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Severe dengue virus (DENV) infection is characterized by exacerbated inflammatory responses that lead to endothelial dysfunction and plasma leakage. We have recently demonstrated that Toll-like receptor 2 (TLR2) on blood monocytes senses DENV infection leading to endothelial activation. Here, we report that non-infectious immature DENV particles, which are released in large numbers by DENV-infected cells, drive endothelial activation via the TLR2 axis. We show that fully immature DENV particles induce a rapid, within 6 hours post-infection, inflammatory response in PBMCs. Furthermore, pharmacological blocking of TLR2/TLR6/CD14 and/or NF-kB prior to exposure of PBMCs to immature DENV reduces the initial production of inter alia TNF-α and IL-1ß by monocytes and prevents endothelial activation. However, prolonged TLR2 block induces TNF-α production and leads to exacerbated endothelial activation, indicating that TLR2-mediated responses play an important role not only in the initiation but also the resolution of inflammation. Altogether, these data indicate that the maturation status of the virus has the potential to influence the kinetics and extent of inflammatory responses during DENV infection.
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Vírus da Dengue , Dengue , Humanos , Receptor 2 Toll-Like , Leucócitos Mononucleares , Receptor 6 Toll-Like , Fator de Necrose Tumoral alfa , NF-kappa B , Inflamação , VírionRESUMO
The raising of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants led to the use of COVID-19 bivalent vaccines, which include antigens of the wild-type (WT) virus, and of the Omicron strain. In this study, we aimed to evaluate the impact of bivalent vaccination on the neutralizing antibody (NAb) response. We enrolled 93 volunteers who had received three or four doses of monovalent vaccines based on the original virus (n = 61), or a booster shot with the bivalent vaccine (n = 32). Serum samples collected from volunteers were subjected to neutralization assays using the WT SARS-CoV-2, and Omicron subvariants. In addition, immunoinformatics to quantify and localize highly conserved NAb epitopes were performed. As main result, we observed that the neutralization titers of samples from individuals vaccinated with the bivalent vaccine were higher for the original virus, in comparison to their capacity of neutralizing the Omicron variant and its subvariants. NAb that recognize epitopes mostly conserved in the WT SARS-CoV-2 were boosted, while those that recognize epitopes mostly present in the Omicron variant, and subvariants were primed. These results indicate that formulation of future vaccines shall consider to target present viruses, and not viruses that no longer circulate.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Humanos , COVID-19/prevenção & controle , SARS-CoV-2/genética , Vacinação , Imunização Secundária , Anticorpos Neutralizantes , Epitopos/genética , Vacinas CombinadasRESUMO
The development of safe and effective vaccine formulations against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a hallmark in the history of vaccines. Here we report a COVID-19 subunit vaccine based on a SARS-CoV-2 Spike protein receptor binding domain (RBD) incorporated into nano-multilamellar vesicles (NMV) associated with monophosphoryl lipid A (MPLA). The results based on immunization of C57BL/6 mice demonstrated that recombinant antigen incorporation into NMVs improved antibody and T-cell responses without inducing toxic effects under both in vitro and in vivo conditions. Administration of RBD-NMV-MPLA formulations modulated antigen avidity and IgG subclass responses, whereas MPLA incorporation improved the activation of CD4+/CD8+ T-cell responses. In addition, immunization with the complete vaccine formulation reduced the number of doses required to achieve enhanced serum virus-neutralizing antibody titers. Overall, this study highlights NMV/MPLA technology, displaying the performance improvement of subunit vaccines against SARS-CoV-2, as well as other infectious diseases.
