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1.
Nat Immunol ; 20(6): 756-764, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110315

RESUMO

Emerging data show that tissue-resident memory T (TRM) cells play an important protective role at murine and human barrier sites. TRM cells in the epidermis of mouse skin patrol their surroundings and rapidly respond when antigens are encountered. However, whether a similar migratory behavior is performed by human TRM cells is unclear, as technology to longitudinally follow them in situ has been lacking. To address this issue, we developed an ex vivo culture system to label and track T cells in fresh skin samples. We validated this system by comparing in vivo and ex vivo properties of murine TRM cells. Using nanobody labeling, we subsequently demonstrated in human ex vivo skin that CD8+ TRM cells migrated through the papillary dermis and the epidermis, below sessile Langerhans cells. Collectively, this work allows the dynamic study of resident immune cells in human skin and provides evidence of tissue patrol by human CD8+ TRM cells.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Memória Imunológica , Pele/imunologia , Animais , Antígenos/imunologia , Linhagem Celular Tumoral , Movimento Celular/imunologia , Epiderme/imunologia , Epiderme/metabolismo , Imunofluorescência , Humanos , Camundongos , Especificidade de Órgãos/imunologia , Anticorpos de Domínio Único/imunologia , Pele/metabolismo , Vacinas de DNA/genética , Vacinas de DNA/imunologia
2.
Immunity ; 54(12): 2859-2876.e7, 2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-34788599

RESUMO

Repeat antigens, such as the Plasmodium falciparum circumsporozoite protein (PfCSP), use both sequence degeneracy and structural diversity to evade the immune response. A few PfCSP-directed antibodies have been identified that are effective at preventing malaria infection, including CIS43, but how these repeat-targeting antibodies might be improved has been unclear. Here, we engineered a humanized mouse model in which B cells expressed inferred human germline CIS43 (iGL-CIS43) B cell receptors and used both vaccination and bioinformatic analysis to obtain variant CIS43 antibodies with improved protective capacity. One such antibody, iGL-CIS43.D3, was significantly more potent than the current best-in-class PfCSP-directed antibody. We found that vaccination with a junctional epitope peptide was more effective than full-length PfCSP at recruiting iGL-CIS43 B cells to germinal centers. Structure-function analysis revealed multiple somatic hypermutations that combinatorically improved protection. This mouse model can thus be used to understand vaccine immunogens and to develop highly potent anti-malarial antibodies.


Assuntos
Subpopulações de Linfócitos B/imunologia , Epitopos/imunologia , Vacinas Antimaláricas/imunologia , Malária/imunologia , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/imunologia , Vacinas de DNA/imunologia , Transferência Adotiva , Animais , Anticorpos Antiprotozoários/metabolismo , Modelos Animais de Doenças , Epitopos/genética , Engenharia Genética , Humanos , Evasão da Resposta Imune , Imunogenicidade da Vacina , Camundongos , Camundongos SCID , Proteínas de Protozoários/genética , Relação Estrutura-Atividade , Vacinação
3.
Immunity ; 54(3): 542-556.e9, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33631118

RESUMO

A combination of vaccination approaches will likely be necessary to fully control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane-anchored pre-fusion stabilized spike (MVA/S) but not secreted S1 induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8+ T cell responses, and conferred protection from SARS-CoV-2 infection and virus replication in the lungs as early as day 2 following intranasal and intratracheal challenge. Single-cell RNA sequencing analysis of lung cells on day 4 after infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities and lowered induction of interferon-stimulated genes. These results demonstrate that MVA/S vaccination induces neutralizing antibodies and CD8+ T cells in the blood and lungs and is a potential vaccine candidate for SARS-CoV-2.


Assuntos
Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Vetores Genéticos/genética , SARS-CoV-2/imunologia , Vacinas de DNA/imunologia , Vaccinia virus/genética , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Antígenos Virais/imunologia , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Vacinas contra COVID-19/genética , Modelos Animais de Doenças , Expressão Gênica , Ordem dos Genes , Imunofenotipagem , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Macaca , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/patologia , Camundongos , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Vacinação/métodos , Vacinas de DNA/genética
4.
Proc Natl Acad Sci U S A ; 121(25): e2322264121, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38865265

