RESUMO
BACKGROUND: Additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines that are safe and effective as primary vaccines and boosters remain urgently needed to combat the coronavirus disease 2019 (COVID-19) pandemic. We describe safety and durability of immune responses following 2 primary doses and a homologous booster dose of an investigational DNA vaccine (INO-4800) targeting full-length spike antigen. METHODS: Three dosage strengths of INO-4800 (0.5 mg, 1.0 mg, and 2.0 mg) were evaluated in 120 age-stratified healthy adults. Intradermal injection of INO-4800 followed by electroporation at 0 and 4 weeks preceded an optional booster 6-10.5 months after the second dose. RESULTS: INO-4800 appeared well tolerated with no treatment-related serious adverse events. Most adverse events were mild and did not increase in frequency with age and subsequent dosing. A durable antibody response was observed 6 months following the second dose; a homologous booster dose significantly increased immune responses. Cytokine-producing T cells and activated CD8+ T cells with lytic potential were significantly increased in the 2.0-mg dose group. CONCLUSIONS: INO-4800 was well tolerated in a 2-dose primary series and homologous booster in all adults, including elderly participants. These results support further development of INO-4800 for use as primary vaccine and booster. CLINICAL TRIALS REGISTRATION: NCT04336410.
Assuntos
COVID-19 , Vacinas de DNA , Adulto , Idoso , Anticorpos Antivirais , Formação de Anticorpos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Imunogenicidade da Vacina , SARS-CoV-2 , Vacinação/efeitos adversos , Vacinas de DNA/efeitos adversosRESUMO
mAbs are a possible adjunct to vaccination and drugs in treatment of influenza virus infection. However, questions remain whether small animal models accurately predict efficacy in humans. We have established the pig, a large natural host animal for influenza, with many physiological similarities to humans, as a robust model for testing mAbs. We show that a strongly neutralizing mAb (2-12C) against the hemagglutinin head administered prophylactically at 15 mg/kg reduced viral load and lung pathology after pandemic H1N1 influenza challenge. A lower dose of 1 mg/kg of 2-12C or a DNA plasmid-encoded version of 2-12C reduced pathology and viral load in the lungs but not viral shedding in nasal swabs. We propose that the pig influenza model will be useful for testing candidate mAbs and emerging delivery platforms prior to human trials.
Assuntos
Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Infecções por Orthomyxoviridae , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/tratamento farmacológico , SuínosRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of COVID-19, resulting in cases of mild to severe respiratory distress and significant mortality. The global outbreak of this novel coronavirus has now infected >20 million people worldwide, with >5 million cases in the United States (11 August 2020). The development of diagnostic and research tools to determine infection and vaccine efficacy is critically needed. We have developed multiple serologic assays using newly designed SARS-CoV-2 reagents for detecting the presence of receptor-binding antibodies in sera. The first assay is surface plasmon resonance (SPR) based and can quantitate both antibody binding to the SARS-CoV-2 spike protein and blocking to the Angiotensin-converting enzyme 2 (ACE2) receptor in a single experiment. The second assay is enzyme-linked immunosorbent assay (ELISA) based and can measure competition and blocking of the ACE2 receptor to the SARS-CoV-2 spike protein with antispike antibodies. The assay is highly versatile, and we demonstrate the broad utility of the assay by measuring antibody functionality of sera from small animals and nonhuman primates immunized with an experimental SARS-CoV-2 vaccine. In addition, we employ the assay to measure receptor blocking of sera from SARS-CoV-2-infected patients. The assay is shown to correlate with pseudovirus neutralization titers. This type of rapid, surrogate neutralization diagnostic can be employed widely to help study SARS-CoV-2 infection and assess the efficacy of vaccines.
