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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.
Appl Microbiol Biotechnol ; 103(21-22): 8875-8888, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31641814

RESUMO

Monoclonal antibodies (mABs) are of great biopharmaceutical importance for the diagnosis and treatment of diseases. However, their production in mammalian expression hosts usually requires extensive production times and is expensive. Escherichia coli has become a new platform for production of functional small antibody fragment variants. In this study, we have used a rhamnose-inducible expression system that allows precise control of protein expression levels. The system was first evaluated for the cytoplasmic production of super folder green fluorescence protein (sfGFP) in various production platforms and then for the periplasmic production of the anti-HIV single-chain variable antibody fragment (scFv) of PGT135. Anti-HIV broadly neutralizing antibodies, like PGT135, have potential for clinical use to prevent HIV transmission, to promote immune responses and to eradicate infected cells. Different concentrations of L-rhamnose resulted in the controlled production of both sfGFP and scFv PGT135 antibody. In addition, by optimizing the culture conditions, the amount of scFv PGT135 antibody that was expressed soluble or as inclusions bodies could be modulated. The proteins were produced in batch bioreactors, with yields of 4.9 g/L for sfGFP and 0.8 g/L for scFv. The functionality of the purified antibodies was demonstrated by their ability to neutralize a panel of different HIV variants in vitro. We expect that this expression system will prove very useful for the development of a more cost-effective production process for proteins and antibody fragments in microbial cells.


Assuntos
Anticorpos Monoclonais/biossíntese , Escherichia coli/metabolismo , Anticorpos Anti-HIV/biossíntese , Infecções por HIV/terapia , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/uso terapêutico , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Reatores Biológicos/microbiologia , Escherichia coli/genética , Expressão Gênica/genética , Anticorpos Anti-HIV/uso terapêutico , HIV-1/imunologia , Regiões Promotoras Genéticas/genética , Anticorpos de Cadeia Única/imunologia
3.
Cells ; 8(2)2019 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-30781676

RESUMO

Expansion of hematopoietic stem cells (HSCs) for therapeutic purposes has been a "holy grail" in the field for many years. Ex vivo expansion of HSCs can help to overcome material shortage for transplantation purposes and genetic modification protocols. In this review, we summarize improved understanding in blood development, the effect of niche and conservative signaling pathways on HSCs in mice and humans, and also advances in ex vivo culturing protocols of human HSCs with cytokines or small molecule compounds. Different expansion protocols have been tested in clinical trials. However, an optimal condition for ex vivo expansion of human HSCs still has not been found yet. Translating and implementing new findings from basic research (for instance by using genetic modification of human HSCs) into clinical protocols is crucial to improve ex vivo expansion and eventually boost stem cell gene therapy.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Nicho de Células-Tronco , Animais , Linhagem da Célula , Autorrenovação Celular , Humanos , Via de Sinalização Wnt
4.
PLoS One ; 13(11): e0205139, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30427829

RESUMO

BACKGROUND: Live, attenuated viral vectors that express HIV-1 antigens are being investigated as an approach to generating durable immune responses against HIV-1 in humans. We recently developed a replication-competent, highly attenuated Ad26 vector that expresses mosaic HIV-1 Env (rcAd26.MOS1.HIV-Env, "rcAd26"). Here we present the results of a first-in-human, placebo-controlled clinical trial to test the safety, immunogenicity and mucosal shedding of rcAd26 given orally. METHODS: Healthy adults were randomly assigned to receive a single oral dose of vaccine or placebo at 5:1 ratio in a dosage escalation of 10^8 to 10^11 rcAd26 VP (nominal doses) at University of Rochester Medical Center, Rochester, NY, USA. Participants were isolated and monitored for reactogenicity for 10 days post-vaccination, and adverse events were recorded up to day 112. Rectal and oropharyngeal secretions were evaluated for shedding of the vaccine. Humoral and cellular immune responses were measured. Household contacts were monitored for secondary vaccine transmission. RESULTS: We enrolled 22 participants and 11 household contacts between February 7 and June 24, 2015. 18 participants received one dose of HIV-1 vaccine and 4 participants received placebo. The vaccine caused only mild to moderate adverse events. No vaccine-related SAEs were observed. No infectious rcAd26 viral particles were detected in rectal or oropharyngeal secretions from any participant. Env-specific ELISA and ELISPOT responses were undetectable. No household contacts developed vaccine-induced HIV-1 seropositivity or vaccine-associated illness. CONCLUSIONS: The highly attenuated rcAd26.MOS1.HIV-Env vaccine was well tolerated up to 10^11 VP in healthy, HIV-1-uninfected adults, though the single dose was poorly immunogenic suggesting the replicative capacity of the vector was too attenuated. There was no evidence of shedding of infectious virus or secondary vaccine transmission following the isolation period. These data suggest the use of less attenuated viral vectors in future studies of live, oral HIV-1 vaccines. TRIAL REGISTRATION: ClinicalTrials.gov NCT02366013.


