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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.
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Vacinas contra COVID-19 , COVID-19 , Humanos , COVID-19/prevenção & controle , SARS-CoV-2/genética , Vacinação , Imunização Secundária , Anticorpos Neutralizantes , Epitopos/genética , Vacinas CombinadasRESUMO
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
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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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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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.
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IMPORTANCE: Several additional COVID-19 vaccine doses were administered in the Brazilian population to prevent the disease caused by the B.1.1.529 (Omicron) variant. The efficacy of a third dose as a booster is already well described. However, it is important to clarify the humoral immune response gain induced by a fourth dose. In this study, we evaluate the effect of the fourth COVID-19 vaccine dose in a diverse Brazilian population, considering a real-life context. Our study reveals that the fourth dose of the COVID-19 vaccine increased the neutralizing antibody response against SARS-CoV-2 Omicron and significantly contributed in the reduction of the disease caused by this variant.
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Vacinas contra COVID-19 , COVID-19 , Humanos , SARS-CoV-2/genética , Brasil , COVID-19/prevenção & controle , Anticorpos Neutralizantes , Anticorpos AntiviraisRESUMO
High-risk Human papillomavirus (HPV) infections represent an important public health issue. Nearly all cervical malignancies are associated with HPV, and a range of other female and male cancers, such as anogenital and oropharyngeal. Aiming to treat HPV-related tumors, our group developed vaccines based on the genetic fusion of the HSV-1 glycoprotein D (gD) with the HPV-16 E7 oncoprotein (gDE7 vaccines). Despite the promising antitumor results reached by gDE7 vaccines in mice, combined therapies may increase the therapeutic effects by improving antitumor responses and halting immune suppressive mechanisms elicited by tumor cells. Considering cancer immunosuppressive mechanisms, indoleamine-2,3-dioxygenase (IDO) enzyme and interleukin-6 (IL-6) stand out in HPV-related tumors. Since IL-6 sustained the constitutive IDO expression, here we evaluated the therapeutic outcomes achieved by the combination of active immunotherapy based on a gDE7 protein-based vaccine with adjuvant treatments involving blocking IDO, either by use of IDO inhibitors or IL-6 knockout mice. C57BL/6 wild-type (WT) and transgenic IL-6-/- mice were engrafted with HPV16-E6/E7-expressing TC-1 cells and treated with 1-methyl-tryptophan isoforms (D-1MT and DL-1MT), capable to inhibit IDO. In vitro, the 1MT isoforms reduced IL-6 gene expression and IL-6 secretion in TC-1 cells. In vivo, the multi-targeted treatment improved the antitumor efficacy of the gDE7-based protein vaccine. Although the gDE7 immunization achieves partial tumor mass control in combination with D-1MT or DL-1MT in WT mice or when administered in IL-6-/- mice, the combination of gDE7 and 1MT in IL-6-/- mice further enhanced the antitumor effects, reaching total tumor rejection. The outcome of the combined therapy was associated with an increased frequency of activated dendritic cells and decreased frequencies of intratumoral polymorphonuclear myeloid-derived suppressor cells and T regulatory cells. In conclusion, the present study demonstrated that IL-6 and IDO negatively contribute to the activation of immune cells, particularly dendritic cells, reducing gDE7 vaccine-induced protective immune responses and, therefore, opening perspectives for the use of combined strategies based on inhibition of IL-6 and IDO as immunometabolic adjuvants for immunotherapies against HPV-related tumors.