Assuntos
Anticorpos Bloqueadores/sangue , Betacoronavirus/imunologia , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Peptidil Dipeptidase A/imunologia , Pneumonia Viral/diagnóstico , Enzima de Conversão de Angiotensina 2 , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Betacoronavirus/isolamento & purificação , COVID-19 , Teste para COVID-19 , Vacinas contra COVID-19 , Infecções por Coronavirus/sangue , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Ensaio de Imunoadsorção Enzimática , Cobaias , Humanos , Imunoglobulina G/sangue , Camundongos , Testes de Neutralização , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/imunologia , Primatas , Coelhos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/imunologia , Ressonância de Plasmônio de Superfície , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologiaRESUMO
We recently developed anti-OspA human immunoglobulin G1 monoclonal antibodies (HuMAbs) that are effective in preventing Borrelia transmission from ticks in a murine model. Here, we investigated a novel approach of DNA-mediated gene transfer of HuMAbs that provide protection against Lyme disease. Plasmid DNA-encoded anti-OspA HuMAbs inoculated in mice achieved a serum antibody concentration of >6 µg/mL. Among mice injected with DNA-encoded monoclonal antibodies, 75%-77% were protected against an acute challenge by Borrelia-infected ticks. Our results represent the first demonstration of employing DNA transfer as a delivery system for antibodies that block transmission of Borrelia in animal models.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Antígenos de Superfície/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , DNA Bacteriano/imunologia , Lipoproteínas/imunologia , Doença de Lyme/transmissão , Animais , Anticorpos Monoclonais Humanizados/genética , Anticorpos Monoclonais Humanizados/uso terapêutico , Antígenos de Superfície/genética , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas/genética , Borrelia burgdorferi , Feminino , Células HEK293 , Humanos , Lipoproteínas/genética , Doença de Lyme/prevenção & controle , Camundongos , Camundongos Endogâmicos C3H , Camundongos SCID , Plasmídeos/imunologia , Carrapatos , TransfecçãoRESUMO
BACKGROUND: Nonlive vaccine approaches that are simple to deliver and stable at room temperature or 2-8°C could be advantageous in controlling future Ebola virus (EBOV) outbreaks. Using an immunopotent DNA vaccine that generates protection from lethal EBOV challenge in small animals and nonhuman primates, we performed a clinical study to evaluate both intramuscular (IM) and novel intradermal (ID) DNA delivery. METHODS: Two DNA vaccine candidates (INO-4201 and INO-4202) targeting the EBOV glycoprotein (GP) were evaluated for safety, tolerability, and immunogenicity in a phase 1 clinical trial. The candidates were evaluated alone, together, or in combination with plasmid-encoded human cytokine interleukin-12 followed by in vivo electroporation using either the CELLECTRA® IM or ID delivery devices. RESULTS: The safety profile of all 5 regimens was shown to be benign, with the ID route being better tolerated. Antibodies to EBOV GP were generated by all 5 regimens with the fastest and steepest rise observed in the ID group. Cellular immune responses were generated with every regimen. CONCLUSIONS: ID delivery of INO-4201 was well tolerated and resulted in 100% seroreactivity after 2 doses and elicited interferon-γ T-cell responses in over 70% of subjects, providing a new approach for EBOV prevention in diverse populations. Clinical Trials Registration. NCT02464670.
Assuntos
Vacinas contra Ebola/efeitos adversos , Vacinas contra Ebola/imunologia , Imunidade Celular/imunologia , Imunidade Humoral/imunologia , Vacinas de DNA/efeitos adversos , Vacinas de DNA/imunologia , Adolescente , Adulto , Anticorpos Antivirais/imunologia , Ebolavirus/imunologia , Eletroporação/métodos , Feminino , Glicoproteínas/imunologia , Voluntários Saudáveis , Doença pelo Vírus Ebola/imunologia , Humanos , Injeções Intradérmicas/métodos , Interleucina-12/imunologia , Masculino , Pessoa de Meia-Idade , Temperatura , Vacinação/métodos , Adulto JovemRESUMO
Background: There remains an important need for prophylactic anti-Ebola virus vaccine candidates that elicit long-lasting immune responses and can be delivered to vulnerable populations that are unable to receive live-attenuated or viral vector vaccines. Methods: We designed novel synthetic anti-Ebola virus glycoprotein (EBOV-GP) DNA vaccines as a strategy to expand protective breadth against diverse EBOV strains and evaluated the impact of vaccine dosing and route of administration on protection against lethal EBOV-Makona challenge in cynomolgus macaques. Long-term immunogenicity was monitored in nonhuman primates for >1 year, followed by a 12-month boost. Results: Multiple-injection regimens of the EBOV-GP DNA vaccine, delivered by intramuscular administration followed by electroporation, were 100% protective against lethal EBOV-Makona challenge. Impressively, 2 injections of a simple, more tolerable, and dose-sparing intradermal administration followed by electroporation generated strong immunogenicity and was 100% protective against lethal challenge. In parallel, we observed that EBOV-GP DNA vaccination induced long-term immune responses in macaques that were detectable for at least 1 year after final vaccination and generated a strong recall response after the final boost. Conclusions: These data support that this simple intradermal-administered, serology-independent approach is likely important for additional study towards the goal of induction of anti-EBOV immunity in multiple at-risk populations.
