RESUMO
A phase 1/2 clinical trial evaluating dosing, safety, immunogenicity, and overall survival on metastatic colorectal cancer (mCRC) patients after immunotherapy with an advanced-generation Ad5 [E1-, E2b-]-CEA(6D) vaccine was performed. We report our extended observations on long-term overall survival and further immune analyses on a subset of treated patients including assessment of cytolytic T cell responses, T regulatory (Treg) to T effector (Teff) cell ratios, flow cytometry on peripheral blood mononuclear cells (PBMCs), and determination of HLA-A2 status. An overall survival of 20 % (median survival 11 months) was observed during long-term follow-up, and no long-term adverse effects were reported. Cytolytic T cell responses increased after immunizations, and cell-mediated immune (CMI) responses were induced whether or not patients were HLA-A2 positive or Ad5 immune. PBMC samples from a small subset of patients were available for follow-up immune analyses. It was observed that the levels of carcinoembryonic antigen (CEA)-specific CMI activity decreased from their peak values during follow-up in five patients analyzed. Preliminary results revealed that activated CD4+ and CD8+ T cells were detected in a post-immunization sample exhibiting high CMI activity. Treg to Teff cell ratios were assessed, and samples from three of five patients exhibited a decrease in Treg to Teff cell ratio during the treatment protocol. Based upon the favorable safety and immunogenicity data obtained, we plan to perform an extensive immunologic and survival analysis on mCRC patients to be enrolled in a randomized/controlled clinical trial that investigates Ad5 [E1-, E2b-]-CEA(6D) as a single agent with booster immunizations.
Assuntos
Vacinas Anticâncer/uso terapêutico , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/terapia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Adenoviridae , Proteínas E1 de Adenovirus/genética , Proteínas E2 de Adenovirus/genética , Adulto , Idoso , Vacinas Anticâncer/imunologia , Células Cultivadas , Neoplasias Colorretais/patologia , Citotoxicidade Imunológica , Feminino , Seguimentos , Humanos , Imunização , Interferon gama/metabolismo , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Oligopeptídeos/genética , Oligopeptídeos/imunologia , Deleção de Sequência/genética , Análise de SobrevidaRESUMO
First-generation, E1-deleted adenovirus subtype 5 (Ad5)-based vectors, although promising platforms for use as cancer vaccines, are impeded in activity by naturally occurring or induced Ad-specific neutralizing antibodies. Ad5-based vectors with deletions of the E1 and the E2b regions (Ad5 [E1-, E2b-]), the latter encoding the DNA polymerase and the pre-terminal protein, by virtue of diminished late phase viral protein expression, were hypothesized to avoid immunological clearance and induce more potent immune responses against the encoded tumor antigen transgene in Ad-immune hosts. Indeed, multiple homologous immunizations with Ad5 [E1-, E2b-]-CEA(6D), encoding the tumor antigen carcinoembryonic antigen (CEA), induced CEA-specific cell-mediated immune (CMI) responses with antitumor activity in mice despite the presence of preexisting or induced Ad5-neutralizing antibody. In the present phase I/II study, cohorts of patients with advanced colorectal cancer were immunized with escalating doses of Ad5 [E1-, E2b-]-CEA(6D). CEA-specific CMI responses were observed despite the presence of preexisting Ad5 immunity in a majority (61.3 %) of patients. Importantly, there was minimal toxicity, and overall patient survival (48 % at 12 months) was similar regardless of preexisting Ad5 neutralizing antibody titers. The results demonstrate that, in cancer patients, the novel Ad5 [E1-, E2b-] gene delivery platform generates significant CMI responses to the tumor antigen CEA in the setting of both naturally acquired and immunization-induced Ad5-specific immunity.