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Infecções por Papillomavirus , Neoplasias do Colo do Útero , Masculino , Humanos , Feminino , Camundongos , Animais , Interleucina-6 , Camundongos Endogâmicos C57BL , Papillomaviridae , Imunoterapia , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismoRESUMO
The active immunotherapy concept relies on the use of vaccines that are capable of inducing antitumor immunity, reversion of the suppressive immunological environment, and long-term memory responses. Previously, antitumor vaccines based on a recombinant plasmid (pgDE7h) or a purified protein (gDE7) led to regression of early-established human papillomavirus (HPV)-associated tumors in a preclinical model. In this work, the anticancer vaccines were combined with cisplatin to treat HPV-induced tumors at advanced growth stages. The antitumor effects were evaluated in terms of tumor regression, induction of specific CD8+ T cells, and immune modulation of the tumor microenvironment. Acute toxicity induced by the treatment was measured by weight loss and histological alterations in the liver and kidneys. Our results revealed that the combination of cisplatin with either one of the tested immunotherapies (pgDE7h or gDE7) led to complete tumor regression in mice. Also, the combined treatment resulted in synergistic effects, particularly among mice immunized with gDE7, including activation of systemic and tumor-infiltrating E7-specific CD8+ T cells, tumor infiltration of macrophages and dendritic cells, and prevention of tumor relapses at different anatomical sites. Furthermore, the protocol allowed the reduction of cisplatin dosage and its intrinsic toxic effects, without reducing antitumor outcomes. These results expand our knowledge of active immunotherapy protocols and open perspectives for alternative treatments of HPV-associated tumors.
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Vacinas Anticâncer/farmacologia , Cisplatino/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/virologia , Infecções por Papillomavirus/complicações , Animais , Camundongos , Camundongos Endogâmicos C57BL , Recidiva Local de Neoplasia/prevenção & controle , Neoplasias/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVES: The aim of this study was to achieve greater specificity of dengue virus (DENV) serological tests based on a recombinant antigen derived from non-structural protein 1 (ΔNS1) with regard to cross-reactive Zika virus (ZIKV) anti-NS1 antibody responses. This is of relevance in endemic regions for the serological discrimination of both DENV and ZIKV, such as Brazil and other tropical countries. METHODS: The ΔNS1 proteins were obtained as recombinant antigens and were evaluated as solid-phase-bound antigens in the ELISA test to detect anti-NS1 IgG antibodies. The performance of the ∆NS1-based DENV IgG ELISA was assessed with both mouse and human serum samples previously exposed to DENV or ZIKV. RESULTS: The ∆NS1-based DENV IgG ELISA detected anti-DENV NS1 IgG without cross-reactivity with ZIKV-positive serum samples. The sensitivity and specificity of the assay determined using samples previously characterized by real-time PCR (qRT-PCR) or plaque reduction neutralization assay (PRNT) were 82% and 93%, respectively. CONCLUSION: The ∆NS1-based DENV IgG ELISA conferred enhanced diagnostic specificity for anti-DENV serological tests and may be particularly useful for serological analyses in endemic regions for both DENV and ZIKV transmission.
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Vírus da Dengue , Dengue , Infecção por Zika virus , Zika virus , Animais , Anticorpos Antivirais , Dengue/diagnóstico , Ensaio de Imunoadsorção Enzimática , Camundongos , Sensibilidade e Especificidade , Proteínas não Estruturais Virais , Infecção por Zika virus/diagnósticoRESUMO
OBJECTIVES: This study was performed to determine whether Dengue virus (DENV) immunochromatographic tests can detect and differentiate nonstructural protein 1 (NS1) from each of the four DENV serotypes and do not cross-react with the Zika virus (ZIKV) NS1 protein. METHODS: We compared the specificity of six NS1-based DENV immunochromatographic tests (point of care) in the detection of NS1 proteins from each of the four DENV serotypes and ZIKV. The tests were performed with NS1 proteins produced in mammalian cells. Cross-reactivity was confirmed with a purified recombinant ZIKV NS1 protein and DENV+ or ZIKV+ human serum samples. RESULTS: Cross-reaction was observed in 2 out of the 6 evaluated tests using cell culture supernatants containing NS1 protein of each tested virus. Cross-reactivity with ZIKV was confirmed with purified recombinant ZIKV NS1 produced in Escherichia coli. Further analyses with serum samples collected from DENV+ or ZIKV+ patients confirmed the cross-reactivity with ZIKV protein in 2 tests. CONCLUSIONS: The detection of the NS1 protein is the basis for several commercially available serological DENV diagnostic tests. The present results emphasize the relevance of testing specificity of presently available NS1-based DENV serological tests and the need of adjustments of tests that cross-react with the ZIKV protein. Our results are particularly relevant for regions where both viruses are endemically found, as in the case of Brazil.