RESUMO

Despite the tremendous clinical potential of nucleic acid-based vaccines, their efficacy to induce therapeutic immune response has been limited by the lack of efficient local gene delivery techniques in the human body. In this study, we develop a hydrogel-based organic electronic device (µEPO) for both transdermal delivery of nucleic acids and in vivo microarrayed cell electroporation, which is specifically oriented toward one-step transfection of DNAs in subcutaneous antigen-presenting cells (APCs) for cancer immunotherapy. The µEPO device contains an array of microneedle-shaped electrodes with pre-encapsulated dry DNAs. Upon a pressurized contact with skin tissue, the electrodes are rehydrated, electrically triggered to release DNAs, and then electroporate nearby cells, which can achieve in vivo transfection of more than 50% of the cells in the epidermal and upper dermal layer. As a proof-of-concept, the µEPO technique is employed to facilitate transdermal delivery of neoantigen genes to activate antigen-specific immune response for enhanced cancer immunotherapy based on a DNA vaccination strategy. In an ovalbumin (OVA) cancer vaccine model, we show that high-efficiency transdermal transfection of APCs with OVA-DNAs induces robust cellular and humoral immune responses, including antigen presentation and generation of IFN-γ+ cytotoxic T lymphocytes with a more than 10-fold dose sparing over existing intramuscular injection (IM) approach, and effectively inhibits tumor growth in rodent animals.


Assuntos
Eletroporação , Imunoterapia , Vacinas de DNA , Animais , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Eletroporação/métodos , Camundongos , Imunoterapia/métodos , Administração Cutânea , Neoplasias/terapia , Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/administração & dosagem , Ovalbumina/imunologia , Ovalbumina/administração & dosagem , Células Apresentadoras de Antígenos/imunologia , Feminino , Camundongos Endogâmicos C57BL , Humanos , Vacinação/métodos
5.
Proc Natl Acad Sci U S A ; 120(44): e2306465120, 2023 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-37871214

RESUMO

Nucleic acid vaccines have shown promising results in the clinic against infectious diseases and cancers. To robustly improve the vaccine efficacy and safety, we developed an approach to increase the intracellular stability of nucleic acids by transiently inhibiting lysosomal function in targeted tissues using sucrose. To achieve efficient and localized delivery of sucrose in animals, we designed a biomimetic lipid nanoparticle (LNP) to target the delivery of sucrose into mouse muscle cells. Using this approach, viral antigen expression in mouse muscle after DNA vaccination was substantially increased and prolonged without inducing local or systemic inflammation or toxicity. The same change in antigen expression would be achieved if the vaccine dose could be increased by 3,000 folds, which is experimentally and clinically impractical due to material restrictions and severe toxicity that will be induced by such a high dose of nucleic acids. The increase in antigen expression augmented the infiltration and activation of antigen-presenting cells, significantly improved vaccine-elicited humoral and T cell responses, and fully protected mice against the viral challenge at a low dose of vaccine. Based on these observations, we conclude that transient inhibition of lysosome function in target tissue by sucrose LNPs is a safe and potent approach to substantially improve nucleic acid-based vaccines.


Assuntos
Nanopartículas , Ácidos Nucleicos , Vacinas de DNA , Vacinas , Animais , Camundongos , Vacinas Baseadas em Ácido Nucleico , Lisossomos , Sacarose
6.
Proc Natl Acad Sci U S A ; 120(5): e2215091120, 2023 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-36696444

RESUMO

A foundational principle of rational vaccinology is that vaccine structure plays a critical role in determining therapeutic efficacy, but in order to establish fundamental, effective, and translatable vaccine design parameters, a highly modular and well-defined platform is required. Herein, we report a DNA dendron vaccine, a molecular nanostructure that consists of an adjuvant DNA strand that splits into multiple DNA branches with a varied number of conjugated peptide antigens that is capable of dendritic cell uptake, immune activation, and potent cancer killing. We leveraged the well-defined architecture and chemical modularity of the DNA dendron to study structure-function relationships that dictate molecular vaccine efficacy, particularly regarding the delivery of immune-activating DNA sequences and antigenic peptides on a single chemical construct. We investigated how adjuvant and antigen placement and number impact dendron cellular uptake and immune activation, in vitro. These parameters also played a significant role in raising a potent and specific immune response against target cancer cells. By gaining this structural understanding of molecular vaccines, DNA dendrons successfully treated a mouse cervical human papillomavirus TC-1 cancer model, in vivo, where the vaccine structure defined its efficacy; the top-performing design effectively reduced tumor burden (<150 mm3 through day 30) and maintained 100% survival through 44 d after tumor inoculation.


