RESUMO

BACKGROUND: SARS-CoV2 virus, responsible for the COVID-19 pandemic, has four structural proteins and 16 nonstructural proteins. S-protein is one of the structural proteins exposed on the virus surface and is the main target for producing neutralizing antibodies and vaccines. The S-protein forms a trimer that can bind the angiotensin-converting enzyme 2 (ACE2) through its receptor binding domain (RBD) for cell entry. AIMS: The goal of this study was to express in HEK293 cells a new RBD recombinant protein in a constitutive and stable manner in order to use it as an alternative immunogen and diagnostic tool for COVID-19. MATERIALS & METHODS: The protein was designed to contain an immunoglobulin signal sequence, an explanded C-terminal section of the RBD, a region responsible for the bacteriophage T4 trimerization inducer, and six histidines in the pCDNA-3.1 plasmid. Following transformation, the cells were selected with geneticin-G418 and purified from serum-fre culture supernatants using Ni2+-agarand size exclusion chromatography. The protein was structurally identified by cross-linking and circular dichroism experiments, and utilized to immunize mice in conjuction with AS03 or alum adjuvants. The mice sera were examined for antibody recognition, receptor-binding inhibition, and virus neutralization, while spleens were evaluated for γ-interferon production in the presence of RBD. RESULTS: The protein released in the culture supernatant of cells, and exhibited a molecular mass of 135 kDa with a secondary structure like the monomeric and trimeric RBD. After purification, it formed a multimeric structure comprising trimers and hexamers, which were able to bind the ACE2 receptor. It generated high antibody titers in mice when combined with AS03 adjuvant (up to 1:50,000). The sera were capable of inhibiting binding of biotin-labeled ACE2 to the virus S1 subunit and could neutralize the entry of the Wuhan virus strain into cells at dilutions up to 1:2000. It produced specific IFN-γ producing cells in immunized mouse splenocytes. DISCUSSION: Our data describe a new RBD containing protein, forming trimers and hexamers, which are able to induce a protective humoral and cellular response against SARS-CoV2. CONCLUSION: These results add a new arsenal to combat COVID-19, as an alternative immunogen or antigen for diagnosis.

Assuntos

Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19 , Proteínas Recombinantes , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Humanos , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/química , Camundongos , Anticorpos Neutralizantes/imunologia , SARS-CoV-2/imunologia , COVID-19/imunologia , COVID-19/prevenção & controle , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Células HEK293 , Enzima de Conversão de Angiotensina 2/metabolismo , Enzima de Conversão de Angiotensina 2/imunologia , Anticorpos Antivirais/imunologia , Vacinas contra COVID-19/imunologia , Camundongos Endogâmicos BALB C , Feminino , Multimerização Proteica , Domínios Proteicos/imunologia , Ligação Proteica

RESUMO

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 Combinadas

RESUMO

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.

Assuntos

Vacinas contra COVID-19 , COVID-19 , Humanos , SARS-CoV-2/genética , Brasil , COVID-19/prevenção & controle , Anticorpos Neutralizantes , Anticorpos Antivirais

RESUMO

Amongst the potential contribution of protein or peptide-display systems to study epitopes with relevant immunological features, the RAD display system stands out as a highly stable scaffold protein that allows the presentation of constrained target peptides. Here, we employed the RAD display system to present peptides derived from the SARS-CoV-2 Spike (S) protein as a tool to detect specific serum antibodies and to generate polyclonal antibodies capable of inhibiting SARS-CoV-2 infectivity in vitro. 44 linear S-derived peptides were genetically fused with the RAD scaffold (RAD-SCoV-epitopes) and screened for antigenicity with sera collected from COVID-19-infected patients. In a second step, selected RAD-SCoV-epitopes were used to immunize mice and generate antibodies. Phenotypic screening showed that some of these antibodies were able to recognize replicating viral particles in VERO CCL-81 and most notably seven of the RAD-SCoV-epitopes were able to induce antibodies that inhibited viral infection. Our findings highlight the RAD display system as an useful platform for the immunological characterization of peptides and a potentially valuable strategy for the design of antigens for peptide-based vaccines, for epitope-specific antibody mapping, and for the development of antibodies for diagnostic and therapeutic purposes.