Assuntos
Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Doença pelo Vírus Ebola/prevenção & controle , Vacinas de DNA/imunologia , Animais , Modelos Animais de Doenças , Vacinas contra Ebola/administração & dosagem , Feminino , Injeções Intramusculares , Macaca fascicularis , Masculino , Vacinas de DNA/administração & dosagemRESUMO
We developed a method of simultaneous vaccination with DNA and protein resulting in robust and durable cellular and humoral immune responses with efficient dissemination to mucosal sites and protection against simian immunodeficiency virus (SIV) infection. To further optimize the DNA-protein coimmunization regimen, we tested a SIVmac251-based vaccine formulated with either of two Toll-like receptor 4 (TLR4) ligand-based liposomal adjuvant formulations (TLR4 plus TLR7 [TLR4+7] or TLR4 plus QS21 [TLR4+QS21]) in macaques. Although both vaccines induced humoral responses of similar magnitudes, they differed in their functional quality, including broader neutralizing activity and effector functions in the TLR4+7 group. Upon repeated heterologous SIVsmE660 challenge, a trend of delayed viral acquisition was found in vaccinees compared to controls, which reached statistical significance in animals with the TRIM-5α-resistant (TRIM-5α R) allele. Vaccinees were preferentially infected by an SIVsmE660 transmitted/founder virus carrying neutralization-resistant A/K mutations at residues 45 and 47 in Env, demonstrating a strong vaccine-induced sieve effect. In addition, the delay in virus acquisition directly correlated with SIVsmE660-specific neutralizing antibodies. The presence of mucosal V1V2 IgG binding antibodies correlated with a significantly decreased risk of virus acquisition in both TRIM-5α R and TRIM-5α-moderate/sensitive (TRIM-5α M/S) animals, although this vaccine effect was more prominent in animals with the TRIM-5α R allele. These data support the combined contribution of immune responses and genetic background to vaccine efficacy. Humoral responses targeting V2 and SIV-specific T cell responses correlated with viremia control. In conclusion, the combination of DNA and gp120 Env protein vaccine regimens using two different adjuvants induced durable and potent cellular and humoral responses contributing to a lower risk of infection by heterologous SIV challenge.IMPORTANCE An effective AIDS vaccine continues to be of paramount importance for the control of the pandemic, and it has been proven to be an elusive target. Vaccine efficacy trials and macaque challenge studies indicate that protection may be the result of combinations of many parameters. We show that a combination of DNA and protein vaccinations applied at the same time provides rapid and robust cellular and humoral immune responses and evidence for a reduced risk of infection. Vaccine-induced neutralizing antibodies and Env V2-specific antibodies at mucosal sites contribute to the delay of SIVsmE660 acquisition, and genetic makeup (TRIM-5α) affects the effectiveness of the vaccine. These data are important for the design of better vaccines and may also affect other vaccine platforms.
Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Produtos do Gene env , Imunidade Humoral , Vacinas contra a SAIDS , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia , Vacinas de DNA , Adjuvantes Imunológicos/farmacologia , Substituição de Aminoácidos , Animais , Produtos do Gene env/genética , Produtos do Gene env/imunologia , Produtos do Gene env/farmacologia , Imunização , Macaca , Mutação de Sentido Incorreto , Vacinas contra a SAIDS/genética , Vacinas contra a SAIDS/imunologia , Vacinas contra a SAIDS/farmacologia , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vírus da Imunodeficiência Símia/genética , Vírus da Imunodeficiência Símia/imunologia , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/imunologia , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vacinas de DNA/farmacologiaRESUMO
HIV sequence diversity and the propensity of eliciting immunodominant responses targeting variable regions of the HIV proteome are hurdles in the development of an effective AIDS vaccine. An HIV-derived conserved element (CE) p24gag plasmid DNA (pDNA) vaccine is able to redirect immunodominant responses to otherwise subdominant and often more vulnerable viral targets. By homology to the HIV immunogen, seven CE were identified in SIV p27Gag Analysis of 31 rhesus macaques vaccinated with full-length SIV gag pDNA showed inefficient induction (58% response rate) of cellular responses targeting these CE. In contrast, all 14 macaques immunized with SIV p27CE pDNA developed robust T cell responses recognizing CE. Vaccination with p27CE pDNA was also critical for the efficient induction and increased the frequency of Ag-specific T cells with cytotoxic potential (granzyme B+ CD107a+) targeting subdominant CE epitopes, compared with the responses elicited by the p57gag pDNA vaccine. Following p27CE pDNA priming, two booster regimens, gag pDNA or codelivery of p27CE+gag pDNA, significantly increased the levels of CE-specific T cells. However, the CE+gag pDNA booster vaccination elicited significantly broader CE epitope recognition, and thus, a more profound alteration of the immunodominance hierarchy. Vaccination with HIV molecules showed that CE+gag pDNA booster regimen further expanded the breadth of HIV CE responses. Hence, SIV/HIV vaccine regimens comprising CE pDNA prime and CE+gag pDNA booster vaccination significantly increased cytotoxic T cell responses to subdominant highly conserved Gag epitopes and maximized response breadth.