Assuntos
Vacinas Anticâncer , Dendrímeros , Vacinas contra Papillomavirus , Neoplasias do Colo do Útero , Vacinas de DNA , Animais , Feminino , Camundongos , Humanos , Dendrímeros/farmacologia , Neoplasias do Colo do Útero/prevenção & controle , DNA , Peptídeos , Vacinas contra Papillomavirus/genética
7.
J Immunol ; 211(11): 1680-1692, 2023 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-37850965

RESUMO

Nucleic acid vaccines, including both RNA and DNA platforms, are key technologies that have considerable promise in combating both infectious disease and cancer. However, little is known about the extrinsic factors that regulate nucleic acid vaccine responses and which may determine their effectiveness. The microbiome is recognized as a significant regulator of immune development and response, whose role in regulating some traditional vaccine platforms has recently been discovered. Using germ-free and specific pathogen-free mouse models in combination with different protein, DNA, and mRNA vaccine regimens, we demonstrate that the microbiome is a significant regulator of nucleic acid vaccine immunogenicity. Although the presence of the microbiome enhances CD8+ T cell responses to mRNA lipid nanoparticle immunization, the microbiome suppresses Ig and CD4+ T cell responses to DNA-prime, DNA-protein-boost immunization, indicating contrasting roles for the microbiome in the regulation of these different nucleic acid vaccine platforms. In the case of mRNA lipid nanoparticle vaccination, germ-free mice display reduced dendritic cell/macrophage activation that may underlie the deficient vaccine response. Our study identifies the microbiome as a relevant determinant of nucleic acid vaccine response with implications for continued therapeutic development and deployment of these vaccines.


Assuntos
Microbiota , Vacinas de DNA , Camundongos , Animais , Vacinas Baseadas em Ácido Nucleico , Linfócitos T CD8-Positivos , DNA , RNA Mensageiro , Imunização Secundária
8.
Proc Natl Acad Sci U S A ; 119(15): e2110987119, 2022 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-35385352

RESUMO

Antigen-specific immunotherapy involves the delivery of self-antigens as proteins or peptides (or using nucleic acids encoding them) to reestablish tolerance. The Endotope platform supports the optimal presentation of endogenously expressed epitopes on appropriate major histocompatibility complex (MHC) class I and II molecules. Using specific epitopes that are disease-relevant (including neoepitopes and mimotopes) and restricted to the subject's MHC haplotypes provides a more focused and tailored way of targeting autoreactive T cells. We evaluated the efficacy of an Endotope DNA vaccine tailored to the nonobese diabetic (NOD) mouse in parallel to one expressing the Proinsulin protein, a central autoantigen in NOD mice, and assessed the influence of several parameters (e.g., route, dosing frequency, disease stage) on diabetes prevention. Secretion of encoded peptides and intradermal delivery of DNA offered more effective disease prevention. Long-term weekly treatments were needed to achieve protection that can persist after discontinuation, likely mediated by regulatory T cells induced by at least one epitope. Although epitopes were presented for at least 2 wk, weekly treatments were needed, at least initially, to achieve significant protection. While Endotope and Proinsulin DNA vaccines were effective at both the prediabetic normoglycemic and dysglycemic stages of disease, Proinsulin provided better protection in the latter stage, particularly in animals with slower progression of disease, and Endotope limited insulitis the most in the earlier stage. Thus, our data support the possibility of applying a precision medicine approach based on tailored epitopes for the treatment of tissue-specific autoimmune diseases with DNA vaccines.


Assuntos
Diabetes Mellitus Tipo 1 , Proinsulina , Vacinas de DNA , Animais , Diabetes Mellitus Tipo 1/prevenção & controle , Epitopos de Linfócito T/imunologia , Camundongos , Camundongos Endogâmicos NOD , Medicina de Precisão , Proinsulina/genética , Proinsulina/imunologia , Vacinação , Vacinas de DNA/imunologia
9.
Proc Natl Acad Sci U S A ; 119(14): e2119093119, 2022 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-35312341

RESUMO

SignificanceUsing SARS-CoV-2 as a relevant case study for infectious disease, we investigate the structure-function relationships that dictate antiviral spherical nucleic acid (SNA) vaccine efficacy. We show that the SNA architecture can be rapidly employed to target COVID-19 through incorporation of the receptor-binding domain, and that the resulting vaccine potently activates human cells in vitro and mice in vivo. Furthermore, when challenged with a lethal viral infection, only mice treated with the SNA vaccine survived. Taken together, this work underscores the importance of rational vaccine design for infectious disease to yield vaccines that elicit more potent immune responses to effectively fight disease.