Assuntos

COVID-19 , Pyrococcus furiosus , Humanos , Animais , Camundongos , Epitopos , Glicoproteína da Espícula de Coronavírus/metabolismo , Pyrococcus furiosus/metabolismo , Anticorpos Antivirais , Proteínas do Envelope Viral , SARS-CoV-2 , Peptídeos/química , Anticorpos Neutralizantes

RESUMO

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

Rabies is a fatal viral zoonosis caused by rabies virus (RABV). RABV infects the central nervous system and triggers acute encephalomyelitis in both humans and animals. Endemic in the Brazilian Northeast region, RABV emergence in distinct wildlife species has been identified as a source of human rabies infection and as such, constitutes a public health concern. Here, we performed post-mortem RABV analyses of 144 encephalic tissues from bats sampled from January to July 2022, belonging to 15 different species. We identified phylogenetically distinct RABV from Phyllostomidae and Molossidae bats circulating in Northeastern Brazil. Phylogenetic clustering revealed the close evolutionary relationship between RABV viruses circulating in bats and variants hosted in white-tufted marmosets, commonly captured to be kept as pets and linked to human rabies cases and deaths in Brazil. Our findings underline the urgent need to implement a phylogenetic-scale epidemiological surveillance platform to track multiple RABV variants which may pose a threat to both humans and animals.

Assuntos

Quirópteros , Vírus da Raiva , Raiva , Animais , Humanos , Callithrix , Vírus da Raiva/genética , Raiva/epidemiologia , Raiva/veterinária , Brasil/epidemiologia , Filogenia

RESUMO

The Zika virus (ZIKV) epidemic brought new discoveries regarding arboviruses, especially flaviviruses, as ZIKV was described as sexually and vertically transmitted. The latter shows severe consequences for the embryo/fetus, such as congenital microcephaly and deficiency of the neural system, currently known as Congenital ZIKV Syndrome (CZS). To better understand ZIKV dynamics in trophoblastic cells present in the first trimester of pregnancy (BeWo, HTR-8, and control cell HuH-7), an experiment of viral kinetics was performed for African MR766 low passage and Asian-Brazilian IEC ZIKV lineages. The results were described independently and demonstrated that the three placental cells lines are permissive and susceptible to ZIKV. We noticed cytopathic effects that are typical in in vitro viral infection in BeWo and HTR-8. Regarding kinetics, MR766lp showed peaks of viral loads in 24 and 48 hpi for all cell types tested, as well as marked cells death after peak production. On the other hand, the HTR-8 lineage inoculated with ZIKV-IEC exhibited increased viral production in 144 hpi, with a peak between 24 and 96 hpi. Furthermore, IEC had peak variations of viral production for BeWo in 144 hpi. Considering such in vitro results, the hypothesis that maternal fetal transmission is probably a way of virus transmission between the mother and the embryo/fetus is maintained.

Assuntos

Infecção por Zika virus , Zika virus , Humanos , Gravidez , Feminino , Placenta , Brasil , Cinética , Linhagem Celular

RESUMO

Zika virus (ZIKV), a mosquito-borne pathogen, is an emerging arbovirus associated with sporadic symptomatic cases of great medical concern, particularly among pregnant women and newborns affected with neurological disorders. Serological diagnosis of ZIKV infection is still an unmet challenge due to the co-circulation of the dengue virus, which shares extensive sequence conservation of structural proteins leading to the generation of cross-reactive antibodies. In this study, we aimed to obtain tools for the development of improved serological tests for the detection of ZIKV infection. Polyclonal sera (pAb) and a monoclonal antibody (mAb 2F2) against a recombinant form of the ZIKV nonstructural protein 1 (NS1) allowed the identification of linear peptide epitopes of the NS1 protein. Based on these findings, six chemically synthesized peptides were tested both in dot blot and ELISA assays using convalescent sera collected from ZIKV-infected patients. Two of these peptides specifically detected the presence of ZIKV antibodies and proved to be candidates for the detection of ZIKV-infected subjects. The availability of these tools opens perspectives for the development of NS1-based serological tests with enhanced sensitivity regarding other flaviviruses.

Assuntos

Proteínas não Estruturais Virais , Infecção por Zika virus , Feminino , Humanos , Recém-Nascido , Gravidez , Anticorpos Monoclonais , Anticorpos Antivirais , Ensaio de Imunoadsorção Enzimática , Peptídeos , Testes Sorológicos , Proteínas não Estruturais Virais/isolamento & purificação , Zika virus

RESUMO

The main coronavirus disease 2019 (COVID-19) vaccine formulations used today are mainly based on the wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein as an antigen. However, new virus variants capable of escaping neutralization activity of serum antibodies elicited in vaccinated individuals have emerged. The Omicron (B.1.1.529) variant caused epidemics in regions of the world in which most of the population has been vaccinated. In this study, we aimed to understand what determines individual's susceptibility to Omicron in a scenario of extensive vaccination. For that purpose, we collected nasopharynx swab (n = 286) and blood samples (n = 239) from flu-like symptomatic patients, as well as their vaccination history against COVID-19. We computed the data regarding vaccine history, COVID-19 diagnosis, COVID-19 serology, and viral genome sequencing to evaluate their impact on the number of infections. As main results, we showed that vaccination in general did not reduce the number of individuals infected by Omicron, even with an increased immune response found among vaccinated, noninfected individuals. Nonetheless, we found that individuals who received the third vaccine dose showed significantly reduced susceptibility to Omicron infections. A relevant evidence that support this finding was the higher virus neutralization capacity of serum samples of most patients who received the third vaccine dose. In summary, this study shows that boosting immune responses after a third vaccine dose reduces susceptibility to COVID-19 caused by the Omicron variant. Results presented in this study are useful for future formulations of COVID-19 vaccination policies.