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COVID-19 , SARS-CoV-2 , Animais , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Vacinas contra COVID-19 , Humanos , Imunidade , Imunoglobulina G , Lipídeos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Glicoproteína da Espícula de Coronavírus , Vacinas de Subunidades AntigênicasRESUMO
BACKGROUND: The irregular use of antiretroviral therapy (ART) and late diagnosis still account for a large part of HIV-associated mortality in people living with HIV (PLHIV). Herein, we describe HIV-associated morbidity among hospitalised HIV/AIDS patients with advanced immunosuppression and assess the comorbidities, laboratory parameters, and immunological markers associated with mortality. METHODS: The cross-sectional study was conducted at the Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD) in Manaus, Brazil. In all, 83 participants aged between 12 and 70 years were enrolled by convenience within 72 h of their hospitalisation. Clinical and laboratory data were obtained from electronic medical records. We prospectively measured the cytokines Th1/Th2/Th17 and inflammatory cytokines IL-8, IL-1ß, and IL-12 using cytometric bead array, and the soluble CD14 using in-house enzyme-linked immunosorbent assay. RESULTS: The HIV/AIDS inpatients presented a scenario of respiratory syndromes as the most prevalent comorbidity. Almost all patients had CD4 T counts below 350 cells/mL and the mortality rate was 20.5%. Pulmonary tuberculosis, neurotoxoplasmosis and oropharyngeal-esophageal candidiasis were the most prevalent opportunistic infections. TB and weight loss were more prevalent in HIV/AIDS inpatients who died. The Mann Whitney analysis showed that those who died had higher platelet distribution width (PDW) on admission, which is suggestive for platelet activation. The Poisson multivariate analysis showed the prevalence of TB, digestive syndrome and increases in IL-8 and lactate dehydrogenase (LDH) associated to death. CONCLUSIONS: The advanced immunosuppression characterized by the opportunistic infections presented in these HIV/AIDS inpatients was the major factor of mortality. The role of platelet activation in worse outcomes of hospitalisation and the IL-8 associated with the context of advanced immunosuppression may be promising markers in the prediction of mortality in HIV/AIDS patients.
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Infecções por HIV , Adolescente , Adulto , Idoso , Biomarcadores , Brasil/epidemiologia , Contagem de Linfócito CD4 , Criança , Estudos Transversais , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Infecções por HIV/epidemiologia , Humanos , Pessoa de Meia-Idade , Morbidade , Centros de Atenção Terciária , Adulto JovemRESUMO
Influenza viruses are respiratory pathogens of public health concern worldwide with up to 650,000 deaths occurring each year. Seasonal influenza virus vaccines are employed to prevent disease, but with limited effectiveness. Development of a universal influenza virus vaccine with the potential to elicit long-lasting, broadly cross-reactive immune responses is necessary for reducing influenza virus prevalence. In this study, we have utilized lipid nanoparticle-encapsulated, nucleoside-modified mRNA vaccines to intradermally deliver a combination of conserved influenza virus antigens (hemagglutinin stalk, neuraminidase, matrix-2 ion channel, and nucleoprotein) and induce strong immune responses with substantial breadth and potency in a murine model. The immunity conferred by nucleoside-modified mRNA-lipid nanoparticle vaccines provided protection from challenge with pandemic H1N1 virus at 500 times the median lethal dose after administration of a single immunization, and the combination vaccine protected from morbidity at a dose of 50 ng per antigen. The broad protective potential of a single dose of combination vaccine was confirmed by challenge with a panel of group 1 influenza A viruses. These findings support the advancement of nucleoside-modified mRNA-lipid nanoparticle vaccines expressing multiple conserved antigens as universal influenza virus vaccine candidates.
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Antígenos Virais/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Nucleosídeos/química , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas Sintéticas/administração & dosagem , Animais , Anticorpos Antivirais/metabolismo , Antígenos Virais/química , Modelos Animais de Doenças , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/química , Vacinas contra Influenza/imunologia , Injeções Intradérmicas , Lipossomos , Camundongos , Células NIH 3T3 , Nanopartículas , Neuraminidase/química , Neuraminidase/genética , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , Infecções por Orthomyxoviridae/imunologia , Vacinas Sintéticas/química , Vacinas Sintéticas/imunologia , Vacinas de mRNARESUMO
Malignant brain tumors are among the most aggressive cancers with poor prognosis and no effective treatment. Recently, we reported the oncolytic potential of Zika virus infecting and destroying the human central nervous system (CNS) tumors in vitro and in immunodeficient mice model. However, translating this approach to humans requires pre-clinical trials in another immunocompetent animal model. Here, we analyzed the safety of Brazilian Zika virus (ZIKVBR) intrathecal injections in three dogs bearing spontaneous CNS tumors aiming an anti-tumoral therapy. We further assessed some aspects of the innate immune and inflammatory response that triggers the anti-tumoral response observed during the ZIKVBR administration in vivo and in vitro. For the first time, we showed that there were no negative clinical side effects following ZIKVBR CNS injections in dogs, confirming the safety of the procedure. Furthermore, the intrathecal ZIKVBR injections reduced tumor size in immunocompetent dogs bearing spontaneous intracranial tumors, improved their neurological clinical symptoms significantly, and extended their survival by inducing the destruction specifically of tumor cells, sparing normal neurons, and activating an immune response. These results open new perspectives for upcoming virotherapy using ZIKV to destroy and induce an anti-tumoral immune response in CNS tumors for which there are currently no effective treatments.