Assuntos
Vacinas contra a AIDS/uso terapêutico , Síndrome de Imunodeficiência Adquirida/terapia , HIV-1/imunologia , Imunidade Celular/imunologia , Vacinas contra a AIDS/genética , Vacinas contra a AIDS/imunologia , Síndrome de Imunodeficiência Adquirida/genética , Síndrome de Imunodeficiência Adquirida/imunologia , Síndrome de Imunodeficiência Adquirida/virologia , Adenoviridae/genética , Adulto , Antígenos Virais/genética , Antígenos Virais/imunologia , Antígenos Virais/uso terapêutico , Feminino , Vetores Genéticos/uso terapêutico , HIV-1/genética , HIV-1/patogenicidade , Humanos , Imunidade Celular/genética , Masculino , Pessoa de Meia-Idade , Replicação Viral/efeitos dos fármacos , Replicação Viral/imunologia , Adulto Jovem , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/uso terapêutico
5.
Artigo em Inglês | MEDLINE | ID: mdl-29423380

RESUMO

To define the bottlenecks that restrict antigen expression after oral administration of viral-vectored vaccines, we tracked vectors derived from the human adenovirus type 5 at whole body, tissue, and cellular scales throughout the digestive tract in a murine model of oral delivery. After intragastric administration of vectors encoding firefly luciferase or a model antigen, detectable levels of transgene-encoded protein or mRNA were confined to the intestine, and restricted to delimited anatomical zones. Expression of luciferase in the form of multiple small bioluminescent foci in the distal ileum, cecum, and proximal colon suggested multiple crossing points. Many foci were unassociated with visible Peyer's patches, implying that transduced cells lay in proximity to villous rather than follicle-associated epithelium, as supported by detection of transgene-encoded antigen in villous epithelial cells. Transgene-encoded mRNA but not protein was readily detected in Peyer's patches, suggesting that post-transcriptional regulation of viral gene expression might limit expression of transgene-encoded antigen in this tissue. To characterize the pathways by which the vector crossed the intestinal epithelium and encountered sentinel cells, a fluorescent-labeled vector was administered to mice by the intragastric route or inoculated into ligated intestinal loops comprising a Peyer's patch. The vector adhered selectively to microfold cells in the follicle-associated epithelium, and, after translocation to the subepithelial dome region, was captured by phagocytes that expressed CD11c and lysozyme. In conclusion, although a large number of crossing events took place throughout the intestine within and without Peyer's patches, multiple firewalls prevented systemic dissemination of vector and suppressed production of transgene-encoded protein in Peyer's patches.