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Cromatografia de Afinidade/métodos , Vírus da Dengue/imunologia , Dengue/virologia , Proteínas não Estruturais Virais/imunologia , Zika virus/imunologia , Anticorpos Antivirais/sangue , Brasil , Reações Cruzadas , Vírus da Dengue/isolamento & purificação , Glicoproteínas/imunologia , Humanos , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
Streptococcus agalactiae or group B Streptococcus (GBS) is a Gram-positive bacterium divided into ten distinct serotypes that colonizes the vaginal and rectal tracts of approximately 30% of women worldwide. GBS is the leading cause of invasive infection in newborns, causing sepsis, pneumoniae and meningitis. The main strategy to prevent GSB infection in newborns includes the use of intrapartum antibiotic therapy, which does not prevent late-onset diseases and may select resistant bacterial strains. We still do not have a vaccine formulation specific for this pathogen approved for human use. Conserved surface proteins are potential antigens that could be targets for recognition by antibodies and activation of cell opsonization. We used a serotype V GBS (GBS-V)-derived recombinant surface protein, rBibA, and evaluated the potential protective role of the induced antigen-specific antibodies after parenteral or mucosal immunizations in C57BL/6 mice. In vitro and in vivo assays demonstrated that vaccine formulations containing BibA combined with different adjuvants induced serum IgG and/or secreted IgA antibodies, leading to enhanced opsonophagocytosis of GBS-V cells and reduced invasion of epithelial cells. One BibA-based vaccine formulation adjuvanted with a nontoxic derivative of the heat-labile toxin produced by enterotoxigenic Escherichia coli (ETEC) strains was capable of inducing protection against vaginal colonization and lethal parenteral challenge with GBS-V. Serum collected from vaccinated mice conferred passive protection against vaginal colonization in naïve mice challenged with GBS-V. Taken together, the present data demonstrate that the BibA protein is a promising antigen for development of a vaccine to protect against GBS infection.
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Infecções Estreptocócicas , Streptococcus agalactiae , Animais , Anticorpos Antibacterianos , Feminino , Imunização , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Infecções Estreptocócicas/prevenção & controleRESUMO
Contagious agalactia (CA) is a serious disease notifiable to the World Organisation for Animal Health (OIE) causing severe economic losses to sheep and goat producers worldwide. Mycoplasma agalactiae, considered as its main etiological agent, inflicts a variety of symptoms in infected animals, including keratoconjunctivitis, mastitis, arthritis, ankylosis, abortions, stillbirths and granular vulvovaginitis. Despite its significance, developing a successful vaccine remains elusive, mostly due to the lack of knowledge about M. agalactiae's pathogenicity factors and pathogenic mechanisms, including its "core" antigens. The aim of this study was to identify, characterize and express antigenic proteins of M. agalactiae as potential vaccine candidates. Predicted proteins of type strain PG2 were analyzed using bioinformatic algorithms to assess their cellular localization and to identify their linear and conformational epitopes for B cells. Out of a total of 156 predicted membrane proteins, three were shortlisted as potential antigenic surface proteins, namely [MAG_1560 (WP_011949336.1), MAG_6130 (WP_011949770.1) and P40 (WP_011949418.1)]. These proteins were expressed in recombinant Escherichia coli strains. Purified proteins were evaluated for their antigenicity using Western blot and ELISA using sera of M. agalactiae-naturally infected and non-infected sheep and goats. All 3 proteins were specifically recognized by the tested sera of M. agalactiae-infected animals. Also, specific rabbit antisera raised against each of these 3 proteins confirm their membrane localization using TritonX-114 phase partioning, Western and colony immunoblotting. In conclusion, our study successfully identified P40 (as proof of concept and validation) and two novel antigenic M. agalactiae proteins as potential candidates for developing effective CA vaccines.