Assuntos
Citotoxicidade Imunológica , Epitopos/imunologia , Produtos do Gene gag/imunologia , Infecções por HIV/prevenção & controle , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vacinas de DNA/imunologia , Vacinas contra a AIDS/imunologia , Animais , Citocinas/imunologia , HIV/imunologia , HIV/fisiologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Esquemas de Imunização , Imunização Secundária/métodos , Macaca mulatta , Vacinas contra a SAIDS/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/química , Vírus da Imunodeficiência Símia/imunologia , Vírus da Imunodeficiência Símia/fisiologia , Vacinas de DNA/administração & dosagemRESUMO
BACKGROUND: Despite preventive vaccines for oncogenic human papillomaviruses (HPVs), cervical intraepithelial neoplasia (CIN) is common, and current treatments are ablative and can lead to long-term reproductive morbidity. We assessed whether VGX-3100, synthetic plasmids targeting HPV-16 and HPV-18 E6 and E7 proteins, delivered by electroporation, would cause histopathological regression in women with CIN2/3. METHODS: Efficacy, safety, and immunogenicity of VGX-3100 were assessed in CIN2/3 associated with HPV-16 and HPV-18, in a randomised, double-blind, placebo-controlled phase 2b study. Patients from 36 academic and private gynaecology practices in seven countries were randomised (3:1) to receive 6 mg VGX-3100 or placebo (1 mL), given intramuscularly at 0, 4, and 12 weeks. Randomisation was stratified by age (<25 vs ≥25 years) and CIN2 versus CIN3 by computer-generated allocation sequence (block size 4). Funder and site personnel, participants, and pathologists were masked to treatment. The primary efficacy endpoint was regression to CIN1 or normal pathology 36 weeks after the first dose. Per-protocol and modified intention-to-treat analyses were based on patients receiving three doses without protocol violations, and on patients receiving at least one dose, respectively. The safety population included all patients who received at least one dose. The trial is registered at ClinicalTrials.gov (number NCT01304524) and EudraCT (number 2012-001334-33). FINDINGS: Between Oct 19, 2011, and July 30, 2013, 167 patients received either VGX-3100 (n=125) or placebo (n=42). In the per-protocol analysis 53 (49·5%) of 107 VGX-3100 recipients and 11 (30·6%) of 36 placebo recipients had histopathological regression (percentage point difference 19·0 [95% CI 1·4-36·6]; p=0·034). In the modified intention-to-treat analysis 55 (48·2%) of 114 VGX-3100 recipients and 12 (30·0%) of 40 placebo recipients had histopathological regression (percentage point difference 18·2 [95% CI 1·3-34·4]; p=0·034). Injection-site reactions occurred in most patients, but only erythema was significantly more common in the VGX-3100 group (98/125, 78·4%) than in the placebo group (24/42, 57·1%; percentage point difference 21·3 [95% CI 5·3-37·8]; p=0·007). INTERPRETATION: VGX-3100 is the first therapeutic vaccine to show efficacy against CIN2/3 associated with HPV-16 and HPV-18. VGX-3100 could present a non-surgical therapeutic option for CIN2/3, changing the treatment outlook for this common disease. FUNDING: Inovio Pharmaceuticals.
