RESUMO
The speed of development, versatility and efficacy of mRNA-based vaccines have been amply demonstrated in the case of SARS-CoV-2. DNA vaccines represent an important alternative since they induce both humoral and cellular immune responses in animal models and in human trials. We tested the immunogenicity and protective efficacy of DNA-based vaccine regimens expressing different prefusion-stabilized Wuhan-Hu-1 SARS-CoV-2 Spike antigens upon intramuscular injection followed by electroporation in rhesus macaques. Different Spike DNA vaccine regimens induced antibodies that potently neutralized SARS-CoV-2 in vitro and elicited robust T cell responses. The antibodies recognized and potently neutralized a panel of different Spike variants including Alpha, Delta, Epsilon, Eta and A.23.1, but to a lesser extent Beta and Gamma. The DNA-only vaccine regimens were compared to a regimen that included co-immunization of Spike DNA and protein in the same anatomical site, the latter of which showed significant higher antibody responses. All vaccine regimens led to control of SARS-CoV-2 intranasal/intratracheal challenge and absence of virus dissemination to the lower respiratory tract. Vaccine-induced binding and neutralizing antibody titers and antibody-dependent cellular phagocytosis inversely correlated with transient virus levels in the nasal mucosa. Importantly, the Spike DNA+Protein co-immunization regimen induced the highest binding and neutralizing antibodies and showed the strongest control against SARS-CoV-2 challenge in rhesus macaques.
Assuntos
Macaca mulatta , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de DNA , Animais , COVID-19/imunologia , COVID-19/terapia , Estudos de Coortes , DNA Viral/imunologia , Modelos Animais de Doenças , Feminino , Imunização Passiva , Leucócitos Mononucleares/imunologia , Camundongos , RNA Mensageiro/análise , SARS-CoV-2/genética , Linfócitos T/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Soroterapia para COVID-19RESUMO
The RV144 vaccine trial revealed a correlation between reduced risk of HIV infection and the level of nonneutralizing-antibody (Ab) responses targeting specific epitopes in the second variable domain (V2) of the HIV gp120 envelope (Env) protein, suggesting this region as a target for vaccine development. To favor induction of V2-specific Abs, we developed a vaccine regimen that included priming with DNA expressing an HIV V1V2 trimeric scaffold immunogen followed by booster immunizations with a combination of DNA and protein in rhesus macaques. Priming vaccination with DNA expressing the HIV recombinant subtype CRF01_AE V1V2 scaffold induced higher and broader V2-specific Ab responses than vaccination with DNA expressing CRF01_AE gp145 Env. Abs recognizing the V2 peptide that was reported as a critical target in RV144 developed only after the priming immunization with V1V2 DNA. The V2-specific Abs showed several nonneutralizing Fc-mediated functions, including ADCP and C1q binding. Importantly, robust V2-specific Abs were maintained upon boosting with gp145 DNA and gp120 protein coimmunization. In conclusion, priming with DNA expressing the trimeric V1V2 scaffold alters the hierarchy of humoral immune responses to V2 region epitopes, providing a method for more efficient induction and maintenance of V2-specific Env Abs associated with reduced risk of HIV infection.IMPORTANCE The aim of this work was to design and test a vaccine regimen focusing the immune response on targets associated with infection prevention. We demonstrated that priming with a DNA vaccine expressing only the HIV Env V1V2 region induces Ab responses targeting the critical region in V2 associated with protection. This work shows that V1V2 scaffold DNA priming immunization provides a method to focus immune responses to the desired target region, in the absence of immune interference by other epitopes. This induced immune responses with improved recognition of epitopes important for protective immunity, namely, V2-specific humoral immune responses inversely correlating with HIV risk of infection in the RV144 trial.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , HIV/imunologia , Imunização/métodos , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/genética , Animais , Anticorpos Neutralizantes/imunologia , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Células HEK293 , Antígenos HIV/química , Antígenos HIV/genética , Antígenos HIV/imunologia , Proteína gp120 do Envelope de HIV/genética , Humanos , Imunização Secundária , Imunogenicidade da Vacina , Macaca mulatta , Conformação Proteica , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas de DNA/imunologiaRESUMO