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Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Mycoplasma agalactiae/química , Testes Sorológicos/métodos , Animais , Antígenos de Bactérias/genética , Epitopos de Linfócito B/imunologia , Feminino , Genoma Bacteriano , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Mycoplasma agalactiae/genética , Mycoplasma agalactiae/imunologia , CoelhosRESUMO
Dengue virus represents the main arbovirus affecting humans, but there are no effective drugs or available worldwide licensed vaccine formulations capable of conferring full protection against the infection. Experimental studies and results generated after the release of the licensed anti-DENV vaccine demonstrated that induction of high-titer neutralizing antibodies does not represent the sole protection correlate and that, indeed, T cell-based immune responses plays a relevant role in the establishment of an immune protective state. In this context, this study aimed to further demonstrate protective features of immune responses elicited in immunocompetent C57BL/6 mice immunized with three plasmids encoding DENV2 nonstructural proteins (NS1, NS3, and NS5), which were subsequently challenged with a DENV2 strain naturally capable of inducing lethal encephalitis in immunocompetent mouse strains. The animals were immunized intramuscularly with the DNA vaccine mix and complete protection was observed among vaccinated mice. Vaccine induced protection correlated with the cytokine profiles expressed by spleen cells and brain-infiltrating mononuclear cells. The results confirm the pivotal role of cellular immune responses targeting nonstructural DENV proteins and validate the experimental model based on a DENV2 strain capable of infecting and killing immunocompetent mice as a tool for the evaluation of protective immunity induced by anti-DENV vaccines.
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Zika virus (ZIKV) is a globally-distributed flavivirus transmitted to humans by Aedes mosquitoes, usually causing mild symptoms that may evolve to severe conditions, including neurological alterations, such as neonatal microcephaly and Guillain-Barré syndrome. Due to the absence of specific and effective preventive methods, we designed a new subunit vaccine based on a DNA vector (pgDNS1-ZIKV) encoding the non-structural protein 1 (NS1) genetically fused to the Herpes Simplex Virus (HSV) glycoprotein D (gD) protein. Recombinant plasmids were replicated in Escherichia coli and the expression of the target protein was confirmed in transfected HEK293 cells. C57BL/6 and AB6 (IFNAR1-/-) mice were i.m. immunized by electroporation in order to evaluate pgDNS1-ZIKV immunogenicity. After two doses, high NS1-specific IgG antibody titers were measured in serum samples collected from pgDNS1-ZIKV-immunized mice. The NS1-specific antibodies were capable to bind the native protein expressed in infected mammalian cells. Immunization with pgDNS1-ZIKV increased both humoral and cellular immune responses regarding mice immunized with a ZIKV NS1 encoding vaccine. Immunization with pgDNS1-ZIKV reduced viremia and morbidity scores leading to enhanced survival of immunodeficient AB6 mice challenged with a lethal virus load. These results give support to the use of ZIKV NS1 as a target antigen and further demonstrate the relevant adjuvant effects of HSV-1 gD.
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[This corrects the article DOI: 10.3389/fmedt.2020.558984.].
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In the present study, we evaluated the immunological responses induced by dengue vaccines under experimental conditions after delivery via a transcutaneous (TC) route. Vaccines against type 2 Dengue virus particles (DENV2 New Guinea C (NGC) strain) combined with enterotoxigenic Escherichia coli (ETEC) heat-labile toxin (LT) were administered to BALB/c mice in a three-dose immunization regimen via the TC route. As a control for the parenteral administration route, other mouse groups were immunized with the same vaccine formulation via the intradermic (ID) route. Our results showed that mice vaccinated either via the TC or ID routes developed similar protective immunity, as measured after lethal challenges with the DENV2 NGC strain. Notably, the vaccine delivered through the TC route induced lower serum antibody (IgG) responses with regard to ID-immunized mice, particularly after the third dose. The protective immunity elicited in TC-immunized mice was attributed to different antigen-specific antibody properties, such as epitope specificity and IgG subclass responses, and cellular immune responses, as determined by cytokine secretion profiles. Altogether, the results of the present study demonstrate the immunogenicity and protective properties of a dengue vaccine delivered through the TC route and offer perspectives for future clinical applications.