Assuntos
Adenovírus Humanos/genética , Adenovírus Humanos/imunologia , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Transgenes/genética , Transgenes/imunologia , Administração Oral , Animais , Feminino , Expressão Gênica , Genes Reporter , Vetores Genéticos/administração & dosagem , Humanos , Imunização , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Camundongos , Especificidade de Órgãos , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/metabolismo , Fagócitos/metabolismo , Transporte Proteico , Vacinação
6.
J Virol ; 92(6)2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29298888

RESUMO

Human and chimpanzee adenovirus vectors are being developed to circumvent preexisting antibodies against common adenovirus vectors such as Ad5. However, baseline immunity to these vectors still exists in human populations. Traditional cloning of new adenovirus vaccine vectors is a long and cumbersome process that takes 2 months or more and that requires rare unique restriction enzyme sites. Here we describe a novel, restriction enzyme-independent method for rapid cloning of new adenovirus vaccine vectors that reduces the total cloning procedure to 1 week. We developed 14 novel adenovirus vectors from rhesus monkeys that can be grown to high titers and that are immunogenic in mice. All vectors grouped with the unusual adenovirus species G and show extremely low seroprevalence in humans. Rapid cloning of novel adenovirus vectors is a promising approach for the development of new vector platforms. Rhesus adenovirus vectors may prove useful for clinical development.IMPORTANCE To overcome baseline immunity to human and chimpanzee adenovirus vectors, we developed 14 novel adenovirus vectors from rhesus monkeys. These vectors are immunogenic in mice and show extremely low seroprevalence in humans. Rhesus adenovirus vectors may prove useful for clinical development.


Assuntos
Adenoviridae , Vacinas contra Adenovirus , Clonagem Molecular , Vetores Genéticos , Imunogenicidade da Vacina/genética , Células A549 , Adenoviridae/genética , Adenoviridae/imunologia , Vacinas contra Adenovirus/genética , Vacinas contra Adenovirus/imunologia , Animais , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Humanos , Macaca mulatta , Camundongos
7.
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
8.
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
9.
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
10.
J Virol ; 86(3): 1623-37, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22130529

RESUMO

The species B human adenoviruses (HAdVs) infect cells upon attaching to CD46 or desmoglein 2 (DSG-2) by one or several of their 12 fiber knob trimers (FKs). To test whether DSG-2 and CD46 simultaneously serve as virus receptors for adenovirus type 3 (Ad3), we performed individual and combined CD46/DSG-2 loss-of-function studies in human lung A549 and 16HBE14o cells. Our results suggest that in these cells, DSG-2 functions as a major attachment receptor for Ad3, whereas CD46 exerts a minor contribution to virus attachment and uptake in the range of ∼10%. However, in other cells the role of CD46 may be more pronounced depending on, e.g., the expression levels of the receptors. To test if avidity allows Ad3/7 to use CD46 as a receptor, we performed gain-of-function studies. The cell surface levels of ectopically expressed CD46 in CHO or human M010119 melanoma cells lacking DSG-2 positively correlated with Ad3/7 infections, while Ad11/35 infections depended on CD46 but less on CD46 levels. Antibody-cross-linked soluble CD46 blocked Ad3/7/11/35 infections, while soluble CD46 alone blocked Ad11/35 but not Ad3/7. Soluble Ad3/7-FKs poorly inhibited Ad3/7 infection of CHO-CD46 cells, illustrating that Ad3/7-FKs bind with low affinity to CD46. This was confirmed by Biacore studies. Ad3/7-FK binding to immobilized CD46 at low density was not detected, unlike that of Ad11/35-FK. At higher CD46 densities, however, Ad3/7-FK bound to CD46 with only 15-fold-higher dissociation constants than those of Ad11/35-FK. These data show that an avidity mechanism for Ad3/7 binding to CD46 leads to infection of CD46-positive cells.


Assuntos
Adenoviridae/imunologia , Afinidade de Anticorpos , Proteína Cofatora de Membrana/imunologia , Adenoviridae/fisiologia , Infecções por Adenoviridae , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Receptores Virais/fisiologia , Ressonância de Plasmônio de Superfície
11.
J Virol ; 84(10): 5336-50, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20237079