Assuntos
Infecções por Papillomavirus/tratamento farmacológico , Vacinas contra Papillomavirus/uso terapêutico , Displasia do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/tratamento farmacológico , Vacinas de DNA/uso terapêutico , Adulto , Vacinas Anticâncer/imunologia , Vacinas Anticâncer/uso terapêutico , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Método Duplo-Cego , Feminino , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/imunologia , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/imunologia , Humanos , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/imunologia , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/imunologia , Infecções por Papillomavirus/virologia , Vacinas contra Papillomavirus/imunologia , Proteínas Repressoras/genética , Proteínas Repressoras/imunologia , Resultado do Tratamento , Neoplasias do Colo do Útero/virologia , Vacinas de DNA/imunologia , Adulto Jovem , Displasia do Colo do Útero/virologiaRESUMO
We have previously shown that macaques vaccinated with DNA vectors expressing SIVmac239 antigens developed potent immune responses able to reduce viremia upon high-dose SIVmac251 challenge. To further improve vaccine-induced immunity and protection, we combined the SIVmac239 DNA vaccine with protein immunization using inactivated SIVmac239 viral particles as protein source. Twenty-six weeks after the last vaccination, the animals were challenged intrarectally at weekly intervals with a titrated dose of the heterologous SIVsmE660. Two of DNA-protein coimmunized macaques did not become infected after 14 challenges, but all controls were infected by 11 challenges. Vaccinated macaques showed modest protection from SIVsmE660 acquisition compared with naïve controls (P = 0.050; stratified for TRIM5α genotype). Vaccinees had significantly lower peak (1.6 log, P = 0.0048) and chronic phase viremia (P = 0.044), with 73% of the vaccinees suppressing viral replication to levels below assay detection during the 40-wk follow-up. Vaccine-induced immune responses associated significantly with virus control: binding antibody titers and the presence of rectal IgG to SIVsmE660 Env correlated with delayed SIVsmE660 acquisition; SIV-specific cytotoxic T cells, prechallenge CD4(+) effector memory, and postchallenge CD8(+) transitional memory cells correlated with control of viremia. Thus, SIVmac239 DNA and protein-based vaccine protocols were able to achieve high, persistent, broad, and effective cellular and humoral immune responses able to delay heterologous SIVsmE660 infection and to provide long-term control of viremia. These studies support a role of DNA and protein-based vaccines for development of an efficacious HIV/AIDS vaccine.
Assuntos
DNA Viral/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vacinas Virais/imunologia , Viremia/prevenção & controle , Vírion/imunologia , Animais , Anticorpos Antivirais/biossíntese , DNA Viral/administração & dosagem , Imunidade Celular , Imunoglobulina G/imunologia , Macaca mulatta , Reto/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/economia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Carga Viral , Vacinas Virais/administração & dosagemRESUMO
The hepatitis C virus (HCV) nonstructural (NS) 5A protein has been shown to promote viral persistence by interfering with both innate and adaptive immunity. At the same time, the HCV NS5A protein has been suggested as a target for antiviral therapy. In this study, we performed a detailed characterization of HCV NS5A immunogenicity in wild-type (wt) and immune tolerant HCV NS5A-transgenic (Tg) C57BL/6J mice. We evaluated how efficiently HCV NS5A-based genetic vaccines could activate strong T cell responses. Truncated and full-length wt and synthetic codon-optimized NS5A genotype 1b genes were cloned into eukaryotic expression plasmids, and the immunogenicity was determined after i.m. immunization in combination with in vivo electroporation. The NS5A-based genetic vaccines primed high Ab levels, with IgG titers of >10(4) postimmunization. With respect to CD8(+) T cell responses, the coNS5A gene primed more potent IFN-γ-producing and lytic cytotoxic T cells in wt mice compared with NS5A-Tg mice. In addition, high frequencies of NS5A-specific CD8(+) T cells were found in wt mice after a single immunization. To test the functionality of the CTL responses, the ability to inhibit growth of NS5A-expressing tumor cells in vivo was analyzed after immunization. A single dose of coNS5A primed tumor-inhibiting responses in both wt and NS5A-Tg mice. Finally, immunization with the coNS5A gene primed polyfunctional NS5A-specific CD8(+) T cell responses. Thus, the coNS5A gene is a promising therapeutic vaccine candidate for chronic HCV infections.
