.C6/55K) that efficiently transcomplements both Ad5 and Ad35 vectors. We further demonstrate that transduction with Ad35 is not hampered by preexisting Ad5 immunity and that Ad35 efficiently infects dendritic cells, smooth muscle cells, and synoviocytes, in contrast to Ad5.


Assuntos
Adenovírus Humanos/imunologia , Adenovírus Humanos/fisiologia , Vetores Genéticos , Replicação Viral , Proteínas E1B de Adenovirus/química , Proteínas E1B de Adenovirus/genética , Adenovírus Humanos/genética , Animais , Anticorpos Antivirais/imunologia , Linhagem Celular , Células Cultivadas , Células Dendríticas/virologia , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Músculo Liso/citologia , Músculo Liso/virologia , Testes de Neutralização , Plasmídeos , Membrana Sinovial/citologia , Membrana Sinovial/virologia , Vacinação , Montagem de Vírus
20.
BMC Mol Biol ; 4: 4, 2003 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-12697054

RESUMO

BACKGROUND: After intravenous delivery of the adenoviral vector into rats or mice, 95-99% of the encoded protein is produced in the hepatocytes. We observed, as have others, that the early expression levels of the vector encoded protein vary, greatly, within a species, from one animal strain to another. This study was initiated to determine the molecular mechanism causing the difference: hepatic transfection, transcription or translation. For this purpose different doses of Ad5 luciferase and Ad5 LacZ were intravenously injected into Brown Norway rats and Wag/Rij rats, two strains that differ by a factor of 10 in encoded protein levels. The proportion of LacZ positive hepatocytes, the adenoviral DNA, specific transgenic RNA and luciferase protein were compared in the two strains. RESULTS: The number of transduced hepatocytes and the amounts of Ad5 DNA in the livers was similar in both strains, whereas the Brown Norway rats produced 8 to 10 times more of both vector encoded proteins and of transgene mRNA than the Wag/Rij rats. CONCLUSIONS: It is concluded that the difference between strains in vector encoded protein expression is due to different transcriptional events. No evidence was obtained to suggest that the differences are related to liver damage influenced by vector toxicity or immune reactions.


Assuntos
Adenoviridae/genética , Heterogeneidade Genética , Terapia Genética/métodos , Adenoviridae/enzimologia , Animais , DNA Recombinante/administração & dosagem , DNA Recombinante/biossíntese , DNA Recombinante/genética , DNA Viral/administração & dosagem , DNA Viral/biossíntese , DNA Viral/genética , Dosagem de Genes , Expressão Gênica/genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Hepatócitos/química , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Injeções Intravenosas , Fígado/enzimologia , Fígado/metabolismo , Fígado/patologia , Fígado/virologia , Luciferases/genética , Masculino , RNA Viral/genética , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos , Transfecção/métodos , Transgenes/genética , beta-Galactosidase/biossíntese , beta-Galactosidase/genética
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