RESUMO
Adeno-associated viral (AAV) vectors are currently being tested in multiple clinical trials for liver-directed gene transfer to treat the bleeding disorders hemophilia A and B and metabolic disorders. The optimal viral capsid for transduction of human hepatocytes has been under active investigation, but results across various models are inconsistent. We tested in vivo transduction in "humanized" mice. Methods to quantitate percent AAV transduced human and murine hepatocytes in chimeric livers were optimized using flow cytometry and confocal microscopy with image analysis. Distinct transduction efficiencies were noted following peripheral vein administration of a self-complementary vector expressing a gfp reporter gene. An engineered AAV3 capsid with two amino acid changes, S663V+T492V (AAV3-ST), showed best efficiency for human hepatocytes (~3-times, ~8-times, and ~80-times higher than for AAV9, AAV8, and AAV5, respectively). AAV5, 8, and 9 were more efficient in transducing murine than human hepatocytes. AAV8 yielded the highest transduction rate of murine hepatocytes, which was 19-times higher than that for human hepatocytes. In summary, our data show substantial differences among AAV serotypes in transduction of human and mouse hepatocytes, are the first to report on AAV5 in humanized mice, and support the use of AAV3-based vectors for human liver gene transfer.
Assuntos
Proteínas do Capsídeo/genética , Dependovirus/genética , Vetores Genéticos/administração & dosagem , Hepatócitos/ultraestrutura , Animais , Células Cultivadas , Dependovirus/metabolismo , Hepatócitos/metabolismo , Humanos , Camundongos , Especificidade de Órgãos , Engenharia de Proteínas , Transdução GenéticaRESUMO
Available COVID-19 vaccine only provide protection for a limited time due in part to the rapid emergence of viral variants with spike protein mutations, necessitating the generation of new vaccines to combat SARS-CoV-2. Two serologically distinct replication-defective chimpanzee-origin adenovirus (Ad) vectors (AdC) called AdC6 and AdC7 expressing early SARS-CoV-2 isolate spike (S) or nucleocapsid (N) proteins, the latter expressed as a fusion protein within herpes simplex virus glycoprotein D (gD), were tested individually or as a mixture in a hamster COVID-19 SARS-CoV-2 challenge model. The S protein expressing AdC (AdC-S) vectors induced antibodies including those with neutralizing activity that in part cross-reacted with viral variants. Hamsters vaccinated with the AdC-S vectors were protected against serious disease and showed accelerated recovery upon SARS-CoV-2 challenge. Protection was enhanced if AdC-S vectors were given together with the AdC vaccines that expressed the gD N fusion protein (AdC-gDN). In contrast hamsters that just received the AdC-gDN vaccines showed only marginal lessening of symptoms compared to control animals. These results indicate that immune response to the N protein that is less variable than the S protein may potentiate and prolong protection achieved by the currently used S protein based genetic COVID-19 vaccines.
Assuntos
COVID-19 , Animais , Cricetinae , Humanos , COVID-19/prevenção & controle , SARS-CoV-2/genética , Vacinas contra COVID-19/genética , Pan troglodytes , Adenoviridae/genética , Nucleocapsídeo , Imunização , Anticorpos Antivirais , Anticorpos NeutralizantesRESUMO
Vaccines that aim to expand tumor-specific CD8(+) T cells have yielded disappointing results in cancer patients although they showed efficacy in transplantable tumor mouse models. Using a system that more faithfully mimics a progressing cancer and its immunoinhibitory microenvironment, we here show that in transgenic mice, which gradually develop adenocarcinomas due to expression of HPV-16 E7 within their thyroid, a highly immunogenic vaccine expressing E7 only induces low E7-specific CD8(+) T-cell responses, which fail to affect the size of the tumors. In contrast, the same type of vaccine expressing E7 fused to herpes simplex virus (HSV)-1 glycoprotein D (gD), an antagonist of the coinhibitory B- and T-lymphocyte attenuator (BTLA)/CD160-herpes virus entry mediator (HVEM) pathways, stimulates potent E7-specific CD8(+) T-cell responses, which can be augmented by repeated vaccination, resulting in initial regression of even large tumor masses in all mice with sustained regression in more than half of them. These results indicate that active immunization concomitantly with blockade of the immunoinhibitory HVEM-BTLA/CD160 pathways through HSV-1 gD may result in sustained tumor regression.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Imunoterapia Ativa/métodos , Neoplasias/terapia , Proteínas E7 de Papillomavirus/imunologia , Animais , Animais Geneticamente Modificados , Biomarcadores , Linfócitos T CD8-Positivos/metabolismo , Regulação da Expressão Gênica , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/imunologia , Neoplasias/metabolismo , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Membro 14 de Receptores do Fator de Necrose Tumoral/genética , Membro 14 de Receptores do Fator de Necrose Tumoral/imunologia , Membro 14 de Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Glândula Tireoide/imunologia , Vacinação/métodosRESUMO
Antibody responses to vaccinations or infections decline upon aging. In this study we tested if metabolic changes in B cells may contribute to attenuation of responses to influenza vaccination in aged humans. Our data show that aging affects mitochondrial functions in B cells leading to increases in mitochondrial reactive oxygen species (MROS) and mitochondrial mass (MM) in some aged B cell subsets and decreases in expression levels of Sirtuin 1 (SIRT1), Forkhead box protein (FOX)O1 and carnitine palmitoyltransferase 1 (CPT-1). Seahorse analyses showed minor defects in glycolysis in the aged B cells after activation but a strong reduction in oxidative phosphorylation. The analyses of the transcriptome revealed further pronounced defects in one-carbon metabolism, a pathway that is essential for amino acid and nucleotide metabolism. Overall our data support the notion that the declining ability of aged B cells to increase their metabolism following activation contributes to the weakened antibody responses of the elderly.