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Neoplasias Encefálicas/complicações , Neoplasias Encefálicas/terapia , Terapia Viral Oncolítica/métodos , Segurança do Paciente , Carga Tumoral , Infecção por Zika virus/complicações , Zika virus/imunologia , Animais , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Citocinas/metabolismo , Modelos Animais de Doenças , Cães , Imunidade , Injeções Espinhais , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/virologia , Monócitos/imunologia , Monócitos/virologia , Neurônios/metabolismo , Neurônios/virologia , Resultado do TratamentoRESUMO
Self-assembling proteins may be generated after the addition of short specific amino acid sequences at both the N- and C-terminal ends. To date, this approach has not been evaluated regarding the impact of self-assembled proteins on the induction of immune responses. In the present study, we report the application of this experimental approach to the immunogenicity of protein antigens by measuring the antibody responses in mice immunized with nanoparticles made with a recombinant form of Zika virus nonstructural protein 1 (∆NS1). The results clearly indicated that ∆NS1-derived nanoparticles (NP-∆NS1) are assembled into a 3-dimensional structure with a high degree of multimerization. While ∆NS1 proved to be a weak immunogen, immunization with NP-∆NS1 enhanced subunit vaccines' immunogenicity with improved longevity in vaccinated mice. Thus, immunization with self-assembled antigens (nanovaccines) represents a new and promising strategy to enhance NS1-specific antibodies' induction based on purified recombinant proteins.
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Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Nanopartículas/química , Proteínas não Estruturais Virais/imunologia , Vacinas Virais/imunologia , Zika virus/imunologia , Animais , Epitopos/imunologia , Feminino , Imunização , Imunoglobulina G/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Chikungunya virus (CHIKV) is responsible for a self-limited illness that can evolve into long-lasting painful joint inflammation. In this study, we report a novel experimental CHIKV vaccine formulation of lipid nanoparticles loaded with a recombinant protein derived from the E2 structural protein. This antigen fragment, designated ∆E2.1, maintained the antigenicity of the native viral protein and was specifically recognized by antibodies induced in CHIKV-infected patients. The antigen has been formulated into nanoparticles consisting of nano-multilamellar vesicles (NMVs) combined with the adjuvant monophosphoryl lipid A (MPLA). The vaccine formulation demonstrated a depot effect, leading to controlled antigen release, and induced strong antibody responses significantly higher than in mice immunized with the purified protein combined with the adjuvant. More relevantly, E2-specific antibodies raised in mice immunized with ∆E2.1-loaded NMV-MPLA neutralized CHIKV under in vitro conditions. Taken together, the results demonstrated that the new nanoparticle-based vaccine formulation represents a promising approach for the development of effective anti-CHIKV vaccines.
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Febre de Chikungunya/imunologia , Vírus Chikungunya/imunologia , Lipossomos/imunologia , Proteínas do Envelope Viral/genética , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/efeitos dos fármacos , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/efeitos dos fármacos , Anticorpos Antivirais/imunologia , Febre de Chikungunya/terapia , Febre de Chikungunya/virologia , Vírus Chikungunya/patogenicidade , Humanos , Lipossomos/química , Lipossomos/farmacologia , Camundongos , Nanopartículas/química , Proteínas do Envelope Viral/farmacologia , Vacinas Virais/imunologiaRESUMO
Respiratory failure (RF) is the main cause of hospital admission in HIV/AIDS patients. This study assessed comorbidities and laboratory parameters in HIV/AIDS inpatients with RF (N = 58) in relation to those without RF (N = 36). Tuberculosis showed a huge relative risk and platelet counts were slightly higher in HIV/AIDS inpatients with RF. A flow cytometry assay for reactive oxygen species (ROS) showed lower levels in platelets of these patients in relation to the healthy subjects. However, when stimulated with adrenaline, ROS levels increased in platelets and platelet-derived microparticles of HIV/AIDS inpatients, which may increase the risk of RF during HIV and tuberculosis (HIV-TB) coinfection.