RESUMO

Human adenovirus serotype 35 (HAdV-35; here referred to as Ad35) causes kidney and urinary tract infections and infects respiratory organs of immunocompromised individuals. Unlike other adenoviruses, Ad35 has a low seroprevalence, which makes Ad35-based vectors promising candidates for gene therapy. Ad35 utilizes CD46 and integrins as receptors for infection of epithelial and hematopoietic cells. Here we show that infectious entry of Ad35 into HeLa cells, human kidney HK-2 cells, and normal human lung fibroblasts strongly depended on CD46 and integrins but not heparan sulfate and variably required the large GTPase dynamin. Ad35 infections were independent of expression of the carboxy-terminal domain of AP180, which effectively blocks clathrin-mediated uptake. Ad35 infections were inhibited by small chemicals against serine/threonine kinase Pak1 (p21-activated kinase), protein kinase C (PKC), sodium-proton exchangers, actin, and acidic organelles. Remarkably, the F-actin inhibitor jasplakinolide, the Pak1 inhibitor IPA-3, or the sodium-proton exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) blocked endocytic uptake of Ad35. Dominant-negative proteins or small interfering RNAs against factors driving macropinocytosis, including the small GTPase Rac1, Pak1, or the Pak1 effector C-terminal binding protein 1 (CtBP1), potently inhibited Ad35 infection. Confocal laser scanning microscopy, electron microscopy, and live cell imaging showed that Ad35 colocalized with fluid-phase markers in large endocytic structures that were positive for CD46, alphanu integrins, and also CtBP1. Our results extend earlier observations with HAdV-3 (Ad3) and establish macropinocytosis as an infectious pathway for species B human adenoviruses in epithelial and hematopoietic cells.


Assuntos
Adenovírus Humanos/fisiologia , Células Epiteliais/virologia , Pinocitose , Internalização do Vírus , Linhagem Celular , Fibroblastos/virologia , Humanos , Integrinas/fisiologia , Proteína Cofatora de Membrana/fisiologia , Receptores Virais/fisiologia
12.
J Gen Virol ; 90(Pt 7): 1600-1610, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19282435

RESUMO

The coxsackievirus-adenovirus receptor (CAR) is the described primary receptor for adenovirus serotype 5 (Ad5), a common human pathogen that has been exploited as a viral vector for gene therapy and vaccination. This study showed that monocytes and dendritic cells (DCs), such as freshly isolated human blood myeloid DCs, plasmacytoid DCs and monocyte-derived DCs, are susceptible to recombinant Ad5 (rAd5) infection despite their lack of CAR expression. Langerhans cells and dermal DCs from skin expressed CAR, but blocking CAR only partly decreased rAd5 infection, together suggesting that other receptor pathways mediate viral entry of these cells. Lactoferrin (Lf), an abundant protein in many bodily fluids known for its antiviral and antibacterial properties, promoted rAd5 infection in all cell populations except plasmacytoid DCs using a CAR-independent process. Lf caused phenotypic differentiation of the DCs, but cell activation played only a minor role in the increase in infection frequencies. The C-type lectin receptor DC-SIGN facilitated viral entry of rAd5-Lf complexes and this was dependent on high-mannose-type N-linked glycans on Lf. These results suggest that Lf present at high levels at mucosal sites can facilitate rAd5 attachment and enhance infection of DCs. A better understanding of the tropism and receptor mechanisms of Ad5 may help explain Ad5 pathogenesis and guide the engineering of improved rAd vectors.


Assuntos
Adenovírus Humanos/fisiologia , Proteínas de Transporte/fisiologia , Moléculas de Adesão Celular/fisiologia , Células Dendríticas/virologia , Lectinas Tipo C/fisiologia , Receptores de Superfície Celular/fisiologia , Receptores Virais/fisiologia , Ligação Viral , Células Cultivadas , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Humanos , Lactoferrina , Monócitos/virologia
13.
J Virol ; 83(1): 479-83, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18945780

RESUMO

The deployment of adenovirus serotype 5 (Ad5)-based vectors is hampered by preexisting immunity. When such vectors are delivered intravenously, hepatocyte transduction is mediated by the hexon-coagulation factor X (FX) interaction. Here, we demonstrate that human sera efficiently block FX-mediated cellular binding and transduction of Ad5-based vectors in vitro. Neutralizing activity correlated well with the ability to inhibit Ad5-mediated liver transduction, suggesting that prescreening patient sera in this manner accurately predicts the efficacy of Ad5-based gene therapies. Neutralization in vitro can be partially bypassed by pseudotyping with Ad45 fiber protein, indicating that a proportion of neutralizing antibodies are directed against the Ad5 fiber.