Assuntos
Controle de Doenças Transmissíveis , Ácidos Nucleicos/imunologia , Vacinas de DNA/imunologia , Animais , Biotecnologia , COVID-19/prevenção & controle , Controle de Doenças Transmissíveis/métodos , Doenças Transmissíveis/etiologia , Doenças Transmissíveis/imunologia , Humanos , Ácidos Nucleicos/química , SARS-CoV-2/imunologia , Desenvolvimento de Vacinas , Vacinas de DNA/genética , Vacinas Virais/genética , Vacinas Virais/imunologia
10.
J Infect Dis ; 229(1): 30-38, 2024 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-37380156

RESUMO

BACKGROUND: Andes virus (ANDV), a rodent-borne hantavirus, causes hantavirus pulmonary syndrome (HPS). The safety and immunogenicity of a novel ANDV DNA vaccine was evaluated. METHODS: Phase 1, double-blind, dose-escalation trial randomly assigned 48 healthy adults to placebo or ANDV DNA vaccine delivered via needle-free jet injection. Cohorts 1 and 2 received 2 mg of DNA or placebo in a 3-dose (days 1, 29, 169) or 4-dose (days 1, 29, 57, 169) schedule, respectively. Cohorts 3 and 4 received 4 mg of DNA or placebo in the 3-dose and 4-dose schedule, respectively. Subjects were monitored for safety and neutralizing antibodies by pseudovirion neutralization assay (PsVNA50) and plaque reduction neutralization test (PRNT50). RESULTS: While 98% and 65% of subjects had at least 1 local or systemic solicited adverse event (AE), respectively, most AEs were mild or moderate; no related serious AEs were detected. Cohorts 2, 3, and 4 had higher seroconversion rates than cohort 1 and seropositivity of at least 80% by day 197, sustained through day 337. PsVNA50 geometric mean titers were highest for cohort 4 on and after day 197. CONCLUSIONS: This first-in-human candidate HPS vaccine trial demonstrated that an ANDV DNA vaccine was safe and induced a robust, durable immune response. Clinical Trials Registration. NCT03682107.


Assuntos
Síndrome Pulmonar por Hantavirus , Orthohantavírus , Vacinas de DNA , Adulto , Humanos , Vacinas de DNA/efeitos adversos , Anticorpos Neutralizantes , DNA , Imunogenicidade da Vacina , Método Duplo-Cego , Anticorpos Antivirais
11.
Mol Cancer ; 23(1): 46, 2024 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-38459592

RESUMO

Nucleic acid vaccines have shown promising potency and efficacy for cancer treatment with robust and specific T-cell responses. Improving the immunogenicity of delivered antigens helps to extend therapeutic efficacy and reduce dose-dependent toxicity. Here, we systematically evaluated chemokine-fused HPV16 E6/E7 antigen to improve the cellular and humoral immune responses induced by nucleotide vaccines in vivo. We found that fusion with different chemokines shifted the nature of the immune response against the antigens. Although a number of chemokines were able to amplify specific CD8 + T-cell or humoral response alone or simultaneously. CCL11 was identified as the most potent chemokine in improving immunogenicity, promoting specific CD8 + T-cell stemness and generating tumor rejection. Fusing CCL11 with E6/E7 antigen as a therapeutic DNA vaccine significantly improved treatment effectiveness and caused eradication of established large tumors in 92% tumor-bearing mice (n = 25). Fusion antigens with CCL11 expanded the TCR diversity of specific T cells and induced the infiltration of activated specific T cells, neutrophils, macrophages and dendritic cells (DCs) into the tumor, which created a comprehensive immune microenvironment lethal to tumor. Combination of the DNA vaccine with anti-CTLA4 treatment further enhanced the therapeutic effect. In addition, CCL11 could also be used for mRNA vaccine design. To summarize, CCL11 might be a potent T cell enhancer against cancer.


Assuntos
Vacinas Anticâncer , Neoplasias , Proteínas Oncogênicas Virais , Vacinas contra Papillomavirus , Vacinas de DNA , Animais , Camundongos , Vacinas Baseadas em Ácido Nucleico , Vacinas de DNA/genética , Vacinas contra Papillomavirus/genética , Neoplasias/genética , Neoplasias/terapia , Linfócitos T CD8-Positivos , Proteínas E7 de Papillomavirus/genética , Proteínas Oncogênicas Virais/genética , Camundongos Endogâmicos C57BL , Microambiente Tumoral
12.
N Engl J Med ; 385(12): e35, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34525286