Assuntos
Vacinas Anticâncer/imunologia , Antígeno Carcinoembrionário/imunologia , Neoplasias Colorretais/imunologia , Vetores Genéticos/imunologia , Linfócitos T/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Adulto , Idoso , Anticorpos Neutralizantes/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Antígeno Carcinoembrionário/genética , Estudos de Coortes , Neoplasias Colorretais/terapia , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos/genética , Humanos , Imunização/métodos , Interferon gama/imunologia , Interferon gama/metabolismo , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Linfócitos T/metabolismo , Fatores de Tempo , Resultado do TratamentoRESUMO
We have developed a COVID-19 vaccine, hAd5 S-Fusion + N-ETSD, that expresses SARS-CoV-2 spike (S) and nucleocapsid (N) proteins with modifications to increase immune responses delivered using a human adenovirus serotype 5 (hAd5) platform. Here, we demonstrate subcutaneous (SC) prime and SC boost vaccination of CD-1 mice with this dual-antigen vaccine elicits T-helper cell 1 (Th1) biased T-cell and humoral responses to both S and N that are greater than those seen with hAd5 S wild type delivering only unmodified S. We then compared SC to intranasal (IN) prime vaccination with SC or IN boosts and show that an IN prime with an IN boost is as effective at generating Th1 biased humoral responses as the other combinations tested, but an SC prime with an IN or SC boost elicits greater T cell responses. Finally, we used a combined SC plus IN (SC + IN) prime with or without a boost and found the SC + IN prime alone to be as effective in generating humoral and T-cell responses as the SC + IN prime with a boost. The finding that SC + IN prime-only delivery has the potential to provide broad immunity-including mucosal immunity-against SARS-CoV-2 supports further testing of this vaccine and delivery approach in animal models of viral challenge.
Assuntos
Vacinas contra COVID-19/administração & dosagem , COVID-19/imunologia , COVID-19/prevenção & controle , SARS-CoV-2/imunologia , Adenoviridae/genética , Administração Intranasal , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , Feminino , Vetores Genéticos , Hipodermóclise , Imunidade Celular/imunologia , Imunidade nas Mucosas/imunologia , Imunização Secundária , Camundongos , Camundongos Endogâmicos , Vacinação/métodosRESUMO
We have developed a dual-antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike protein (S-Fusion) and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to increase the potential for MHC class II responses. The vaccine antigens are delivered by a human adenovirus serotype 5 platform, hAd5 [E1-, E2b-, E3-], previously demonstrated to be effective in the presence of Ad immunity. Vaccination of rhesus macaques with the hAd5 S-Fusion + N-ETSD vaccine by subcutaneous prime injection followed by two oral boosts elicited neutralizing anti-S IgG and T helper cell 1-biased T-cell responses to both S and N that protected the upper and lower respiratory tracts from high titer (1 x 106 TCID50) SARS-CoV-2 challenge. Notably, viral replication was inhibited within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge.
Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adenovírus Humanos/genética , Adenovírus Humanos/imunologia , Adenovírus Humanos/metabolismo , Administração Oral , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Vacinas contra COVID-19/administração & dosagem , Citocinas/sangue , Imunização Secundária/métodos , Imunoglobulina G/sangue , Pulmão/virologia , Macaca mulatta , Nariz/virologia , Fosfoproteínas/imunologia , Domínios Proteicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Vacinação , Replicação Viral/imunologiaRESUMO
Adenovirus serotype 5 (Ad5) has been widely used in clinical trials because it expresses inserted transgenes robustly and augments the innate immune response. Strategies to improve Ad5 vectors that can circumvent Ad5 immunity have become a critical issue, especially for use as a cancer immunotherapeutic in which repeated immunization is required. In this study, we constructed a novel Ad5 vector with unique deletions of the viral DNA polymerase and the pre-terminal protein region (Ad5 [E1-, E2b-]). This vector contains the carcinoembryonic antigen (CEA) gene insert and is designed to induce cell-mediated immunity (CMI) against the tumor-associated target. The CEA immunogenicity and in vivo anti-tumor effects of repeated immunizations with Ad5 [E1-, E2b-]-CEA compared with those observed with current generation Ad5 [E1-]-CEA were tested in Ad5 pre-immunized mice. We report that Ad5-immune mice immunized multiple times with Ad5 [E1-, E2b-]-CEA induced CEA-specific CMI responses that were significantly increased over those detected in Ad5-immune mice immunized multiple times with a current generation Ad5 [E1-]-CEA. Ad5 immune mice bearing CEA-expressing tumors that were treated with Ad5 [E1-, E2b-]-CEA had increased anti-tumor response as compared with Ad5 [E1-]-CEA treated mice. These results demonstrate that Ad5 [E1-, E2b-]-CEA can induce CMI immune responses which result in tumor growth inhibition despite the presence of pre-existing Ad5 immunity. Multiple re-immunizations using the same vector platform are now possible with the novel Ad5 [E1-, E2b-] platform.