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Vacinas contra Dengue/administração & dosagem , Vírus da Dengue/imunologia , Dengue/prevenção & controle , Administração Cutânea , Animais , Anticorpos Antivirais/sangue , Dengue/sangue , Dengue/imunologia , Dengue/virologia , Vacinas contra Dengue/genética , Vacinas contra Dengue/imunologia , Vírus da Dengue/genética , Humanos , Imunização , Imunoglobulina G/sangue , Injeções Intradérmicas , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Targeting dendritic cells (DCs) by means of monoclonal antibodies (mAbs) capable of binding their surface receptors (DEC205 and DCIR2) has previously been shown to enhance the immunogenicity of genetically fused antigens. This approach has been repeatedly demonstrated to enhance the induced immune responses to passenger antigens and thus represents a promising therapeutic and/or prophylactic strategy against different infectious diseases. Additionally, under experimental conditions, chimeric αDEC205 or αDCIR2 mAbs are usually administered via an intraperitoneal (i.p.) route, which is not reproducible in clinical settings. In this study, we characterized the delivery of chimeric αDEC205 or αDCIR2 mAbs via an intradermal (i.d.) route, compared the elicited humoral immune responses, and evaluated the safety of this potential immunization strategy under preclinical conditions. As a model antigen, we used type 2 dengue virus (DENV2) nonstructural protein 1 (NS1). The results show that the administration of chimeric DC-targeting mAbs via the i.d. route induced humoral immune responses to the passenger antigen equivalent or superior to those elicited by i.p. immunization with no toxic effects to the animals. Collectively, these results clearly indicate that i.d. administration of DC-targeting chimeric mAbs presents promising approaches for the development of subunit vaccines, particularly against DENV and other flaviviruses.
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In this study we evaluated the association of high hydrostatic pressure (HHP) and alkaline pH as a minimally denaturing condition for the solubilization of inclusion bodies (IBs) generated by recombinant proteins expressed by Escherichia coli strains. The method was successfully applied to a recombinant form of the dengue virus (DENV) non-structural protein 1 (NS1). The minimal pH for IBs solubilization at 1 bar was 12 while a pH of 10 was sufficient for solubilization at HHP: 2.4 kbar for 90 min and 0.4 kbar for 14 h 30 min. An optimal refolding condition was achieved by compression of IBs at HHP and pH 10.5 in the presence of arginine, oxidized and reduced glutathiones, providing much higher yields (up to 8-fold) than association of HHP and GdnHCl via an established protocol. The refolded NS1, 109 ± 9.5 mg/L bacterial culture was recovered mainly as monomer and dimer, corresponding up to 90% of the total protein and remaining immunologically active. The proposed conditions represent an alternative for the refolding of immunologically active recombinant proteins expressed as IBs.
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Vírus da Dengue/química , Redobramento de Proteína , Proteínas não Estruturais Virais/química , Vírus da Dengue/genética , Concentração de Íons de Hidrogênio , Pressão Hidrostática , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas não Estruturais Virais/genéticaRESUMO
Dengue fever has become a global threat, causing millions of infections every year. An effective vaccine against all four serotypes of dengue virus (DENV) has not been developed yet. Among the different vaccination strategies available today, DNA vaccines are safe and practical, but currently induce relatively weak immune responses in humans. In order to improve immunogenicity, antigens may be targeted to dendritic cells (DCs), the main antigen presenting cells and orchestrators of the adaptive immune response, inducing T and B cell activation. It was previously shown that a DNA vaccine encoding a fusion protein comprised of an antigen and a single-chain Fv antibody (scFv) specific for the DC endocytic receptor DEC205 induced strong immune responses to the targeted antigen. In this work, we evaluate this strategy to improve the immunogenicity of dengue virus (DENV) proteins. Plasmids encoding the scFv αDEC205, or an isotype control (scFv ISO), fused to the DENV2 envelope protein domain III (EDIII) were generated, and EDIII specific immune responses were evaluated in immunized mice. BALB/c mice were intramuscularly (i.m.) immunized three times with plasmid DNAs encoding either scDEC-EDIII or scISO-EDIII followed by electroporation. Analyses of the antibody responses indicated that EDIII fusion with scFv targeting the DEC205 receptor significantly enhanced serum anti-EDIII IgG titers that inhibited DENV2 infection. Similarly, mice immunized with the scDEC-EDIII plasmid developed a robust CD4+ T cell response to the targeted antigen, allowing the identification of two linear epitopes recognized by the BALB/c haplotype. Taken together, these results indicate that targeting DENV2 EDIII protein to DCs using a DNA vaccine encoding the scFv αDEC205 improves both antibody and CD4+ T cell responses. This strategy opens perspectives for the use of DNA vaccines that encode antigens targeted to DCs as a strategy to increase immunogenicity.