Assuntos
Envelhecimento/imunologia , Formação de Anticorpos , Linfócitos B/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Metabolismo Energético , Feminino , Humanos , MasculinoRESUMO
This chapter reviews the performance of viral vectors based on adenoviruses or adeno-associated virus as vaccine carriers for infectious diseases. Replication-defective adenovirus vectors based on multiple human or non-human serotypes have consistently induced potent transgene product-specific B and T cell responses and are increasingly being explored in human clinical trials. The immunogenicity of most vectors based on adeno-associated virus vectors has been poor with the exception of a recently described hybrid vector from rhesus macaques that due to its ability to induce potent responses in mice warrant further investigation.
Assuntos
Adenoviridae/genética , Dependovirus/genética , Portadores de Fármacos , Vetores Genéticos , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Imunidade Adaptativa , Animais , Linfócitos B/imunologia , Ensaios Clínicos como Assunto , Humanos , Linfócitos T/imunologiaRESUMO
We analyzed gene expression profiles of young and aged mouse CD8+ T cells specific for the nucleoprotein (NP) of influenza A/PR8/34 virus. CD8+ T cells were stimulated either by the NP antigen expressed in its native form or fused into the herpes virus (HSV)-1 glycoprotein D (gD) protein, which blocks signaling through the immunoinhibitory B and T lymphocyte attenuator (BTLA) and CD160 pathways. We show that NP-specific CD8+ T cells from aged mice exhibit numerous differences in gene expression compared to NP-specific CD8+ T cells from young mice, including a significant reduction of expression in genes involved in T cell receptor (TcR) and CD28 signaling. We also show that these changes can be reversed in a sub-population (~50%) of the aged mice by a BTLA/CD160 checkpoint blockade. These results suggest that BTLA/CD160 checkpoint blockade has potential value as a vaccine additive to induce better CD8+ T cell responses in the aged.
Assuntos
Envelhecimento/fisiologia , Antígenos CD/metabolismo , Linfócitos T CD8-Positivos/fisiologia , Regulação da Expressão Gênica/imunologia , Receptores Imunológicos/metabolismo , Transcriptoma/fisiologia , Animais , Antígenos CD/genética , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Vírus da Influenza A , Vacinas contra Influenza/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/prevenção & controle , Receptores Imunológicos/genética , VacinaçãoRESUMO
The goal of these studies was to test whether adeno-associated virus (AAV) capsid-specific CD8(+) T cells cause loss of hepatic AAV-mediated gene expression in experimental animals. Mice immunized with adenoviral vectors expressing AAV capsid or with AAV vectors developed CD8(+) T cells in blood, lymphatic tissues, and liver to epitopes shared between AAV2 and AAV8, and serotype-specific neutralizing antibodies. At the height of the T cells' effector phase, mice were infused with a heterologous AAV vector expressing human factor IX under a hepatocyte-specific promoter. Despite the presence of lytic CD8(+) T cells in the liver, hepatic Factor IX expression was sustained and comparable in AAV-preimmune and naïve animals. These results suggest that, in mice, pre-existing CD8(+) T cells to AAV capsid do not affect the longevity of AAV-mediated hepatic gene transfer. These results are in contrast to the outcome of a recent gene therapy trial of hemophilia B patients who were treated by hepatic gene transfer of AAV2 vectors expressing Factor IX. The loss of Factor IX expression, accompanied by a rise in liver enzymes and detectable frequencies of circulating AAV capsid-specific T cells, suggested T-cell-mediated destruction of transduced hepatocytes following reactivation of AAV-specific T cells upon AAV transfer.