RESUMO

BACKGROUND: Although Zika virus (ZIKV) infection is typically self-limiting, other associated complications such as congenital birth defects and the Guillain-Barré syndrome are well described. There are no approved vaccines against ZIKV infection. METHODS: In this phase 1, open-label clinical trial, we evaluated the safety and immunogenicity of a synthetic, consensus DNA vaccine (GLS-5700) encoding the ZIKV premembrane and envelope proteins in two groups of 20 participants each. The participants received either 1 mg or 2 mg of vaccine intradermally, with each injection followed by electroporation (the use of a pulsed electric field to introduce the DNA sequence into cells) at baseline, 4 weeks, and 12 weeks. RESULTS: The median age of the participants was 38 years, and 60% were women; 78% were White and 22% Black; in addition, 30% were Hispanic. At the interim analysis at 14 weeks (i.e., after the third dose of vaccine), no serious adverse events were reported. Local reactions at the vaccination site (e.g., injection-site pain, redness, swelling, and itching) occurred in approximately 50% of the participants. After the third dose of vaccine, binding antibodies (as measured on enzyme-linked immunosorbent assay) were detected in all the participants, with geometric mean titers of 1642 and 2871 in recipients of 1 mg and 2 mg of vaccine, respectively. Neutralizing antibodies developed in 62% of the samples on Vero-cell assay. On neuronal-cell assay, there was 90% inhibition of ZIKV infection in 70% of the serum samples and 50% inhibition in 95% of the samples. The intraperitoneal injection of postvaccination serum protected 103 of 112 IFNAR knockout mice (bred with deletion of genes encoding interferon-α and interferon-ß receptors) (92%) that were challenged with a lethal dose of ZIKV-PR209 strain; none of the mice receiving baseline serum survived the challenge. Survival was independent of the neutralization titer. CONCLUSIONS: In this phase 1, open-label clinical trial, a DNA vaccine elicited anti-ZIKV immune responses. Further studies are needed to better evaluate the safety and efficacy of the vaccine. (Funded by GeneOne Life Science and others; ZIKA-001 ClinicalTrials.gov number, NCT02809443.).


Assuntos
Anticorpos Neutralizantes/sangue , Imunogenicidade da Vacina , Vacinas de DNA , Vacinas Virais/imunologia , Infecção por Zika virus/prevenção & controle , Zika virus/imunologia , Adulto , Animais , Anticorpos Antivirais/sangue , Feminino , Humanos , Injeções Intradérmicas/efeitos adversos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Linfócitos T/fisiologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/efeitos adversos , Vacinas de DNA/imunologia , Infecção por Zika virus/imunologia
13.
Cancer Immunol Immunother ; 73(12): 248, 2024 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-39358555

RESUMO

BACKGROUND: Tumor-reactive T cells play a crucial role in anti-tumor responses, but T cells induced by DNA vaccination are time-consuming processes and exhibit limited anti-tumor efficacy. Therefore, we evaluated the anti-tumor effectiveness of reactive T cells elicited by electroporation (EP)-mediated DNA vaccine targeting epidermal growth factor receptor variant III (pEGFRvIII plasmid), in conjunction with adoptive cell therapy (ACT), involving the transfer of lymphocytes from a pEGFRvIII EP-vaccinated healthy donor. METHODS: The validation of the established pEGFRvIII plasmid and EGFRvIII-positive cell model was confirmed through immunofluorescence and western blot analysis. Flow cytometry and cytotoxicity assays were performed to evaluate the functionality of antigen-specific reactive T cells induced by EP-mediated pEGFRvIII vaccines, ACT, or their combination. The anti-tumor effectiveness of EP-mediated pEGFRvIII vaccines alone or combined with ACT was evaluated in the B16F10-EGFRvIII tumor model. RESULTS: EP-mediated pEGFRvIII vaccines elicited serum antibodies and a robust cellular immune response in both healthy and tumor-bearing mice. However, this response only marginally inhibited early-stage tumor growth in established tumor models. EP-mediated pEGFRvIII vaccination followed by adoptive transfer of lymphocytes from vaccinated healthy donors led to notable anti-tumor efficacy, attributed to the synergistic action of antigen-specific CD4+ Th1 cells supplemented by ACT and antigen-specific CD8+ T cells elicited by the EP-mediated DNA vaccination. CONCLUSIONS: Our preclinical studies results demonstrate an enhanced anti-tumor efficacy of EP-mediated DNA vaccination boosted with adoptively transferred, vaccinated healthy donor-derived allogeneic lymphocytes.


Assuntos
Vacinas Anticâncer , Eletroporação , Vacinas de DNA , Animais , Vacinas de DNA/imunologia , Eletroporação/métodos , Camundongos , Vacinas Anticâncer/imunologia , Camundongos Endogâmicos C57BL , Imunoterapia Adotiva/métodos , Feminino , Humanos , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Modelos Animais de Doenças , Linhagem Celular Tumoral , Células Alógenas/imunologia , Receptores ErbB/imunologia
14.
Cancer Immunol Immunother ; 73(9): 178, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38954031