Assuntos
Adenoviridae/genética , Antígeno Carcinoembrionário/imunologia , Imunoterapia/métodos , Neoplasias Experimentais/terapia , Adenoviridae/imunologia , Proteínas E1 de Adenovirus/genética , Proteínas E2 de Adenovirus/genética , Animais , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/imunologia , Antígeno Carcinoembrionário/genética , Linhagem Celular , Linhagem Celular Tumoral , Deleção de Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Humanos , Imunização/métodos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Resultado do TratamentoRESUMO
Lassa virus (LASV), the causative agent of Lassa fever (LF), was first identified in 1969. Since then, outbreaks in the endemic countries of Nigeria, Liberia, and Sierra Leone occur on an annual basis resulting in a case-fatality rate of 15-70% in hospitalized patients. There is currently no licensed vaccine and there are limited animal models to test vaccine efficacy. An estimated 37.7 million people are at risk of contracting LASV; therefore, there is an urgent need for the development of a safe, effective vaccine against LASV infection. The LF endemic countries are also inflicted with HIV, Ebola, and malaria infections. The safety in immunocompromised populations must be considered in LASV vaccine development. The novel adenovirus vector-based platform, Ad5 (E1-,E2b-) has been used in clinical trial protocols for treatment of immunocompromised individuals, has been shown to exhibit high stability, low safety risk in humans, and induces a strong cell-mediated and pro-inflammatory immune response even in the presence of pre-existing adenovirus immunity. To this nature, our lab has developed an Ad5 (E1-,E2b-) vector-based vaccine expressing the LASV-NP or LASV-GPC. We found that guinea pigs vaccinated with two doses of Ad5 (E1-,E2b-) LASV-NP and Ad5 (E1-,E2b-) LASV-GPC were protected against lethal LASV challenge. The Ad5 (E1-,E2b-) LASV-NP and LASV-GPC vaccine represents a potential vaccine candidate against LF.
Assuntos
Adenoviridae/genética , Vetores Genéticos/genética , Febre Lassa/imunologia , Febre Lassa/prevenção & controle , Vacinas Virais/uso terapêutico , Animais , Chlorocebus aethiops , Ensaio de Imunoadsorção Enzimática , Feminino , Cobaias , Vírus Lassa/imunologia , Vírus Lassa/patogenicidade , Células Vero , Vacinas Virais/imunologiaRESUMO
Phenotypic heterogeneity of human carcinoma lesions, including heterogeneity in expression of tumor-associated antigens (TAAs), is a well-established phenomenon. Carcinoembryonic antigen (CEA), MUC1, and brachyury are diverse TAAs, each of which is expressed on a wide range of human tumors. We have previously reported on a novel adenovirus serotype 5 (Ad5) vector gene delivery platform (Ad5 [E1-, E2b-]) in which regions of the early 1 (E1), early 2 (E2b), and early 3 (E3) genes have been deleted. The unique deletions in this platform result in a dramatic decrease in late gene expression, leading to a marked reduction in host immune response to the vector. Ad5 [E1-, E2b-]-CEA vaccine (ETBX-011) has been employed in clinical studies as an active vaccine to induce immune responses to CEA in metastatic colorectal cancer patients. We report here the development of novel recombinant Ad5 [E1-, E2b-]-brachyury and-MUC1 vaccine constructs, each capable of activating antigen-specific human T cells in vitro and inducing antigen-specific CD4+ and CD8+ T cells in vaccinated mice. We also describe the use of a combination of the three vaccines (designated Tri-Ad5) of Ad5 [E1-, E2b-]-CEA, Ad5 [E1-, E2b-]-brachyury and Ad5 [E1-, E2b-]-MUC1, and demonstrate that there is minimal to no "antigenic competition" in in vitro studies of human dendritic cells, or in murine vaccination studies. The studies reported herein support the rationale for the application of Tri-Ad5 as a therapeutic modality to induce immune responses to a diverse range of human TAAs for potential clinical studies.