Assuntos
Adenoviridae/imunologia , Fator X/metabolismo , Vetores Genéticos/imunologia , Hepatócitos/virologia , Soros Imunes/metabolismo , Linhagem Celular Tumoral , Terapia Genética/métodos , Humanos , Testes de Neutralização , Transdução Genética , Ligação Viral
14.
Nature ; 457(7225): 87-91, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18997770

RESUMO

A recombinant adenovirus serotype 5 (rAd5) vector-based vaccine for HIV-1 has recently failed in a phase 2b efficacy study in humans. Consistent with these results, preclinical studies have demonstrated that rAd5 vectors expressing simian immunodeficiency virus (SIV) Gag failed to reduce peak or setpoint viral loads after SIV challenge of rhesus monkeys (Macaca mulatta) that lacked the protective MHC class I allele Mamu-A*01 (ref. 3). Here we show that an improved T-cell-based vaccine regimen using two serologically distinct adenovirus vectors afforded substantially improved protective efficacy in this challenge model. In particular, a heterologous rAd26 prime/rAd5 boost vaccine regimen expressing SIV Gag elicited cellular immune responses with augmented magnitude, breadth and polyfunctionality as compared with the homologous rAd5 regimen. After SIV(MAC251) challenge, monkeys vaccinated with the rAd26/rAd5 regimen showed a 1.4 log reduction of peak and a 2.4 log reduction of setpoint viral loads as well as decreased AIDS-related mortality as compared with control animals. These data demonstrate that durable partial immune control of a pathogenic SIV challenge for more than 500 days can be achieved by a T-cell-based vaccine in Mamu-A*01-negative rhesus monkeys in the absence of a homologous Env antigen. These findings have important implications for the development of next-generation T-cell-based vaccine candidates for HIV-1.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Macaca mulatta/imunologia , Macaca mulatta/virologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Adenoviridae/genética , Animais , Anticorpos Antivirais/imunologia , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Humanos , Testes de Neutralização , Vacinas contra a SAIDS/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/mortalidade , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vacinação , Carga Viral
15.
J Immunol ; 181(6): 4188-98, 2008 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-18768876

RESUMO

A critical goal of vaccine development for a wide variety of pathogens is the induction of potent and durable mucosal immunity. However, it has been assumed that this goal would be difficult to achieve by systemic vaccination due to the anatomic and functional distinctness of the systemic and mucosal immune systems and the resultant compartmentalization of immune responses. In this study, we show that Ag-specific CD8(+) T lymphocytes traffic efficiently to mucosal surfaces following systemic vaccination. Intramuscular immunization with recombinant adenovirus (rAd) vector-based vaccines expressing SIV Gag resulted in potent, durable, and functional CD8(+) T lymphocyte responses at multiple mucosal effector sites in both mice and rhesus monkeys. In adoptive transfer studies in mice, vaccine-elicited systemic CD8(+) T lymphocytes exhibited phenotypic plasticity, up-regulated mucosal homing integrins and chemokine receptors, and trafficked rapidly to mucosal surfaces. Moreover, the migration of systemic CD8(+) T lymphocytes to mucosal compartments accounted for the vast majority of Ag-specific mucosal CD8(+) T lymphocytes induced by systemic vaccination. Thus, i.m. vaccination can overcome immune compartmentalization and generate robust mucosal CD8(+) T lymphocyte memory. These data demonstrate that the systemic and mucosal immune systems are highly coordinated following vaccination.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/transplante , Movimento Celular/imunologia , Epitopos de Linfócito T/imunologia , Imunidade nas Mucosas , Adenovírus Humanos/genética , Adenovírus Humanos/imunologia , Animais , Linfócitos T CD8-Positivos/virologia , Movimento Celular/genética , Epitopos de Linfócito T/biossíntese , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Humanos , Imunidade Celular/genética , Imunidade nas Mucosas/genética , Memória Imunológica/genética , Injeções Intramusculares , Cinética , Macaca mulatta , Camundongos , Camundongos Endogâmicos C57BL , Fase de Repouso do Ciclo Celular/genética , Fase de Repouso do Ciclo Celular/imunologia , Regulação para Cima/genética , Regulação para Cima/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Replicação Viral/genética , Replicação Viral/imunologia
16.
J Virol ; 82(14): 6829-37, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18448519