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Micropartículas Derivadas de Células/metabolismo , Infecções por HIV/sangue , HIV/imunologia , Espécies Reativas de Oxigênio/sangue , Insuficiência Respiratória/complicações , Fármacos Anti-HIV/uso terapêutico , Biomarcadores/sangue , Plaquetas , Citometria de Fluxo , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Humanos , Insuficiência Respiratória/sangueRESUMO
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was confirmed in Brazil in February 2020, the first cases were followed by an increase in the number of cases throughout the country, resulting in an important public health crisis that requires fast and coordinated responses. OBJECTIVES: The objective of this work is to describe the isolation and propagation properties of SARS-CoV-2 isolates from the first confirmed cases of coronavirus disease 2019 (COVID-19) in Brazil. METHODS: After diagnosis in patients that returned from Italy to the São Paulo city in late February by RT-PCR, SARS-CoV-2 isolates were obtained in cell cultures and characterised by full genome sequencing, electron microscopy and in vitro replication properties. FINDINGS: The virus isolate was recovered from nasopharyngeal specimen, propagated in Vero cells (E6, CCL-81 and hSLAM), with clear cytopathic effects, and characterised by full genome sequencing, electron microscopy and in vitro replication properties. Virus stocks - viable (titre 2.11 × 106 TCID50/mL, titre 1.5 × 106 PFUs/mL) and inactivated from isolate SARS.CoV2/SP02.2020.HIAE.Br were prepared and set available to the public health authorities and the scientific community in Brazil and abroad. MAIN CONCLUSION: We believe that the protocols for virus growth and studies here described and the distribution initiative may constitute a viable model for other developing countries, not only to help a rapid effective pandemic response, but also to facilitate and support basic scientific research.
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Betacoronavirus/isolamento & purificação , Infecções por Coronavirus , Pandemias , Pneumonia Viral , Animais , Brasil , COVID-19 , Chlorocebus aethiops , Humanos , SARS-CoV-2 , Células VeroRESUMO
BACKGROUND: Proteins in inclusion bodies (IBs) present native-like secondary structures. However, chaotropic agents at denaturing concentrations, which are widely used for IB solubilization and subsequent refolding, unfold these secondary structures. Removal of the chaotropes frequently causes reaggregation and poor recovery of bioactive proteins. High hydrostatic pressure (HHP) and alkaline pH are two conditions that, in the presence of low level of chaotropes, have been described as non-denaturing solubilization agents. In the present study we evaluated the strategy of combination of HHP and alkaline pH on the solubilization of IB using as a model an antigenic form of the zika virus (ZIKV) non-structural 1 (NS1) protein. RESULTS: Pressure-treatment (2.4 kbar) of NS1-IBs at a pH of 11.0 induced a low degree of NS1 unfolding and led to solubilization of the IBs, mainly into monomers. After dialysis at pH 8.5, NS1 was refolded and formed soluble oligomers. High (up to 68 mg/liter) NS1 concentrations were obtained by solubilization of NS1-IBs at pH 11 in the presence of arginine (Arg) with a final yield of approximately 80% of total protein content. The process proved to be efficient, quick and did not require further purification steps. Refolded NS1 preserved biological features regarding reactivity with antigen-specific antibodies, including sera of ZIKV-infected patients. The method resulted in an increase of approximately 30-fold over conventional IB solubilization-refolding methods. CONCLUSIONS: The present results represent an innovative non-denaturing protein refolding process by means of the concomitant use of HHP and alkaline pH. Application of the reported method allowed the recovery of ZIKV NS1 at a condition that maintained the antigenic properties of the protein.