Assuntos
Linfócitos T CD8-Positivos/imunologia , DNA Viral/imunologia , Hepacivirus/imunologia , Vacinas de DNA/imunologia , Vacinas contra Hepatite Viral/imunologia , Proteínas não Estruturais Virais/imunologia , Animais , Especificidade de Anticorpos , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/metabolismo , Vacinas Anticâncer , Códon/genética , Citotoxicidade Imunológica , DNA Viral/síntese química , DNA Viral/genética , Genes Sintéticos , Antígenos H-2/imunologia , Hepacivirus/genética , Anticorpos Anti-Hepatite C/biossíntese , Anticorpos Anti-Hepatite C/genética , Anticorpos Anti-Hepatite C/imunologia , Imunização , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Ativação Linfocitária , Linfocinas/metabolismo , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Especificidade do Receptor de Antígeno de Linfócitos T , Linfócitos T Citotóxicos/imunologia , Vacinas contra Hepatite Viral/uso terapêutico , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
IL-12 is a 70-kDa heterodimeric cytokine composed of the p35 and p40 subunits. To maximize cytokine production from plasmid DNA, molecular steps controlling IL-12p70 biosynthesis at the posttranscriptional and posttranslational levels were investigated. We show that the combination of RNA/codon-optimized gene sequences and fine-tuning of the relative expression levels of the two subunits within a cell resulted in increased production of the IL-12p70 heterodimer. We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit. This posttranslational regulation mediated by the p40 subunit is conserved in mammals. Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ~1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences. Such optimized DNA plasmids also produced significantly higher levels of systemic bioactive IL-12 upon in vivo DNA delivery in mice compared with plasmids expressing the wild type sequences. A single therapeutic injection of an optimized murine IL-12 DNA plasmid showed significantly more potent control of tumor development in the B16 melanoma cancer model in mice. Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.
Assuntos
Subunidade p35 da Interleucina-12/biossíntese , Subunidade p40 da Interleucina-12/biossíntese , Multimerização Proteica , Animais , Humanos , Imunoterapia , Subunidade p35 da Interleucina-12/genética , Subunidade p35 da Interleucina-12/imunologia , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/imunologia , Macaca mulatta , Melanoma/genética , Melanoma/imunologia , Melanoma/metabolismo , Melanoma/terapia , Camundongos , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/terapia , Estabilidade Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
BACKGROUND: We explored the concept of heterologous prime/boost vaccination using 2 therapeutic vaccines currently in clinical development aimed at treating chronically infected hepatitis C virus (HCV) patients: prime with a DNA-based vaccine expressing HCV genotype 1a NS3/4A proteins (ChronVac-C) and boost with a modified vaccinia virus Ankara vaccine expressing genotype 1b NS3/4/5B proteins (MVATG16643). METHODS: Two ChronVac-C immunizations 4 weeks apart were delivered intramuscularly in combination with in vivo electroporation and subsequently 5 or 12 weeks later boosted by 3 weekly subcutaneous injections of MVATG16643. Two mouse strains were used, and we evaluated quality, magnitude, and functionality of the T cells induced. RESULTS: DNA prime/MVA boost regimen induced significantly higher levels of interferon γ (IFN-γ) or interleukin 2 (IL-2) ELISpot responses compared with each vaccine alone, independent of the time of analysis and the time interval between vaccinations. Both CD8⺠and CD4⺠T-cell responses as well as the spectrum of epitopes recognized was improved. A significant increase in polyfunctional IFN-γ/tumor necrosis factor α (TNF-α)/CD107a⺠CD8⺠T cells was detected following ChronVac-C/MVATG16643 vaccination (from 3% to 25%), and prime/boost was the only regimen that activated quadrifunctional T cells (IFN-γ/TNF-α/CD107a/IL-2). In vivo functional protective capacity of DNA prime/MVA boost was demonstrated in a Listeria-NS3-1a challenge model. CONCLUSIONS: We provide a proof-of-concept that immunogenicity of 2 HCV therapeutic vaccines can be improved using their combination, which merits further clinical development.
Assuntos
Formação de Anticorpos , Hepatite C/prevenção & controle , Vacinação/métodos , Vacinas contra Hepatite Viral/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Genótipo , Hepacivirus , Hepatite C/imunologia , Imunização Secundária , Interferon gama/sangue , Interleucina-2/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fator de Necrose Tumoral alfa/sangue , Vacinas de DNA/imunologia , Vacinas contra Hepatite Viral/genéticaRESUMO
The global coronavirus disease 2019 (COVID-19) pandemic has claimed more than 5 million lives. Emerging variants of concern (VOCs) continually challenge viral control. Directing vaccine-induced humoral and cell-mediated responses to mucosal surfaces may enhance vaccine efficacy. Here we investigate the immunogenicity and protective efficacy of optimized synthetic DNA plasmids encoding wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (pS) co-formulated with the plasmid-encoded mucosal chemokine cutaneous T cell-attracting chemokine (pCTACK; CCL27). pCTACK-co-immunized animals exhibit increased spike-specific antibodies at the mucosal surface and increased frequencies of interferon gamma (IFNγ)+ CD8+ T cells in the respiratory mucosa. pCTACK co-immunization confers 100% protection from heterologous Delta VOC challenge. This study shows that mucosal chemokine adjuvants can direct vaccine-induced responses to specific immunological sites and have significant effects on heterologous challenge. Further study of this unique chemokine-adjuvanted vaccine approach in the context of SARS-CoV-2 vaccines is likely important.