RESUMO

The development of a subunit vaccine for smallpox represents a potential strategy to avoid the safety concerns associated with replication-competent vaccinia virus. Preclinical studies to date with subunit smallpox vaccine candidates, however, have been limited by incomplete information regarding protective antigens and the requirement for multiple boost immunizations to afford protective immunity. Here we explore the protective efficacy of replication-incompetent, recombinant adenovirus serotype 35 (rAd35) vectors expressing the vaccinia virus intracellular mature virion (IMV) antigens A27L and L1R and extracellular enveloped virion (EEV) antigens A33R and B5R in a murine vaccinia virus challenge model. A single immunization with the rAd35-L1R vector effectively protected mice against a lethal systemic vaccinia virus challenge. The rAd35-L1R vector also proved more efficacious than the combination of four rAd35 vectors expressing A27L, L1R, A33R, and B5R. Moreover, serum containing L1R-specific neutralizing antibodies afforded postexposure prophylaxis after systemic vaccinia virus infection. In contrast, the combination of rAd35-L1R and rAd35-B5R vectors was required to protect mice against a lethal intranasal vaccinia virus challenge, suggesting that both IMV- and EEV-specific immune responses are important following intranasal infection. Taken together, these data demonstrate that different protective antigens are required based on the route of vaccinia virus challenge. These studies also suggest that rAd vectors warrant further assessment as candidate subunit smallpox vaccines.


Assuntos
Vacina Antivariólica/imunologia , Varíola/imunologia , Varíola/prevenção & controle , Vírus Vaccinia/imunologia , Adenoviridae/genética , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/uso terapêutico , Peso Corporal , Quimioprevenção/métodos , Vírus da Ectromelia/genética , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Vacina Antivariólica/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia
17.
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
18.
Cell ; 132(3): 397-409, 2008 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-18267072

RESUMO

Adenoviruses are used extensively as gene transfer agents, both experimentally and clinically. However, targeting of liver cells by adenoviruses compromises their potential efficacy. In cell culture, the adenovirus serotype 5 fiber protein engages the coxsackievirus and adenovirus receptor (CAR) to bind cells. Paradoxically, following intravascular delivery, CAR is not used for liver transduction, implicating alternate pathways. Recently, we demonstrated that coagulation factor (F)X directly binds adenovirus leading to liver infection. Here, we show that FX binds to the Ad5 hexon, not fiber, via an interaction between the FX Gla domain and hypervariable regions of the hexon surface. Binding occurs in multiple human adenovirus serotypes. Liver infection by the FX-Ad5 complex is mediated through a heparin-binding exosite in the FX serine protease domain. This study reveals an unanticipated function for hexon in mediating liver gene transfer in vivo.


Assuntos
Adenovírus Humanos/fisiologia , Proteínas do Capsídeo/metabolismo , Fator X/metabolismo , Fígado/virologia , Transdução Genética , Internalização do Vírus , Adenovírus Humanos/química , Adenovírus Humanos/classificação , Animais , Proteínas do Capsídeo/química , Proteínas de Transporte/metabolismo , Microscopia Crioeletrônica , Fator X/química , Hepatócitos/virologia , Humanos , Imageamento Tridimensional , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Filogenia , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Ressonância de Plasmônio de Superfície , Varfarina/farmacologia
19.
Infect Immun ; 76(4): 1709-18, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18212075