Assuntos
Bioquímica/métodos , Corpos de Inclusão/química , Proteínas não Estruturais Virais/química , Zika virus/metabolismo , Álcalis/química , Pressão Hidrostática , Corpos de Inclusão/genética , Corpos de Inclusão/metabolismo , Redobramento de Proteína , Estrutura Secundária de Proteína , Solubilidade , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Zika virus/química , Zika virus/genéticaRESUMO
Background: Zika virus (ZIKV) infections have been linked to different levels of clinical outcomes, ranging from mild rash and fever to severe neurological complications and congenital malformations. Methods: We investigated the clinical and immunological response, focusing on the immune mediators profile in 95 acute ZIKV-infected adult patients from Campinas, Brazil. These patients included 6 pregnant women who later delivered during the course of this study. Clinical observations were recorded during hospitalization. Levels of 45 immune mediators were quantified using multiplex microbead-based immunoassays. Results: Whereas 11.6% of patients had neurological complications, 88.4% displayed mild disease of rash and fever. Several immune mediators were specifically higher in ZIKV-infected patients, and levels of interleukin 10, interferon gamma-induced protein 10 (IP-10), and hepatocyte growth factor differentiated between patients with or without neurological complications. Interestingly, higher levels of interleukin 22, monocyte chemoattractant protein 1, TNF-α, and IP-10 were observed in ZIKV-infected pregnant women carrying fetuses with fetal growth-associated malformations. Notably, infants with congenital central nervous system deformities had significantly higher levels of interleukin 18 and IP-10 but lower levels of hepatocyte growth factor than those without such abnormalities born to ZIKV-infected mothers. Conclusions: This study identified several key markers for the control of ZIKV pathogenesis. This will allow a better understanding of the molecular mechanisms of ZIKV infection in patients.
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Citocinas/sangue , Malformações do Sistema Nervoso/epidemiologia , Complicações Infecciosas na Gravidez/epidemiologia , Infecção por Zika virus/epidemiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Brasil/epidemiologia , Criança , Feminino , Retardo do Crescimento Fetal/virologia , Humanos , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Malformações do Sistema Nervoso/virologia , Gravidez , Complicações Infecciosas na Gravidez/virologia , Resultado da Gravidez , Carga Viral , Adulto Jovem , Zika virus , Infecção por Zika virus/complicaçõesRESUMO
DNA vaccines have failed to induce satisfactory immune responses in humans. Several mechanisms of double-stranded DNA (dsDNA) sensing have been described, and modulate DNA vaccine immunogenicity at many levels. We hypothesized that the immunogenicity of DNA vaccines in humans is suppressed by APOBEC (apolipoprotein B (APOB) mRNA-editing, catalytic polypeptide)-mediated plasmid degradation. We showed that plasmid sensing via STING (stimulator of interferon (IFN) genes) and TBK-1 (TANK-binding kinase 1) leads to IFN-ß induction, which results in APOBEC3A mRNA upregulation through a mechanism involving protein kinase C signaling. We also showed that murine APOBEC2 expression in HEK293T cells led to a 10-fold reduction in intracellular plasmid levels and plasmid-encoded mRNA, and a 2.6-fold reduction in GFP-expressing cells. A bicistronic DNA vaccine expressing an immunogen and an APOBEC2-specific shRNA efficiently silenced APOBEC2 both in vitro and in vivo, increasing the frequency of induced IFN-γ-secreting T cells. Our study brings new insights into the intracellular machinery involved in dsDNA sensing and how to modulate it to improve DNA vaccine immunogenicity in humans.
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Apolipoproteínas B/metabolismo , Citidina Desaminase/metabolismo , HIV-1/fisiologia , Proteínas Musculares/metabolismo , Proteínas/metabolismo , Linfócitos T/imunologia , Vacinas de DNA/imunologia , Desaminases APOBEC , Animais , Antígenos Virais/genética , Apolipoproteínas B/genética , Citidina Desaminase/genética , Células HEK293 , Antígenos HLA-DR/genética , Humanos , Imunomodulação , Interferon beta/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Proteínas Musculares/genética , Fragmentos de Peptídeos/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/genética , Edição de RNA , RNA Interferente Pequeno/genética , Transdução de Sinais/genética , Transgenes/genética , Vacinas de DNA/genéticaRESUMO
T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity.