An efficacious human immunodeficiency virus (HIV) vaccine will likely require induction of both mucosal and systemic immune responses. We compared the immunogenicity and protective efficacy of two mucosal/systemic vaccine regimens and investigated their effects on the rectal microbiome. Rhesus macaques were primed twice mucosally with replication-competent adenovirus type 5 host range mutant (Ad5hr)-simian immunodeficiency virus (SIV) recombinants and boosted twice intramuscularly with ALVAC-SIV recombinant plus SIV gp120 protein or with DNA for SIV genes and rhesus interleukin-12 plus SIV gp120 protein. Controls received empty Ad5hr vector and alum adjuvant only. Both regimens elicited strong, comparable mucosal and systemic cellular and humoral immunity. Prevaccination rectal microbiomes of males and females differed and significantly changed over the course of immunization, most strongly in females after Ad5hr immunizations. Following repeated low-dose intrarectal SIV challenges, both vaccine groups exhibited modestly but significantly reduced acute viremia. Male and female controls exhibited similar acute viral loads; however, vaccinated females, but not males, exhibited lower levels of acute viremia, compared to same-sex controls. Few differences in adaptive immune responses were observed between the sexes. Striking differences in correlations of the rectal microbiome of males and females with acute viremia and immune responses associated with protection were seen and point to effects of the microbiome on vaccine-induced immunity and viremia control. Our study clearly demonstrates direct effects of a mucosal SIV vaccine regimen on the rectal microbiome and validates our previously reported SIV vaccine-induced sex bias. Sex and the microbiome are critical factors that should not be overlooked in vaccine design and evaluation.IMPORTANCE Differences in HIV pathogenesis between males and females, including immunity postinfection, have been well documented, as have steroid hormone effects on the microbiome, which is known to influence mucosal immune responses. Few studies have applied this knowledge to vaccine trials. We investigated two SIV vaccine regimens combining mucosal priming immunizations and systemic protein boosting. We again report a vaccine-induced sex bias, with female rhesus macaques but not males displaying significantly reduced acute viremia. The vaccine regimens, especially the mucosal primes, significantly altered the rectal microbiome. The greatest effects were in females. Striking differences between female and male macaques in correlations of prevalent rectal bacteria with viral loads and potentially protective immune responses were observed. Effects of the microbiome on vaccine-induced immunity and viremia control require further study by microbiome transfer. However, the findings presented highlight the critical importance of considering effects of sex and the microbiome in vaccine design and evaluation.
Assuntos
Imunização Secundária/métodos , Macaca mulatta/imunologia , Microbiota/efeitos dos fármacos , Reto/microbiologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Viremia/imunologia , Vacinas contra a AIDS/imunologia , Adenoviridae/genética , Animais , Feminino , Imunidade Humoral , Imunidade nas Mucosas , Masculino , Microbiota/fisiologia , Reto/imunologia , Vacinas contra a SAIDS/imunologiaRESUMO
Human interleukin-12 (hIL-12) is a heparin-binding cytokine whose activity was previously shown to be enhanced by heparin and other sulfated glycosaminoglycans. The current study investigated the mechanisms by which heparin increases hIL-12 activity. Using multiple human cell types, including natural killer cells, an IL-12 indicator cell line, and primary peripheral blood mononuclear and T cells, along with bioactivity, flow cytometry, and isothermal titration calorimetry assays, we found that heparin-dependent modulation of hIL-12 function correlates with several of heparin's biophysical characteristics, including chain length, sulfation level, and concentration. Specifically, only heparin molecules longer than eight saccharide units enhanced hIL-12 activity. Furthermore, heparin molecules with three sulfate groups per disaccharide unit outperformed heparin molecules with one or two sulfate groups per disaccharide unit in terms of enhanced hIL-12 binding and activity. Heparin also significantly reduced the EC50 value of hIL-12 by up to 11.8-fold, depending on the responding cell type. Cytokine-profiling analyses revealed that heparin affected the level, but not the type, of cytokines produced by lymphocytes in response to hIL-12. Interestingly, although murine IL-12 also binds heparin, heparin did not enhance its activity. Using the gathered data, we propose a model of hIL-12 stabilization in which heparin serves as a co-receptor enhancing the interaction between heterodimeric hIL-12 and its receptor subunits. The results of this study provide a foundation for further investigation of heparin's interactions with IL-12 family cytokines and for the use of heparin as an immunomodulatory agent.