Assuntos

Vacinas contra COVID-19 , COVID-19 , Humanos , Anticorpos Neutralizantes , Anticorpos Antivirais , COVID-19/prevenção & controle , Teste para COVID-19 , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/imunologia , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus

RESUMO

Individuals with chronic obstructive pulmonary disease (COPD) are more susceptible to exacerbation crisis triggered by secondary lung infections due to the dysfunction of antiviral signaling, principally via suppression of IFN-γ. Although the probiotic is known for controlling pulmonary inflammation in COPD, the influence of the Lactobacillus rhamnosus (Lr) on antiviral signaling in bronchial epithelium exposed to cigarette smoke extract (CSE) and viruses, remains unknown. Thus, the present study investigated the Lr effect on the antiviral signaling and the secretion of inflammatory mediators from bronchial epithelial cells (16HBE cells) exposed to CSE and SARS-CoV-2. The 16HBE cells were cultured, treated with Lr, stimulated with CSE, and infected with SARS-CoV-2. The cellular viability was evaluated using the MTT assay and cytotoxicity measured by lactate dehydrogenase (LDH) activity. The viral load, TLR2, TLR3, TLR4, TLR7, TLR8, MAVS, MyD88, and TRIF were quantified using specific PCR. The pro-inflammatory mediators were measured by a multiplex biometric immunoassay, and angiotensin converting enzyme 2 (ACE2) activity, NF-κB, RIG-I, MAD5, and IRF3 were measured using specific ELISA kits. Lr decreased viral load, ACE2, pro-inflammatory mediators, TLR2, TLR4, NF-κB, TLR3, TLR7, and TLR8 as well as TRIF and MyD88 expression in CSE and SARS-CoV-2 -exposed 16HBE cells. Otherwise, RIG-I, MAD5, IRF3, IFN-γ, and the MAVS expression were restored in 16HBE cells exposed to CSE and SARS-CoV-2 and treated with Lr. Lr induces antiviral signaling associated to IFN-γ secreting viral sensors and attenuates cytokine storm associated to NF-κB in bronchial epithelial cells, supporting its emerging role in prevention of COPD exacerbation.

Assuntos

COVID-19 , Fumar Cigarros , Lacticaseibacillus rhamnosus , Doença Pulmonar Obstrutiva Crônica , Humanos , SARS-CoV-2/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Fumar Cigarros/efeitos adversos , Receptor 4 Toll-Like/metabolismo , Receptor 2 Toll-Like , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 7 Toll-Like/metabolismo , Receptor 8 Toll-Like/metabolismo , COVID-19/metabolismo , Células Epiteliais/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Antivirais/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo

RESUMO

Reliable serological tests for the detection of SARS-CoV-2 antibodies among infected or vaccinated individuals are important for epidemiological and clinical studies. Low-cost approaches easily adaptable to high throughput screenings, such as Enzyme-Linked Immunosorbent Assays (ELISA) or electrochemiluminescence immunoassay (ECLIA), can be readily validated using different SARS-CoV-2 antigens. A total of 1,119 serum samples collected between March and July of 2020 from health employees and visitors to the University Hospital at the University of São Paulo were screened with the Elecsys® Anti-SARS-CoV-2 immunoassay (Elecsys) (Roche Diagnostics) and three in-house ELISAs that are based on different antigens: the Nucleoprotein (N-ELISA), the Receptor Binding Domain (RBD-ELISA), and a portion of the S1 protein (ΔS1-ELISA). Virus neutralization test (CPE-VNT) was used as the gold standard to validate the serological assays. We observed high sensitivity and specificity values with the Elecsys (96.92% and 98.78%, respectively) and N-ELISA (93.94% and 94.40%, respectively), compared with RBD-ELISA (90.91% sensitivity and 88.80% specificity) and the ΔS1-ELISA (77.27% sensitivity and 76% specificity). The Elecsys® proved to be a reliable SARS-CoV-2 serological test. Similarly, the recombinant SARS-CoV-2 N protein displayed good performance in the ELISA tests. The availability of reliable diagnostic tests is critical for the precise determination of infection rates, particularly in countries with high SARS-CoV-2 infection rates, such as Brazil. Collectively, our results indicate that the development and validation of new serological tests based on recombinant proteins may provide new alternatives for the SARS-CoV-2 diagnostic market.