Assuntos
Adenoviridae/genética , Vacinas contra Adenovirus/uso terapêutico , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/uso terapêutico , Imunoterapia , Neoplasias/terapia , Proteínas E1 de Adenovirus/genética , Proteínas E1 de Adenovirus/imunologia , Proteínas E2 de Adenovirus/genética , Proteínas E2 de Adenovirus/imunologia , Animais , Antígenos de Neoplasias/genética , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/imunologia , Células Dendríticas/imunologia , Feminino , Citometria de Fluxo , Vetores Genéticos/administração & dosagem , Humanos , Imunização , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T/imunologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The effectiveness of recombinant Adenovirus serotype 5 (Ad5) vectors to induce immune responses against targeted antigens has been limited by the presence of pre-existing or Ad5 vaccine induced anti-vector immunity. The Ad5 [E1-, E2b-] platform, a recombinant Ad5 with additional deletions, has been previously reported by us to induce immune responses in the presence of Ad5 immunity. In an Ad5 immune non-human primate (NHP) model, an Ad5 [E1-, E2b-] construct expressing HIV-1 Gag induced immune responses in the presence of pre-existing Ad5 immunity. In the present study we expand on these prior observations by comparing the cell mediated immune (CMI) responses induced by Ad5 [E1-, E2b-]-SIV-gag/nef in Ad5 naïve and Ad5 immune NHP. Additionally, NHP were immunized with an Ad5 [E1-, E2b-]-HIV-pol construct following two homologous administrations of Ad5 [E1-, E2b-]-SIV-gag/nef to determine if an immune response could be induced against a third antigen in the presence of vaccine induced Ad5 immunity. Positive CMI responses, as assessed by interferon-gamma (IFN-γ) secreting lymphocytes, were induced against all three antigens. These CMI responses increased over a course of multiple immunizations and the response profiles observed in Ad5 naïve and Ad5 immune NHP were similar. No influence of the major histocompatibility complex on CMI responses was observed. These data indicate that the new Ad5 [E1-, E2b-] platform based vaccine could be used for homologous vaccination regimes to induce robust CMI responses in the presence of Ad5 vector immunity.
Assuntos
Adenoviridae/genética , Adenoviridae/imunologia , Portadores de Fármacos , Vetores Genéticos , Vacinas contra a SAIDS/imunologia , Animais , Feminino , Deleção de Genes , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Produtos do Gene nef/genética , Produtos do Gene nef/imunologia , Produtos do Gene pol/genética , Produtos do Gene pol/imunologia , Interferon gama/metabolismo , Linfócitos/imunologia , Macaca mulatta , Masculino , Vacinas contra a SAIDS/administração & dosagem , Vacinas contra a SAIDS/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Vaccines against emerging pathogens such as the 2009 H1N1 pandemic virus can benefit from current technologies such as rapid genomic sequencing to construct the most biologically relevant vaccine. A novel platform (Ad5 [E1-, E2b-]) has been utilized to induce immune responses to various antigenic targets. We employed this vector platform to express hemagglutinin (HA) and neuraminidase (NA) genes from 2009 H1N1 pandemic viruses. Inserts were consensuses sequences designed from viral isolate sequences and the vaccine was rapidly constructed and produced. Vaccination induced H1N1 immune responses in mice, which afforded protection from lethal virus challenge. In ferrets, vaccination protected from disease development and significantly reduced viral titers in nasal washes. H1N1 cell mediated immunity as well as antibody induction correlated with the prevention of disease symptoms and reduction of virus replication. The Ad5 [E1-, E2b-] should be evaluated for the rapid development of effective vaccines against infectious diseases.