Assuntos
Heparina/farmacologia , Interleucina-12/farmacologia , Animais , Fenômenos Biofísicos , Calorimetria , Citocinas/biossíntese , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Células HEK293 , Heparina/química , Heparitina Sulfato/metabolismo , Humanos , Interleucina-12/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Receptores de Interleucina-2/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
T follicular helper (TFH) cells are fundamental in germinal center (GC) maturation and selection of antigen-specific B cells within secondary lymphoid organs. GC-resident TFH cells have been fully characterized in human immunodeficiency virus (HIV) infection. However, the role of GC TFH cells in GC B cell responses following various simian immunodeficiency virus (SIV) vaccine regimens in rhesus macaques (RMs) has not been fully investigated. We characterized GC TFH cells of RMs over the course of a mucosal/systemic vaccination regimen to elucidate GC formation and SIV humoral response generation. Animals were mucosally primed twice with replicating adenovirus type 5 host range mutant (Ad5hr)-SIV recombinants and systemically boosted with ALVAC-SIVM766Gag/Pro/gp120-TM and SIVM766&CG7V gD-gp120 proteins formulated in alum hydroxide (ALVAC/Env) or DNA encoding SIVenv/SIVGag/rhesus interleukin 12 (IL-12) plus SIVM766&CG7V gD-gp120 proteins formulated in alum phosphate (DNA&Env). Lymph nodes were biopsied in macaque subgroups prevaccination and at day 3, 7, or 14 after the 2nd Ad5hr-SIV prime and the 2nd vector/Env boost. Evaluations of GC TFH and GC B cell dynamics including correlation analyses supported a significant role for early GC TFH cells in providing B cell help during initial phases of GC formation. GC TFH responses at day 3 post-mucosal priming were consistent with generation of Env-specific memory B cells in GCs and elicitation of prolonged Env-specific humoral immunity in the rectal mucosa. GC Env-specific memory B cell responses elicited early post-systemic boosting correlated significantly with decreased viremia postinfection. Our results highlight the importance of early GC TFH cell responses for robust GC maturation and generation of long-lasting SIV-specific humoral responses at mucosal and systemic sites. Further investigation of GC TFH cell dynamics should facilitate development of an efficacious HIV vaccine.IMPORTANCE The modest HIV protection observed in the human RV144 vaccine trial associated antibody responses with vaccine efficacy. T follicular helper (TFH) cells are CD4+ T cells that select antibody secreting cells with high antigenic affinity in germinal centers (GCs) within secondary lymphoid organs. To evaluate the role of TFH cells in eliciting prolonged virus-specific humoral responses, we vaccinated rhesus macaques with a combined mucosal prime/systemic boost regimen followed by repeated low-dose intrarectal challenges with SIV, mimicking human exposure to HIV-1. Although the vaccine regimen did not prevent SIV infection, decreased viremia was observed in the immunized macaques. Importantly, vaccine-induced TFH responses elicited at day 3 postimmunization and robust GC maturation were strongly associated. Further, early TFH-dependent SIV-specific B cell responses were also correlated with decreased viremia. Our findings highlight the contribution of early vaccine-induced GC TFH responses to elicitation of SIV-specific humoral immunity and implicate their participation in SIV control.
Assuntos
Vírus da Imunodeficiência Símia/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/fisiologia , Animais , Anticorpos Antivirais/imunologia , Formação de Anticorpos , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Centro Germinativo/imunologia , Imunidade Humoral/imunologia , Imunização/métodos , Linfonodos/imunologia , Macaca mulatta/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vacinação/métodos , Viremia/imunologiaRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1006902.].
RESUMO
B cell follicles in secondary lymphoid tissues represent an immune privileged sanctuary for AIDS viruses, in part because cytotoxic CD8+ T cells are mostly excluded from entering the follicles that harbor infected T follicular helper (TFH) cells. We studied the effects of native heterodimeric IL-15 (hetIL-15) treatment on uninfected rhesus macaques and on macaques that had spontaneously controlled SHIV infection to low levels of chronic viremia. hetIL-15 increased effector CD8+ T lymphocytes with high granzyme B content in blood, mucosal sites and lymph nodes, including virus-specific MHC-peptide tetramer+ CD8+ cells in LN. Following hetIL-15 treatment, multiplexed quantitative image analysis (histo-cytometry) of LN revealed increased numbers of granzyme B+ T cells in B cell follicles and SHIV RNA was decreased in plasma and in LN. Based on these properties, hetIL-15 shows promise as a potential component in combination immunotherapy regimens to target AIDS virus sanctuaries and reduce long-term viral reservoirs in HIV-1 infected individuals. TRIAL REGISTRATION: ClinicalTrials.gov NCT02452268.