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Vacinas contra a AIDS/imunologia , Antígenos Virais/imunologia , Linfócitos T CD4-Positivos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , HIV-1/imunologia , Imunidade Celular/imunologia , Vacinas de DNA/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Sequência Conservada/imunologia , ELISPOT , Feminino , Citometria de Fluxo , Vetores Genéticos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Infecções por HIV/prevenção & controle , Antígenos HLA-DR/imunologia , Humanos , Interferon gama/efeitos dos fármacos , Interferon gama/metabolismo , Interleucina-2/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos Endogâmicos BALB C , Plasmídeos , Ligação Proteica/imunologia , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The unprecedented global impact caused by SARS-CoV-2 imposed huge health and economic challenges, highlighting the urgent need for safe and effective vaccines. The receptor-binding domain (RBD) of SARS-CoV-2 is the major target for neutralizing antibodies and for vaccine formulations. Nonetheless, the low immunogenicity of the RBD requires the use of alternative strategies to enhance its immunological properties. Here, we evaluated the use of a subunit vaccine antigen generated after the genetic fusing of the RBD with a mouse IgG antibody. Subcutaneous administration of RBD-IgG led to the extended presence of the protein in the blood of immunized animals and enhanced RBD-specific IgG titers. Furthermore, RBD-IgG immunized mice elicited increased virus neutralizing antibody titers, measured both with pseudoviruses and with live original (Wuhan) SARS-CoV-2. Immunized K18-hACE2 mice were fully resistant to the lethal challenge of the Wuhan SARS-CoV-2, demonstrated by the control of body-weight loss and virus loads in their lungs and brains. Thus, we conclude that the genetic fusion of the RBD with an IgG molecule enhanced the immunogenicity of the antigen and the generation of virus-neutralizing antibodies, supporting the use of IgG chimeric antigens as an approach to improve the performance of SARS-CoV-2 subunit vaccines.
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Regulatory T cells (Tregs) may impede cancer vaccine efficacy in hematologic malignancies and cancer. CCR4 antagonists, an emergent class of Treg inhibitor, have been shown to block recruitment of Tregs mediated by CCL22 and CCL17. Our aim was to demonstrate the ability of a CCR4 antagonist (a small chemical molecule identified in silico) when combined with vaccines to break peripheral tolerance controlled by Tregs, a prerequisite for the induction of CD8(+) T cells against self Ags. Immunization of transgenic or normal mice expressing tumor-associated self Ags (Her2/neu, OVA, gp100) with a CCR4 antagonist combined with various vaccines led to the induction of effector CD8(+) T cells and partial inhibition of tumor growth expressing self Ags in both prophylactic and therapeutic settings. The CCR4 antagonist was more efficient than cyclophosphamide to elicit anti-self CD8(+) T cells. We also showed that the population of Tregs expressing CCR4 corresponded to memory (CD44(high)) and activated (ICOS(+)) Tregs, an important population to be targeted to modulate Treg activity. CCR4 antagonist represents a competitive class of Treg inhibitor able to induce functional anti-self CD8(+) T cells and tumor growth inhibition when combined with vaccines. High expression of CCR4 on human Tregs also supports the clinical development of this strategy.
Assuntos
Autoantígenos/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Vacinas Anticâncer/administração & dosagem , Neoplasias/terapia , Receptores CCR4/antagonistas & inibidores , Evasão Tumoral/efeitos dos fármacos , Animais , Antígenos de Neoplasias/imunologia , Antineoplásicos/administração & dosagem , Autoantígenos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Terapia Combinada , Modelos Animais de Doenças , Feminino , Humanos , Fatores Imunológicos/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Especificidade do Receptor de Antígeno de Linfócitos T/efeitos dos fármacos , Especificidade do Receptor de Antígeno de Linfócitos T/imunologia , Evasão Tumoral/imunologiaRESUMO
BACKGROUND: Nucleic acid-based vaccines have been studied for the past four decades, but the approval of the first messenger RNA (mRNA) vaccines during the COVID-19 pandemic opened renewed perspectives for the development of similar vaccines against different infectious diseases. Presently available mRNA vaccines are based on non-replicative mRNA, which contains modified nucleosides encased in lipid vesicles, allowing for entry into the host cell cytoplasm, and reducing inflammatory reactions. An alternative immunization strategy employs self-amplifying mRNA (samRNA) derived from alphaviruses, but lacks viral structural genes. Once incorporated into ionizable lipid shells, these vaccines lead to enhanced gene expression, and lower mRNA doses are required to induce protective immune responses. In the present study, we tested a samRNA vaccine formulation based on the SP6 Venezuelan equine encephalitis (VEE) vector incorporated into cationic liposomes (dimethyldioctadecyl ammonium bromide and a cholesterol derivative). Three vaccines were generated that encoded two reporter genes (GFP and nanoLuc) and the Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5). METHODS: Transfection assays were performed using Vero and HEK293T cells, and the mice were immunized via the intradermal route using a tattooing device. RESULTS: The liposome-replicon complexes showed high transfection efficiencies with in vitro cultured cells, whereas tattooing immunization with GFP-encoding replicons demonstrated gene expression in mouse skin up to 48 h after immunization. Mice immunized with liposomal PfRH5-encoding RNA replicons elicited antibodies that recognized the native protein expressed in P. falciparum schizont extracts, and inhibited the growth of the parasite in vitro. CONCLUSION: Intradermal delivery of cationic lipid-encapsulated samRNA constructs is a feasible approach for developing future malaria vaccines.