RESUMO

Intracranial tumors present a significant therapeutic challenge due to their physiological location. Immunotherapy presents an attractive method for targeting these intracranial tumors due to relatively low toxicity and tumor specificity. Here we show that SCIB1, a TRP-2 and gp100 directed ImmunoBody® DNA vaccine, generates a strong TRP-2 specific immune response, as demonstrated by the high number of TRP2-specific IFNγ spots produced and the detection of a significant number of pentamer positive T cells in the spleen of vaccinated mice. Furthermore, vaccine-induced T cells were able to recognize and kill B16HHDII/DR1 cells after a short in vitro culture. Having found that glioblastoma multiforme (GBM) expresses significant levels of PD-L1 and IDO1, with PD-L1 correlating with poorer survival in patients with the mesenchymal subtype of GBM, we decided to combine SCIB1 ImmunoBody® with PD-1 immune checkpoint blockade to treat mice harboring intracranial tumors expressing TRP-2 and gp100. Time-to-death was significantly prolonged, and this correlated with increased CD4+ and CD8+ T cell infiltration in the tissue microenvironment (TME). However, in addition to PD-L1 and IDO, the GBM TME was found to contain a significant number of immunoregulatory T (Treg) cell-associated transcripts, and the presence of such cells is likely to significantly affect clinical outcome unless also tackled.


Assuntos
Neoplasias Encefálicas , Vacinas Anticâncer , Inibidores de Checkpoint Imunológico , Receptor de Morte Celular Programada 1 , Vacinas de DNA , Animais , Feminino , Humanos , Camundongos , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/terapia , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral , Glioblastoma/imunologia , Glioblastoma/terapia , Glioblastoma/tratamento farmacológico , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunoterapia/métodos , Oxirredutases Intramoleculares , Camundongos Endogâmicos C57BL , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Vacinas de DNA/imunologia , Vacinas de DNA/uso terapêutico , Masculino , Criança , Pessoa de Meia-Idade
15.
BMC Biotechnol ; 24(1): 71, 2024 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-39350162

RESUMO

BACKGROUND: Human papillomavirus type 16 (HPV-16) infection is strongly associated with considerable parts of cervical, neck, and head cancers. Performed investigations have had moderate clinical success, so research to reach an efficient vaccine has been of great interest. In the present study, the immunization potential of a newly designed HPV-16 construct was evaluated in a mouse model. RESULTS: Initially, a construct containing HPV-16 mutant (m) E6/E7 fusion gene was designed and antigen produced in two platforms (i.e., DNA vaccine and recombinant protein). Subsequently, the immunogenicity of these platforms was investigated in five mice) C57BL/6 (groups based on several administration strategies. Three mice groups were immunized recombinant protein, DNA vaccine, and a combination of them, and two other groups were negative controls. The peripheral blood mononuclear cells (PBMCs) proliferation, Interleukin-5 (IL-5) and interferon-γ (IFN-γ) cytokines, IgG1 and IgG2a antibody levels were measured. After two weeks, TC-1 tumor cells were injected into all mice groups, and subsequently further analysis of tumor growth and metastasis and mice survival were performed according to the schedule. Overall, the results obtained from in vitro immunology and tumor cells challenging assays indicated the potential of the mE6/E7 construct as an HPV16 therapeutic vaccine candidate. The results demonstrated a significant increase in IFN-γ cytokine (P value < 0.05) in the Protein/Protein (D) and DNA/Protein (E) groups. This finding was in agreement with in vivo assays. Control groups show a 10.5-fold increase (P value < 0.001) and (C) DNA/DNA group shows a 2.5-fold increase (P value < 0.01) in tumor growth compared to D and E groups. Also, a significant increase in survival of D and E (P value < 0.001) and C (P value < 0.01) groups were observed. CONCLUSIONS: So, according to the findings, the recombinant protein could induce stronger protection compared to the DNA vaccine form. Protein/Protein and DNA/Protein are promising administration strategies for presenting this construct to develop an HPV-16 therapeutic vaccine candidate.


Assuntos
Papillomavirus Humano 16 , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas Virais , Proteínas E7 de Papillomavirus , Vacinas contra Papillomavirus , Proteínas Repressoras , Vacinas de DNA , Animais , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/imunologia , Camundongos , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Vacinas de DNA/imunologia , Vacinas de DNA/genética , Vacinas de DNA/administração & dosagem , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/imunologia , Vacinas contra Papillomavirus/imunologia , Vacinas contra Papillomavirus/genética , Vacinas contra Papillomavirus/administração & dosagem , Feminino , Infecções por Papillomavirus/prevenção & controle , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/imunologia , Modelos Animais de Doenças , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia
16.
Annu Rev Genet ; 50: 595-618, 2016 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-27893966

RESUMO

Plastid-made biopharmaceuticals treat major metabolic or genetic disorders, including Alzheimer's, diabetes, hypertension, hemophilia, and retinopathy. Booster vaccines made in chloroplasts prevent global infectious diseases, such as tuberculosis, malaria, cholera, and polio, and biological threats, such as anthrax and plague. Recent advances in this field include commercial-scale production of human therapeutic proteins in FDA-approved cGMP facilities, development of tags to deliver protein drugs to targeted human cells or tissues, methods to deliver precise doses, and long-term stability of protein drugs at ambient temperature, maintaining their efficacy. Codon optimization utilizing valuable information from sequenced chloroplast genomes enhanced expression of eukaryotic human or viral genes in chloroplasts and offered unique insights into translation in chloroplasts. Support from major biopharmaceutical companies, development of hydroponic production systems, and evaluation by regulatory agencies, including the CDC, FDA, and USDA, augur well for advancing this novel concept to the clinic and revolutionizing affordable healthcare.


