RESUMO
Ebola virus (EBOV) causes severe hemorrhagic fever for which there is no approved treatment or preventive vaccine. Immunological correlates of protective immunity against EBOV disease are not well understood. However, non-human primate studies have associated protection of experimental vaccines with binding and neutralizing antibodies to the EBOV glycoprotein (GP) as well as EBOV GP-specific CD4(+) and CD8(+) T cells. In this report a full length, unmodified Zaire EBOV GP gene from the 2014 EBOV Makona strain (EBOV/Mak) was cloned into a baculovirus vector. Recombinant EBOV/Mak GP was produced in Sf9 insect cells as glycosylated trimers and, when purified, formed spherical 30-40 nm particles. In mice, EBOV/Mak GP co-administered with the saponin adjuvant Matrix-M was significantly more immunogenic, as measured by virus neutralization titers and anti-EBOV/Mak GP IgG as compared to immunization with AlPO4 adjuvanted or non-adjuvanted EBOV/Mak GP. Similarly, antigen specific T cells secreting IFN-γ were induced most prominently by EBOV/Mak GP with Matrix-M. Matrix-M also enhanced the frequency of antigen-specific germinal center B cells and follicular helper T (TFH) cells in the spleen in a dose-dependent manner. Immunization with EBOV/Mak GP with Matrix-M was 100% protective in a lethal viral challenge murine model; whereas no protection was observed with the AlPO4 adjuvant and only 10% (1/10) mice were protected in the EBOV/Mak GP antigen alone group. Matrix-M adjuvanted vaccine induced a rapid onset of specific IgG and neutralizing antibodies, increased frequency of multifunctional CD4+ and CD8(+) T cells, specific TFH cells, germinal center B cells, and persistence of EBOV GP-specific plasma B cells in the bone marrow. Taken together, the addition of Matrix-M adjuvant to the EBOV/Mak GP nanoparticles enhanced both B and T-cell immune stimulation which may be critical for an Ebola subunit vaccine with broad and long lasting protective immunity.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Vacinas contra Ebola/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Nanopartículas , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Ebolavirus , Centro Germinativo/citologia , CamundongosRESUMO
Ovarian carcinoma is the fifth leading cause of death among women in the United States. Persistent activation of STAT3 is frequently detected in ovarian carcinoma. STAT3 is activated by Janus family kinases (JAK) via cytokine receptors, growth factor receptor, and non-growth factor receptor tyrosine kinases. Activation of STAT3 mediates tumor cell proliferation, survival, motility, invasion, and angiogenesis, and recent work demonstrates that STAT3 activation suppresses antitumor immune responses and supports tumor-promoting inflammation. We hypothesized that therapeutic targeting of the JAK/STAT3 pathway would inhibit tumor growth by direct effects on ovarian carcinoma cells and by inhibition of cells in the tumor microenvironment (TME). To test this, we evaluated the effects of a small-molecule JAK inhibitor, AZD1480, on cell viability, apoptosis, proliferation, migration, and adhesion of ovarian carcinoma cells in vitro. We then evaluated the effects of AZD1480 on in vivo tumor growth and progression, gene expression, tumor-associated matrix metalloproteinase (MMP) activity, and immune cell populations in a transgenic mouse model of ovarian carcinoma. AZD1480 treatment inhibited STAT3 phosphorylation and DNA binding, and migration and adhesion of cultured ovarian carcinoma cells and ovarian tumor growth rate, volume, and ascites production in mice. In addition, drug treatment led to altered gene expression, decreased tumor-associated MMP activity, and fewer suppressor T cells in the peritoneal TME of tumor-bearing mice than control mice. Taken together, our results show pharmacologic inhibition of the JAK2/STAT3 pathway leads to disruption of functions essential for ovarian tumor growth and progression and represents a promising therapeutic strategy.
Assuntos
Analgésicos/farmacologia , Janus Quinases/metabolismo , Neoplasias Ovarianas/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Analgésicos/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Análise por Conglomerados , Modelos Animais de Doenças , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Integrina alfaVbeta3/genética , Integrina alfaVbeta3/metabolismo , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Transgênicos , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Pirimidinas/administração & dosagem , Pirimidinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Immunization with vaccinia virus (VACV), the virus comprising the smallpox vaccine, induces memory CD8(+) T cells that protect from subsequent infections with smallpox in humans or the related ectromelia virus (ECTV) in mice. Memory CD8(+) T cells largely mediate these effects by expanding into secondary effectors that secrete the antiviral cytokine interferon-γ (IFN-γ) and induce cytolysis via releasing factors such as perforin, which permeabilizes target cells. We show that protection from ECTV infection after VACV immunization depends on the initial memory cell frequency and ability of expanded secondary effectors to kill infected targets in a perforin-dependent manner. Although IFN-γ is essential for antiviral protection, it can be produced by either secondary effectors or concomitant primary effector CD8(+) T cells recruited to the response. Thus, during lethal virus challenge, memory CD8(+) T cells are required for cytolytic killing of infected cells, but primary effectors can play important roles by producing IFN-γ.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Citotoxicidade Imunológica , Vírus da Ectromelia/imunologia , Interferon gama/metabolismo , Vacina Antivariólica/imunologia , Subpopulações de Linfócitos T/imunologia , Vírus/imunologia , Animais , Memória Imunológica , Camundongos , Vacina Antivariólica/administração & dosagemRESUMO
Direct presentation (DP) and cross presentation (CP) on MHC I by professional APCs are defined by the internal or external source of the Ag, respectively. Although some Ags are substrates for both DP and CP, others are only substrates for DP. The reasons for this difference remain largely unknown. In this study, we studied in tissue culture and also in vivo, the effects of altering the length and sequence of the amino acid chains flanking an MHC class I restricted determinant (the chicken OVA OVA(258-265), SIINFEKL) that is normally a good substrate for both DP and CP. We demonstrate that CP but not DP strictly requires flanking N and C-terminal extensions of minimal length. Furthermore, we show that removal but not replacement of just one amino acid 22 residues downstream from the determinant is sufficient to strongly affect CP without affecting either protein stability or DP. Thus, our work shows that the flanking residues of an antigenic determinant can differentially affect CP and DP, and that features of the Ag other than half-life can have a major impact in CP. Our studies may have implications for understanding CP in viral infections and possibly for the design of new vaccines.