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IMPORTANCE: The emergence of SARS-CoV-2 had a major impact across the world. It is true that the collaboration of scientists from all over the world resulted in a rapid response against COVID-19, mainly with the development of vaccines against the disease. However, many viral genetic variants that threaten vaccines have emerged. Our study reveals highly conserved antigenic regions in the vaccines have emerged. Our study reveals highly conserved antigenic regions in the spike protein in all variants of concern (Alpha, Beta, Gamma, Delta, and Omicron) as well as in the wild-type virus. Such immune targets can be used to fight future SARS-CoV-2 variants.
Assuntos
COVID-19 , Médicos , Vacinas , Humanos , SARS-CoV-2/genéticaRESUMO
Dengue is an infectious disease of global health concern that continues to require surveillance. Serological testing has been used to investigate dengue-infected patients, but specificity is affected by the co-circulation of ZIKA virus (ZIKV), which shares extensive antigen similarities. The goal of this study was the development of a specific dengue virus (DENV) IgG ELISA based on a multi-epitope NS1-based antigen for antibody detection. The multi-epitope protein (T-ΔNS1), derived from a fragment of the NS1-protein of the four DENV serotypes, was expressed in Escherichia coli and purified via affinity chromatography. The antigenicity and specificity were evaluated with sera of mice infected with DENV-1-4 or ZIKV or after immunization with the recombinant ΔNS1 proteins. The performance of the T-ΔNS1-based IgG ELISA was also determined with human serum samples. The results demonstrate that the DENV T-ΔNS1 was specifically recognized by the serum IgG of dengue-infected mice or humans but showed no or reduced reactivity with ZIKV-infected subjects. Based on the available set of clinical samples, the ELISA based on the DENV T-ΔNS1 achieved 77.42% sensitivity and 88.57% specificity. The results indicate that the T-ΔNS1 antigen is a promising candidate for the development of specific serological analysis.
RESUMO
By following simple protein engineering steps, recombinant proteins with promising applications in the field of drug delivery can be assembled in the form of functional materials of increasing complexity, either as nanoparticles or nanoparticle-leaking secretory microparticles. Among the suitable strategies for protein assembly, the use of histidine-rich tags in combination with coordinating divalent cations allows the construction of both categories of material out of pure polypeptide samples. Such molecular crosslinking results in chemically homogeneous protein particles with a defined composition, a fact that offers soft regulatory routes towards clinical applications for nanostructured protein-only drugs or for protein-based drug vehicles. Successes in the fabrication and final performance of these materials are expected, irrespective of the protein source. However, this fact has not yet been fully explored and confirmed. By taking the antigenic RBD domain of the SARS-CoV-2 spike glycoprotein as a model building block, we investigated the production of nanoparticles and secretory microparticles out of the versions of recombinant RBD produced by bacteria (Escherichia coli), insect cells (Sf9), and two different mammalian cell lines (namely HEK 293F and Expi293F). Although both functional nanoparticles and secretory microparticles were effectively generated in all cases, the technological and biological idiosyncrasy of each type of cell factory impacted the biophysical properties of the products. Therefore, the selection of a protein biofabrication platform is not irrelevant but instead is a significant factor in the upstream pipeline of protein assembly into supramolecular, complex, and functional materials.