Assuntos
Interleucina-15/uso terapêutico , Linfonodos/metabolismo , RNA Viral/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Vírus da Imunodeficiência Símia/genética , Linfócitos T Citotóxicos/efeitos dos fármacos , Adjuvantes Imunológicos/uso terapêutico , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/patologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/patologia , Feminino , Centro Germinativo/efeitos dos fármacos , Centro Germinativo/patologia , Imunoterapia/métodos , Interleucina-15/química , Interleucina-15/metabolismo , Linfonodos/patologia , Linfonodos/virologia , Contagem de Linfócitos , Macaca mulatta , Masculino , Multimerização Proteica , RNA Viral/análise , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/metabolismo , Linfócitos T Citotóxicos/patologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/patologiaRESUMO
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
The common γ-chain cytokine interleukin-15 (IL-15) plays a significant role in regulating innate and adaptive lymphocyte homeostasis and can stimulate anti-tumor activity of leukocytes. We have previously shown that the circulating IL-15 in the plasma is the heterodimeric form (hetIL-15), produced upon co-expression of IL-15 and IL-15 Receptor alpha (IL-15Rα) polypeptides in the same cell, heterodimerization of the two chains and secretion. We investigated the pharmacokinetic and pharmacodynamic profile and toxicity of purified human hetIL-15 cytokine upon injection in rhesus macaques. We compared the effects of repeated hetIL-15 administration during a two-week dosing cycle, using different subcutaneous dosing schemata, i.e. fixed doses of 0.5, 5 and 50⯵g/kg or a doubling step-dose scheme ranging from 2 to 64⯵g/kg. Following a fixed-dose regimen, dose-dependent peak plasma IL-15 levels decreased significantly between the first and last injection. The trough plasma IL-15 levels measured at 48â¯h after injections were significantly higher after the first dose, compared to subsequent doses. In contrast, following the step-dose regimen, the systemic exposure increased by more than 1 log between the first injection given at 2⯵g/kg and the last injection given at 64⯵g/kg, and the trough levels were comparable after each injection. Blood lymphocyte cell count, proliferation, and plasma IL-18 levels peaked at day 8 when hetIL-15 was provided at fixed doses, and at the end of the cycle following a step-dose regimen, suggesting that sustained expansion of target cells requires increasing doses of cytokine. Macaques treated with a 50⯵g/kg dose showed moderate and transient toxicity, including fever, signs of capillary leak syndrome and renal dysfunction. In contrast, these effects were mild or absent using the step-dose regimen. The results provide a new method of optimal administration of this homeostatic cytokine and may have applications for the delivery of other cytokines.
Assuntos
Citocinas/sangue , Subunidade alfa de Receptor de Interleucina-15/metabolismo , Interleucina-15/sangue , Interleucina-15/farmacocinética , Animais , Subunidade alfa de Receptor de Interleucina-15/imunologia , Linfócitos , Macaca mulatta , Absorção SubcutâneaRESUMO
Post-transcriptional regulatory mechanisms of several complex and simple retroviruses and retroelements have been elucidated, with the exception of the gammaretrovirus family. We found that, similar to the other retroviruses, gag gene expression of MuLV and XMRV depends on post-transcriptional regulation mediated via an RNA sequence overlapping the pro-pol open reading frame, termed the Post-Transcriptional Element (PTE). PTE function can be replaced by heterologous RNA export elements, e.g. CTE of simian type D retroviruses. Alternatively, Gag particle production is achieved using an RNA/codon optimized gag gene. PTE function is transferable and can replace HIV Rev-RRE-regulated expression of HIV gag. Analysis of PTE by SHAPE revealed a highly structured RNA comprising seven stem-loop structures, with the 5' and 3' stem-loops forming an essential bipartite signal. MuLV and XMRV PTE share 98% identity and have highly similar RNA structures, with changes mostly located to single-stranded regions. PTE identification strongly suggests that all retroviruses and retroelements share common strategies of post-transcriptional gene regulation to produce Gag. Expression depends on complex RNA structures embedded within retroviral mRNA, in coding regions or the 3' untranslated region. These specific structures serve as recognition signals for either cellular or viral proteins.