Assuntos
COVID-19 , Vacinas Virais , Adjuvantes Imunológicos/farmacologia , Animais , Anticorpos Antivirais , Linfócitos T CD8-Positivos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Quimiocinas , Humanos , SARS-CoV-2/genética , Vacinas Virais/genéticaRESUMO
The enhanced transmissibility and immune evasion associated with emerging SARS-CoV-2 variants demands the development of next-generation vaccines capable of inducing superior protection amid a shifting pandemic landscape. Since a portion of the global population harbors some level of immunity from vaccines based on the original Wuhan-Hu-1 SARS-CoV-2 sequence or natural infection, an important question going forward is whether this immunity can be boosted by next-generation vaccines that target emerging variants while simultaneously maintaining long-term protection against existing strains. Here, we evaluated the immunogenicity of INO-4800, our synthetic DNA vaccine candidate for COVID-19 currently in clinical evaluation, and INO-4802, a next-generation DNA vaccine designed to broadly target emerging SARS-CoV-2 variants, as booster vaccines in nonhuman primates. Rhesus macaques primed over one year prior with the first-generation INO-4800 vaccine were boosted with either INO-4800 or INO-4802 in homologous or heterologous prime-boost regimens. Both boosting schedules led to an expansion of T cells and antibody responses which were characterized by improved neutralizing and ACE2 blocking activity across wild-type SARS-CoV-2 as well as multiple variants of concern. These data illustrate the durability of immunity following vaccination with INO-4800 and additionally support the use of either INO-4800 or INO-4802 in prime-boost regimens.
Assuntos
COVID-19 , Vacinas de DNA , Vacinas Virais , Animais , Formação de Anticorpos , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , SARS-CoV-2 , VacinaçãoRESUMO
HIV Envelope (Env) is the main vaccine target for induction of neutralizing antibodies. Stabilizing Env into native-like trimer (NLT) conformations is required for recombinant protein immunogens to induce autologous neutralizing antibodies(nAbs) against difficult to neutralize HIV strains (tier-2) in rabbits and non-human primates. Immunizations of mice with NLTs have generally failed to induce tier-2 nAbs. Here, we show that DNA-encoded NLTs fold properly in vivo and induce autologous tier-2 nAbs in mice. DNA-encoded NLTs also uniquely induce both CD4 + and CD8 + T-cell responses as compared to corresponding protein immunizations. Murine neutralizing antibodies are identified with an advanced sequencing technology. The structure of an Env-Ab (C05) complex, as determined by cryo-EM, identifies a previously undescribed neutralizing Env C3/V5 epitope. Beyond potential functional immunity gains, DNA vaccines permit in vivo folding of structured antigens and provide significant cost and speed advantages for enabling rapid evaluation of new HIV vaccines.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Vacinas de DNA/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/administração & dosagem , Animais , Anticorpos Neutralizantes/ultraestrutura , Antígenos Virais/imunologia , Linhagem Celular Tumoral , Microscopia Crioeletrônica , ELISPOT , Epitopos/imunologia , Células HEK293 , Anticorpos Anti-HIV/ultraestrutura , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Camundongos Endogâmicos BALB C , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/virologia , Vacinação/métodos , Vacinas de DNA/administração & dosagem , Produtos do Gene env do Vírus da Imunodeficiência Humana/químicaRESUMO
Monoclonal antibody therapy has played an important role against SARS-CoV-2. Strategies to deliver functional, antibody-based therapeutics with improved in vivo durability are needed to supplement current efforts and reach underserved populations. Here, we compare recombinant mAbs COV2-2196 and COV2-2130, which compromise clinical cocktail Tixagevimab/Cilgavimab, with optimized nucleic acid-launched forms. Functional profiling of in vivo-expressed, DNA-encoded monoclonal antibodies (DMAbs) demonstrated similar specificity, broad antiviral potency and equivalent protective efficacy in multiple animal challenge models of SARS-CoV-2 prophylaxis compared to protein delivery. In PK studies, DNA-delivery drove significant serum antibody titers that were better maintained compared to protein administration. Furthermore, cryo-EM studies performed on serum-derived DMAbs provide the first high-resolution visualization of in vivo-launched antibodies, revealing new interactions that may promote cooperative binding to trimeric antigen and broad activity against VoC including Omicron lineages. These data support the further study of DMAb technology in the development and delivery of valuable biologics.