RESUMO

Prime-boost vaccination regimens with heterologous antigen delivery systems have indicated that redirection of the immune response is feasible. We showed earlier that T-cell responses to circumsporozoite (CS) protein improved significantly when the protein is primed with recombinant adenovirus serotype 35 coding for CS (rAd35.CS). The current study was designed to answer the question whether such an effect can be extended to liver-stage antigens (LSA) of Plasmodium falciparum such as LSA-1. Studies with mice have demonstrated that the LSA-1 protein induces strong antibody response but a weak T-cell immunity. We first identified T-cell epitopes in LSA-1 by use of intracellular gamma interferon (IFN-gamma) staining and confirmed these epitopes by means of enzyme-linked immunospot assay and pentamer staining. We show that a single immunization with rAd35.LSA-1 induced a strong antigen-specific IFN-gamma CD8(+) T-cell response but no measurable antibody response. In contrast, vaccinations with the adjuvanted recombinant LSA-1 protein induced remarkably low cellular responses but strong antibody responses. Finally, both priming and boosting of the adjuvanted protein by rAd35 resulted in enhanced T-cell responses without impairing the level of antibody responses induced by the protein immunizations alone. Furthermore, the incorporation of rAd35 in the vaccination schedule led to a skewing of LSA-1-specific antibody responses toward a Th1-type immune response. Our results show the ability of rAd35 to induce potent T-cell immunity in combination with protein in a prime-boost schedule without impairing the B-cell response.


Assuntos
Adenoviridae , Antígenos de Protozoários/imunologia , Linfócitos B/imunologia , Imunização Secundária , Vacinas Antimaláricas/imunologia , Plasmodium falciparum/metabolismo , Linfócitos T/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/química , Relação Dose-Resposta Imunológica , Mapeamento de Epitopos , Epitopos de Linfócito T , Feminino , Malária Falciparum/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium falciparum/química , Vacinas Sintéticas/imunologia
20.
Vaccine ; 25(49): 8338-45, 2007 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-17977629

RESUMO

Studies were performed with an inactivated vaccine against the mosquito-borne flavivirus, West Nile virus (WNV). The mammalian cell line, PER.C6, was selected as the platform for WNV growth since both the neurovirulent strains NY99 and ISR98 that cause epidemics in humans and high mortality in geese, respectively, could be propagated to high titers (10(9) to 10(10)TCID(50)/ml) on these cells. Based on the high DNA homology of the WNV envelope (E) protein and non-structural protein 5 (NS5), and identical neurovirulence in mice and geese, we concluded that NY99 and ISR98 viruses are closely related and therefore vaccine studies were performed with ISR98 as a model for NY99. A robust challenge model in domestic geese was set up resulting in 100% mortality within 7 days of intracranial challenge with 500 TCID(50) WNV. Geese were used to assess the efficacy and safety of an inactivated WNV vaccine produced on PER.C6 cells. Efficacy studies demonstrated 91.4% (53/58) protection of geese compared to no protection (0/13) in geese receiving a sham vaccine. A follow-up study in 1800 geese showed that the vaccine was safe with a survival rate of 96.6% (95% lower CL 95.7%). Initial studies on the correlates of protection induced by the vaccine indicate an important role for antibodies since geese were protected when injected intra-cranial with a mixture of serum from vaccinated, non-challenged geese and WNV. In all, these results provide a scientific basis for the development of an inactivated WNV vaccine based on NY99 produced on PER.C6 cells for human and equine use.


Assuntos
Gansos/virologia , Doenças das Aves Domésticas/prevenção & controle , Vacinas de Produtos Inativados/efeitos adversos , Vacinas de Produtos Inativados/uso terapêutico , Febre do Nilo Ocidental/veterinária , Vacinas contra o Vírus do Nilo Ocidental/efeitos adversos , Vacinas contra o Vírus do Nilo Ocidental/uso terapêutico , Animais , Animais Lactentes , Linhagem Celular , Humanos , Dose Letal Mediana , Camundongos , Doenças das Aves Domésticas/virologia , Retina/citologia , Resultado do Tratamento , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Replicação Viral , Febre do Nilo Ocidental/mortalidade , Febre do Nilo Ocidental/prevenção & controle , Febre do Nilo Ocidental/virologia , Vacinas contra o Vírus do Nilo Ocidental/administração & dosagem , Vacinas contra o Vírus do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/fisiologia
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