Assuntos
Regulação Viral da Expressão Gênica , Vírus da Leucemia Murina de Moloney/genética , RNA Mensageiro/química , RNA Viral/química , Sequências Reguladoras de Ácido Ribonucleico , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/genética , Produtos do Gene gag/genética , Produtos do Gene gag/metabolismo , Células HEK293 , Células HeLa , Humanos , Vírus da Leucemia Murina de Moloney/metabolismo , Conformação de Ácido Nucleico , RNA Mensageiro/metabolismo , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Interleukin-15 (IL-15), a 114-amino acid cytokine related to IL-2, regulates immune homeostasis and the fate of many lymphocyte subsets. We reported that, in the blood of mice and humans, IL-15 is present as a heterodimer associated with soluble IL-15 receptor α (sIL-15Rα). Here, we show efficient production of this noncovalently linked but stable heterodimer in clonal human HEK293 cells and release of the processed IL-15·sIL-15Rα heterodimer in the medium. Purification of the IL-15 and sIL-15Rα polypeptides allowed identification of the proteolytic cleavage site of IL-15Rα and characterization of multiple glycosylation sites. Administration of the IL-15·sIL-15Rα heterodimer reconstituted from purified subunits resulted in sustained plasma IL-15 levels and in robust expansion of NK and T cells in mice, demonstrating pharmacokinetics and in vivo bioactivity superior to single chain IL-15. These identified properties of heterodimeric IL-15 provide a strong rationale for the evaluation of this molecule for clinical applications.
Assuntos
Subunidade alfa de Receptor de Interleucina-15/metabolismo , Interleucina-15/metabolismo , Complexos Multiproteicos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Feminino , Glicosilação , Células HEK293 , Humanos , Immunoblotting , Interleucina-15/química , Interleucina-15/genética , Subunidade alfa de Receptor de Interleucina-15/química , Subunidade alfa de Receptor de Interleucina-15/genética , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Complexos Multiproteicos/administração & dosagem , Complexos Multiproteicos/farmacocinética , Ligação Proteica , Multimerização Proteica , Proteólise , Solubilidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacosRESUMO
To identify the most promising vaccine candidates for combinatorial strategies, we compared five SIV vaccine platforms including recombinant canary pox virus ALVAC, replication-competent adenovirus type 5 host range mutant RepAd, DNA, modified vaccinia Ankara (MVA), peptides and protein in distinct combinations. Three regimens used viral vectors (prime or boost) and two regimens used plasmid DNA. Analysis at necropsy showed that the DNA-based vaccine regimens elicited significantly higher cellular responses against Gag and Env than any of the other vaccine platforms. The T cell responses induced by most vaccine regimens disseminated systemically into secondary lymphoid tissues (lymph nodes, spleen) and effector anatomical sites (including liver, vaginal tissue), indicative of their role in viral containment at the portal of entry. The cellular and reported humoral immune response data suggest that combination of DNA and viral vectors elicits a balanced immunity with strong and durable responses able to disseminate into relevant mucosal sites.
Assuntos
Imunidade Celular , Vírus da Imunodeficiência Símia/imunologia , Vacinas Virais/imunologia , Animais , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos , Células Cultivadas , Feminino , Macaca mulattaRESUMO
Combinatorial HIV/SIV vaccine approaches targeting multiple arms of the immune system might improve protective efficacy. We compared SIV-specific humoral immunity induced in rhesus macaques by five vaccine regimens. Systemic regimens included ALVAC-SIVenv priming and Env boosting (ALVAC/Env); DNA immunization; and DNA plus Env co-immunization (DNA&Env). RepAd/Env combined mucosal replication-competent Ad-env priming with systemic Env boosting. A Peptide/Env regimen, given solely intrarectally, included HIV/SIV peptides followed by MVA-env and Env boosts. Serum antibodies mediating neutralizing, phagocytic and ADCC activities were induced by ALVAC/Env, RepAd/Env and DNA&Env vaccines. Memory B cells and plasma cells were maintained in the bone marrow. RepAd/Env vaccination induced early SIV-specific IgA in rectal secretions before Env boosting, although mucosal IgA and IgG responses were readily detected at necropsy in ALVAC/Env, RepAd/Env, DNA&Env and DNA vaccinated animals. Our results suggest that combined RepAd priming with ALVAC/Env or DNA&Env regimen boosting might induce potent, functional, long-lasting systemic and mucosal SIV-specific antibodies.