Assuntos
Produtos Biológicos , COVID-19 , Ácidos Nucleicos , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Antivirais/farmacologia , Antivirais/uso terapêutico , COVID-19/prevenção & controle , DNA , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines may target epitopes that reduce durability or increase the potential for escape from vaccine-induced immunity. Using synthetic vaccinology, we have developed rationally immune-focused SARS-CoV-2 Spike-based vaccines. Glycans can be employed to alter antibody responses to infection and vaccines. Utilizing computational modeling and in vitro screening, we have incorporated glycans into the receptor-binding domain (RBD) and assessed antigenic profiles. We demonstrate that glycan-coated RBD immunogens elicit stronger neutralizing antibodies and have engineered seven multivalent configurations. Advanced DNA delivery of engineered nanoparticle vaccines rapidly elicits potent neutralizing antibodies in guinea pigs, hamsters, and multiple mouse models, including human ACE2 and human antibody repertoire transgenics. RBD nanoparticles induce high levels of cross-neutralizing antibodies against variants of concern with durable titers beyond 6 months. Single, low-dose immunization protects against a lethal SARS-CoV-2 challenge. Single-dose coronavirus vaccines via DNA-launched nanoparticles provide a platform for rapid clinical translation of potent and durable coronavirus vaccines.
Assuntos
Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Nanopartículas/administração & dosagem , SARS-CoV-2/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Vacinas contra COVID-19/química , Vacinas contra COVID-19/genética , Cricetinae , Epitopos , Cobaias , Imunogenicidade da Vacina , Camundongos , Nanopartículas/química , Vacinas Baseadas em Ácido Nucleico/administração & dosagem , Vacinas Baseadas em Ácido Nucleico/química , Vacinas Baseadas em Ácido Nucleico/genética , Vacinas Baseadas em Ácido Nucleico/imunologia , Polissacarídeos/química , Polissacarídeos/genética , Polissacarídeos/imunologia , SARS-CoV-2/química , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Potência de VacinaRESUMO
Electropermeabilization of mammalian cells is a technique that has been used for the delivery of therapeutics, such as DNA plasmids or DNA vaccines. Typically, delivery via electropermeabilization occurs through injection of the substance into the tissue of interest followed by the insertion of electrodes at the site and the application of brief electrical pulses. Here we detail a novel and innovative contactless electropermeabilization method to deliver DNA plasmids to dermal tissue in vivo. This process has the advantage of eliminating the insertion of additional needles that serve as electrodes to facilitate the application of electric pulses in conventional electroporation processes. Plasmid encoding GFP was injected into guinea pig skin and pulsed with the novel contactless electropermeabilization method. Three days following treatment, robust GFP expression was observed on the skin of pulsed animals. Strong humoral immune responses were also achieved when a DNA vaccine expressing the influenza antigen NP was delivered and pulsed using the novel device in comparison to naked injection alone. This delivery method has the advantage of being contactless and suggests that gene transfer via this mode warrants further development.
Assuntos
Antígenos/biossíntese , Derme/fisiologia , Eletroporação/métodos , Expressão Gênica , Permeabilidade , Vacinação/métodos , Vacinas de DNA/farmacocinética , Animais , Antígenos/genética , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Cobaias , Plasmídeos , Vacinas de DNA/administração & dosagemRESUMO
The ongoing COVID-19 pandemic has resulted in significant global morbidity and mortality on a scale similar to the influenza pandemic of 1918. Over the course of the last few months, a number of SARS-CoV-2 variants have been identified against which vaccine-induced immune responses may be less effective. These "variants-of-concern" have garnered significant attention in the media, with discussion around their impact on the future of the pandemic and the ability of leading COVID-19 vaccines to protect against them effectively. To address concerns about emerging SARS-CoV-2 variants affecting vaccine-induced immunity, we investigated the neutralisation of representative 'G614', '501Y.V1' and '501Y.V2' virus isolates using sera from ferrets that had received prime-boost doses of the DNA vaccine, INO-4800. Neutralisation titres against G614 and 501Y.V1 were comparable, but titres against the 501Y.V2 variant were approximately 4-fold lower, similar to results reported with other nucleic acid vaccines and supported by in silico biomolecular modelling. The results confirm that the vaccine-induced neutralising antibodies generated by INO-4800 remain effective against current variants-of-concern, albeit with lower neutralisation titres against 501Y.V2 similar to other leading nucleic acid-based vaccines.