Assuntos
Hemaglutininas/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/farmacologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Neuraminidase/imunologia , Infecções por Orthomyxoviridae/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Animais , Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Consenso , Furões , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Hemaglutininas/genética , Humanos , Imunidade Celular/imunologia , Vírus da Influenza A Subtipo H1N1/genética , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Influenza Humana/virologia , Camundongos , Líquido da Lavagem Nasal/imunologia , Neuraminidase/genética , Infecções por Orthomyxoviridae/prevenção & controle , Vacinação/métodos , Replicação Viral/genética , Replicação Viral/imunologia , Eliminação de Partículas ViraisRESUMO
Adenovirus vectors have been shown to be highly effective as vaccine platforms capable of inducing both humoral and cell mediated immune (CMI) responses. An Ad serotype 5 vector containing unique deletions in the E2b region (Ad5 [E1-, E2b-]) has been reported to have several advantages over conventional Adenovirus serotype 5 (Ad5) vectors deleted in only the E1 region (Ad5 [E1-]), including increased carrying capacity and diminished viral late gene expression. Here, we evaluated a novel Ad5 [E1-, E2b-] vector utilizing the E.C7 cell line for viral packaging. Its' effectiveness as a potential vaccine platform as compared to the currently utilized Ad5 [E1-]-based platform was assessed in both Ad5 naïve and Ad5 immune mice. We employed the HIV-1 Gag gene as the antigenic transgene expressed by the novel vector. Cellular expression of the Gag was confirmed by Western Blot analysis. Dose response studies using three intradermal immunizations of 10(7) to 10(10) virus particles (VP) of each construct revealed that immunization with 10(10)VP resulted in the maximum immunological response. Multiple immunizations of Ad naïve BALB/c mice with an Ad5 [E1-, E2b]-gag vaccine resulted in higher ELISpot CMI responses as compared to mice immunized with an Ad5 [E1-]-gag vaccine. More importantly, multiple immunizations of Ad5 immune BALB/c mice with an Ad5 [E1-, E2b]-gag vaccine resulted in significant increases in ELISpot CMI responses when compared to Ad5 immune mice vaccinated with an Ad5 [E1-]-gag vector. Preliminary studies in three Ad5 immune non-human primates (NHP) demonstrated that vaccination with Ad5 [E1-, E2b-]-gag-induced elevated levels of interferon-gamma and IL-2 secreting lymphocytes as assessed by ELISpot assays. These studies indicate that the novel Ad5 [E1-, E2b-] viral vector can be utilized as a potential vaccine platform to induce elevated CMI responses as compared to current generation Ad5 [E1-] viral vectors even in the presence of pre-existing Ad5 immunity.
Assuntos
Adenoviridae/imunologia , Antígenos Virais/imunologia , Vetores Genéticos , Vacinas de DNA/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Adenoviridae/genética , Animais , Antígenos Virais/genética , Antígenos Virais/metabolismo , Relação Dose-Resposta Imunológica , Deleção de Genes , Engenharia Genética , Humanos , Imunidade Celular , Imunização Secundária , Interferon gama/metabolismo , Interleucina-2/metabolismo , Ativação Linfocitária/genética , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
Recombinant Adenovirus serotype 5 (Ad5) vectors have been used as vaccine platforms in numerous animal and human clinical studies. The immune response induced by Ad5 vaccines can be mitigated due to pre-existing Ad5 immunity. We previously reported the use of a novel Ad5 platform to induce cellular immune responses (CMI) against HIV-1 Gag in Ad5 hyper immune mice. Here, the effectiveness of the Ad5 [E1-, E2b-] vaccine platform was evaluated using a triad mixture of HIV-1 Gag, Pol, and Nef as antigenic transgenes. Broad CMI was induced following vaccination with the HIV-1 expressing vectors in Ad5 naïve and Ad5 immunized mice. A mixture of the three vaccines induced CMI against each transgene product even in the presence of hyper Ad5 immunity. These studies revealed that CMI responses to immunization with Ad5 [E1-, E2b-]-gag, Ad5 [E1-, E2b-]-pol or Ad5 [E1-, E2b-]-nef vectors were transgene specific and did not induce CMI responses against irrelevant antigens such as carcinoembryonic antigen (CEA), herpes simplex virus glycoprotein B (HSV), cytomegalovirus (CMV) or influenza virus antigens. We are evaluating this recombinant triad viral vector as an HIV-1 vaccine in a non-human primate model and the data indicate that the vaccine is worthy of clinical evaluation.