Assuntos
Imunidade nas Mucosas/imunologia , Vacinas contra a SAIDS/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vacinação/métodos , Vacinas contra a AIDS/administração & dosagem , Vacinas contra a AIDS/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Quimioterapia Combinada , ELISPOT , Produtos do Gene env/imunologia , Humanos , Imunoglobulina A/sangue , Imunoglobulina A/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Macaca mulatta , Vacinas contra a SAIDS/administração & dosagem , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Fatores de Tempo , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologiaRESUMO
IL-15 is an important cytokine for the function of the immune system, but the form(s) of IL-15 produced in the human body are not fully characterized. Coexpression of the single-chain IL-15 and the IL-15 receptor alpha (IL-15Rα) in the same cell allows for efficient production, surface display, and eventual cleavage and secretion of the bioactive IL-15/IL-15Rα heterodimer in vivo, whereas the single-chain IL-15 is poorly secreted and unstable. This observation led to the hypothesis that IL-15 is produced and secreted only as a heterodimer with IL-15Rα. We purified human IL-15/IL-15Rα complexes from overproducing human cell lines and developed an ELISA specifically measuring the heterodimeric form of IL-15. Analysis of sera from melanoma patients after lymphodepletion revealed the presence of circulating IL-15/IL-15Rα complexes in amounts similar to the total IL-15 quantified by a commercial IL-15 ELISA that detects both the single-chain and the heterodimeric forms of the cytokine. Therefore, in lymphodepleted cancer patients, the serum IL-15 is exclusively present in its heterodimeric form. Analysis of the form of IL-15 present in either normal or lymphodepleted mice agrees with the human data. These results have important implications for development of assays and materials for clinical applications of IL-15.
Assuntos
Subunidade alfa de Receptor de Interleucina-15/sangue , Subunidade alfa de Receptor de Interleucina-15/imunologia , Interleucina-15/sangue , Interleucina-15/química , Animais , Dimerização , Feminino , Glicosilação , Células HEK293 , Humanos , Interleucina-15/imunologia , Depleção Linfocítica/métodos , Linfócitos/citologia , Linfócitos/imunologia , Melanoma/sangue , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Cutâneas/sangue , Neoplasias Cutâneas/imunologia , SolubilidadeRESUMO
Interleukin-12 is a heterodimeric, pro-inflammatory cytokine that is a key driver of cell-mediated immunity. Clinical interest in IL-12 is significant due to its potent anti-tumor activity and efficacy in controlling certain infectious diseases such as Leishmaniasis and Listeria infection. For clinical applications, the ease of production and purification of IL-12 and the associated cost continues to be a consideration. In this context, we report a simple and effective heparin-affinity based purification of recombinant human IL-12 (hIL-12) from the serum-free supernatants of stable IL-12-transduced HEK293 cells. Fractionation of culture supernatants on heparin Sepharose columns revealed that hIL-12 elutes as a single peak in 500 mM NaCl. Coomassie staining and Western blot analysis showed that hIL-12 eluted in 500 mM NaCl is homogeneous. Purity of hIL-12 was ascertained by RP-HPLC and ESI-MS analysis, and found to be â¼98%. Western blot analysis, using monoclonal antibodies, demonstrated that the crucial inter-subunit disulfide bond linking the p35 and p40 subunits is intact in the purified hIL-12. Results of far UV circular dichroism, steady-state tryptophan fluorescence, and differential scanning calorimetry experiments suggest that purified hIL-12 is in its stable native conformation. Enzyme linked immunosorbent assays (ELISAs) and bioactivity studies demonstrate that hIL-12 is obtained in high yields (0.31±0.05 mg/mL of the culture medium) and is also fully bioactive. Isothermal titration calorimetry data show that IL-12 exhibits a moderate binding affinity (Kd(app)=69±1 µM) to heparin. The purification method described in this study is expected to provide greater impetus for research on the role of heparin in the regulation of the function of IL-12. In addition, the results of this study provide an avenue to obtain high amounts of IL-12 required for structural studies which are aimed at the development of novel IL-12-based therapeutics.