Assuntos
Cloroplastos/genética , Engenharia Genética/métodos , Proteínas Recombinantes/farmacologia , Vacinas de DNA/farmacologia , Administração Oral , Doença de Alzheimer/tratamento farmacológico , Animais , Expressão Gênica , Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Hemofilia A/tratamento farmacológico , Humanos , Hipertensão/tratamento farmacológico , Plantas Geneticamente Modificadas , Plastídeos/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética
17.
J Virol ; 97(2): e0169422, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36719241

RESUMO

Viral subunit vaccines contain the specific antigen deemed most important for development of protective immune responses. Typically, the chosen antigen is a surface protein involved in cellular entry of the virus, and neutralizing antibodies may prevent this. For influenza, hemagglutinin (HA) is thus a preferred antigen. However, the natural trimeric form of HA is often not considered during subunit vaccine development. Here, we have designed a vaccine format that maintains the trimeric HA conformation while targeting antigen toward major histocompatibility complex class II (MHCII) molecules or chemokine receptors on antigen-presenting cells (APC) for enhanced immunogenicity. Results demonstrated that a single DNA vaccination induced strong antibody and T-cell responses in mice. Importantly, a single DNA vaccination also protected mice from lethal challenges with influenza viruses H1N1 and H5N1. To further evaluate the versatility of the format, we developed MHCII-targeted HA from influenza A/California/04/2009(H1N1) as a protein vaccine and benchmarked this against Pandemrix and Flublok. These vaccine formats are different, but similar immune responses obtained with lower vaccine doses indicated that the MHCII-targeted subunit vaccine has an immunogenicity and efficacy that warrants progression to larger animals and humans. IMPORTANCE Subunit vaccines present only selected viral proteins to the immune system and allow for safe and easy production. Here, we have developed a novel vaccine where influenza hemagglutinin is presented in the natural trimeric form and then steered toward antigen-presenting cells for increased immunogenicity. We demonstrate efficient induction of antibodies and T-cell responses, and demonstrate that the vaccine format can protect mice against influenza subtypes H1N1, H5N1, and H7N1.


Assuntos
Vacinas contra Influenza , Infecções por Orthomyxoviridae , Animais , Camundongos , Anticorpos Antivirais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1 , Virus da Influenza A Subtipo H5N1 , Vírus da Influenza A Subtipo H7N1 , Vacinas contra Influenza/imunologia , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/prevenção & controle , Estações do Ano , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de DNA/imunologia , Células Apresentadoras de Antígenos/imunologia , Linfócitos T/imunologia
18.
J Med Virol ; 96(2): e29452, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38314852

RESUMO

The continuous evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been accompanied by the emergence of viral mutations that pose a great challenge to existing vaccine strategies. It is not fully understood with regard to the role of mutations on the SARS-CoV-2 spike protein from emerging viral variants in T cell immunity. In the current study, recombinant eukaryotic plasmids were constructed as DNA vaccines to express the spike protein from multiple SARS-CoV-2 strains. These DNA vaccines were used to immunize BALB/c mice, and cross-T cell responses to the spike protein from these viral strains were quantitated using interferon-γ (IFN-γ) Elispot. Peptides covering the full-length spike protein from different viral strains were used to detect epitope-specific IFN-γ+ CD4+ and CD8+ T cell responses by fluorescence-activated cell sorting. SARS-CoV-2 Delta and Omicron BA.1 strains were found to have broad T cell cross-reactivity, followed by the Beta strain. The landscapes of T cell epitopes on the spike protein demonstrated that at least 30 mutations emerging from Alpha to Omicron BA.5 can mediate the escape of T cell immunity. Omicron and its sublineages have 19 out of these 30 mutations, most of which are new, and a few are inherited from ancient circulating variants of concerns. The cross-T cell immunity between SARS-CoV-2 prototype strain and Omicron strains can be attributed to the T cell epitopes located in the N-terminal domain (181-246 aa [amino acids], 271-318 aa) and C-terminal domain (1171-1273 aa) of the spike protein. These findings provide in vivo evidence for optimizing vaccine manufacturing and immunization strategies for current or future viral variants.


Assuntos
COVID-19 , Vacinas de DNA , Animais , Camundongos , Humanos , Epitopos de Linfócito T/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Imunidade Celular , Mutação , Interferon gama , Anticorpos Antivirais , Anticorpos Neutralizantes
19.
J Med Virol ; 96(6): e29749, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38888113

RESUMO

Human immunodeficiency virus (HIV) infection is still a global public health issue, and the development of an effective prophylactic vaccine inducing potent neutralizing antibodies remains a significant challenge. This study aims to explore the inflammation-related proteins associated with the neutralizing antibodies induced by the DNA/rTV vaccine. In this study, we employed the Olink chip to analyze the inflammation-related proteins in plasma in healthy individuals receiving HIV candidate vaccine (DNA priming and recombinant vaccinia virus rTV boosting) and compared the differences between neutralizing antibody-positive (nab + ) and -negative(nab-) groups. We identified 25 differentially expressed factors and conducted enrichment and correlation analysis on them. Our results revealed that significant expression differences in artemin (ARTN) and C-C motif chemokine ligand 23 (CCL23) between nab+ and -nab- groups. Notably, the expression of CCL23 was negatively corelated to the ID50 of neutralizing antibodies and the intensity of the CD4+ T cell responses. This study enriches our understanding of the immune picture induced by the DNA/rTV vaccine, and provides insights for future HIV vaccine development.


Assuntos
Vacinas contra a AIDS , Anticorpos Neutralizantes , Anticorpos Anti-HIV , Infecções por HIV , HIV-1 , Proteômica , Vaccinia virus , Humanos , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Vaccinia virus/imunologia , Vaccinia virus/genética , Anticorpos Anti-HIV/sangue , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , HIV-1/genética , Adulto , Vacinas contra a AIDS/imunologia , Masculino , Infecções por HIV/imunologia , Vacinas de DNA/imunologia , Feminino , Voluntários Saudáveis , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/administração & dosagem , Plasma/imunologia , Adulto Jovem
20.
Microb Pathog ; 195: 106871, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39163919

RESUMO

The H9N2 avian influenza virus (AIV) is spreading worldwide. Presence of H9N2 virus tends to increase the chances of infection with other pathogens which can lead to more serious economic losses. In a previous study, a regulated delayed lysis Salmonella vector was used to deliver a DNA vaccine named pYL233 encoding M1 protein, mosaic HA protein and chicken GM-CSF adjuvant. To further increase its efficiency, chitosan as a natural adjuvant was applied in this study. The purified plasmid pYL233 was coated with chitosan to form a DNA containing nanoparticles (named CS233) by ionic gel method and immunized by intranasal boost immunization in birds primed by oral administration with Salmonella strain. The CS233 DNA nanoparticle has a particle size of about 150 nm, with an encapsulation efficiency of 93.2 ± 0.12 % which protected the DNA plasmid from DNase I digestion and could be stable for a period of time at 37°. After intranasal boost immunization, the CS233 immunized chickens elicited higher antibody response, elevated CD4+ T cells and CD8+ T cells activation and increased T-lymphocyte proliferation, as well as increased productions of IL-4 and IFN-γ. After challenge, chickens immunized with CS233 resulted in the lowest levels of pulmonary virus titer and viral shedding as compared to the other challenge groups. The results showed that the combination of intranasal immunization with chitosan-coated DNA vaccine and oral immunization with regulatory delayed lytic Salmonella strain could enhance the immune response and able to provide protection against H9N2 challenge.


Assuntos
Administração Intranasal , Anticorpos Antivirais , Galinhas , Quitosana , Imunidade Celular , Vírus da Influenza A Subtipo H9N2 , Vacinas contra Influenza , Influenza Aviária , Plasmídeos , Vacinas de DNA , Eliminação de Partículas Virais , Animais , Vírus da Influenza A Subtipo H9N2/imunologia , Vírus da Influenza A Subtipo H9N2/genética , Vacinas de DNA/imunologia , Vacinas de DNA/administração & dosagem , Influenza Aviária/prevenção & controle , Influenza Aviária/imunologia , Galinhas/imunologia , Vacinas contra Influenza/imunologia , Vacinas contra Influenza/administração & dosagem , Anticorpos Antivirais/sangue , Plasmídeos/genética , Nanopartículas , Imunização Secundária , Linfócitos T CD8-Positivos/imunologia , Adjuvantes Imunológicos/administração & dosagem , Interferon gama , Interleucina-4 , Adjuvantes de Vacinas , Doenças das Aves Domésticas/prevenção & controle , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia , Linfócitos T CD4-Positivos/imunologia , Salmonella/imunologia , Salmonella/genética
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