Assuntos
Células HEK293/metabolismo , Interleucina-12/genética , Interleucina-12/isolamento & purificação , Sequência de Aminoácidos , Sítios de Ligação , Western Blotting , Cromatografia de Afinidade , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Heparina/metabolismo , Humanos , Interleucina-12/química , Interleucina-12/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Regulação para CimaRESUMO
BACKGROUND: An HIV-1 DNA vaccine composed of seven highly conserved, structurally important elements (Conserved Elements, CE) of HIV p24Gag was tested in a phase I randomized, double-blind clinical trial (HVTN 119, NCT03181789) in people without HIV. A CE prime- CE+full-length p55Gag boost DNA vaccine was compared to p55Gag DNA vaccination only. METHODS: Two groups (n=25 each) received 4 DNA vaccinations [2xCE prime- 2xCE+p55Gag boost or 4x p55Gag] by intramuscular injection/electroporation, including IL-12 DNA adjuvant. The placebo group (n=6) received saline. Participants were followed for safety and tolerability. Immunogenicity was assessed for T cell and antibody responses. RESULTS: Both regimens were safe and generally well-tolerated. The p24CE vaccine was immunogenic (29% CD4+ and 4% CD8+ responders) and was significantly boosted by CE+p55Gag (64% CD4+, p=0.037; 42% CD8+, p=0.004). CE+p55Gag induced CD4+ responses to 5 of 7 CE, compared to only 2 CE by p55Gag DNA alone, with a higher reponse to CE5 in 30% of individuals (p=0.006). CE+p55Gag induced significantly higher mean CD4+ CE Tcell breadth (0.68 vs 0.22 CE; p=0.029) and a strong trend for increased CD4+ and CD8+ T-cell breadth (1.14 vs. 0.52 CE; p=0.051) compared to p55Gag alone. Both groups developed high p55Gag T-cell (91% each) and p24Gag antibody (91% vs. 80%) responses. p24CE vaccine-induced CD4+ CE T-cell responses correlated (p=0.007) with p24Gag antibody responses. CONCLUSION: The combination CE/CE+p55Gag DNA vaccine induced T-cell immune responses to conserved regions in p24Gag resulting in significant increases in breadth and epitope recognition throughout p55Gag. Vaccines able to focus immune responses by priming responses to highly conserved regions could be part of a comprehensive HIV vaccine strategy. CLINICAL TRIALS: gov NCT03181789 Study URL: https://www. CLINICALTRIALS: gov/search?term=NCT03181789 FUNDING. HIV vaccine trial network (HVTN), NIAID/NIH.
RESUMO
Kaposi's sarcoma-associated herpesvirus (KSHV [human herpesvirus 8; HHV-8]) open reading frame 57 (ORF57) is a viral early protein participating in posttranscriptional regulatory events, such as splicing, RNA stabilization, and protein expression. Recent data suggest that ORF57 recruits the transcription and export (TREX) complex to viral RNA and exports these transcripts to the cytoplasm. In this study, we show that although ORF57 promotes expression of a selection of KSHV viral intronless RNAs, it is not a bona fide export factor.
Assuntos
Núcleo Celular/metabolismo , Herpesvirus Humano 8/genética , Processamento Pós-Transcricional do RNA/genética , RNA Viral/metabolismo , Proteínas Virais/genética , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Northern Blotting , Oligonucleotídeos/genética , Fases de Leitura Aberta/genética , Proteínas Virais/metabolismoRESUMO
We employed a dose-escalation regimen in rhesus macaques to deliver glycosylated IL-7, a cytokine critical for development and maintenance of T lymphocytes. IL-7 increased proliferation and survival of T cells and triggered several chemokines and cytokines. Induction of CXCL13 in lymph nodes (LNs) led to a remarkable increase of B cells in the LNs, proliferation of germinal center follicular T helper cells and elevated IL-21 levels suggesting an increase in follicle activity. Transcriptomics analysis showed induction of IRF-7 and Flt3L, which was linked to increased frequency of circulating plasmacytoid dendritic cells (pDCs) on IL-7 treatment. These pDCs expressed higher levels of CCR7, homed to LNs, and were associated with upregulation of type-1 interferon gene signature and increased production of IFN-α2a on TLR stimulation. Superior effects and dose-sparing advantage was observed by the step-dose regimen. Thus, IL-7 treatment leads to systemic effects involving both lymphoid and myeloid compartments.