Assuntos

COVID-19 , Anticorpos Antivirais , Brasil/epidemiologia , COVID-19/diagnóstico , Técnicas de Laboratório Clínico/métodos , Hospitais , Humanos , Estudos Retrospectivos , SARS-CoV-2 , Sensibilidade e Especificidade

Assuntos

COVID-19 , SARS-CoV-2 , Evolução Biológica , Humanos , SARS-CoV-2/genética

RESUMO

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.

Assuntos

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/diagnóstico

RESUMO

This study aims to describe the sociodemographic determinants associated with exposure to Zika Virus (ZIKV) in pregnant women during the 2015-2016 epidemic in Salvador, Brazil. METHODS: We recruited women who gave birth between October 2015 and January 2016 to a cross-sectional study at a referral maternity hospital in Salvador, Brazil. We collected information on their demographic, socioeconomic, and clinical characteristics, and evaluated their ZIKV exposure using a plaque reduction neutralization test. Logistic regression was then used to assess the relationship between these social determinants and ZIKV exposure status. RESULTS: We included 469 pregnant women, of whom 61% had a positive ZIKV result. Multivariate analysis found that lower education (adjusted Prevalence Rate [aPR] 1.21; 95%CI 1.04-1.35) and food insecurity (aPR 1.17; 95%CI 1.01-1.30) were positively associated with ZIKV exposure. Additionally, age was negatively associated with the infection risk (aPR 0.99; 95%CI 0.97-0.998). CONCLUSION: Eve after controlling for age, differences in key social determinants, as education and food security, were associated with the risk of ZIKV infection among pregnant women in Brazil. Our findings elucidate risk factors that can be targeted by future interventions to reduce the impact of ZIKV infection in this vulnerable population.

Assuntos

Complicações Infecciosas na Gravidez/epidemiologia , Complicações Infecciosas na Gravidez/virologia , Fatores Socioeconômicos , Infecção por Zika virus/economia , Infecção por Zika virus/epidemiologia , Adulto , Brasil/epidemiologia , Estudos Transversais , Feminino , Humanos , Gravidez , Complicações Infecciosas na Gravidez/economia , Fatores de Risco

RESUMO

This study aims to describe the sociodemographic determinants associated with exposure to Zika Virus (ZIKV) in pregnant women during the 2015–2016 epidemic in Salvador, Brazil. Methods We recruited women who gave birth between October 2015 and January 2016 to a cross-sectional study at a referral maternity hospital in Salvador, Brazil. We collected information on their demographic, socioeconomic, and clinical characteristics, and evaluated their ZIKV exposure using a plaque reduction neutralization test. Logistic regression was then used to assess the relationship between these social determinants and ZIKV exposure status. Results We included 469 pregnant women, of whom 61% had a positive ZIKV result. Multivariate analysis found that lower education (adjusted Prevalence Rate [aPR] 1.21; 95%CI 1.04–1.35) and food insecurity (aPR 1.17; 95%CI 1.01–1.30) were positively associated with ZIKV exposure. Additionally, age was negatively associated with the infection risk (aPR 0.99; 95%CI 0.97–0.998). Conclusion Eve after controlling for age, differences in key social determinants, as education and food security, were associated with the risk of ZIKV infection among pregnant women in Brazil. Our findings elucidate risk factors that can be targeted by future interventions to reduce the impact of ZIKV infection in this vulnerable population.

RESUMO

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.

RESUMO

This article aims to review the present status of anti-flavivirus subunit vaccines, both those at the experimental stage and those already available for clinical use. Aspects regarding development of vaccines to Yellow Fever virus, (YFV), Dengue virus (DENV), West Nile virus (WNV), Zika virus (ZIKV), and Japanese encephalitis virus (JEV) are highlighted, with particular emphasis on purified recombinant proteins generated in bacterial cells. Currently licensed anti-flavivirus vaccines are based on inactivated, attenuated, or virus-vector vaccines. However, technological advances in the generation of recombinant antigens with preserved structural and immunological determinants reveal new possibilities for the development of recombinant protein-based vaccine formulations for clinical testing. Furthermore, novel proposals for multi-epitope vaccines and the discovery of new adjuvants and delivery systems that enhance and/or modulate immune responses can pave the way for the development of successful subunit vaccines. Nonetheless, advances in this field require high investments that will probably not raise interest from private pharmaceutical companies and, therefore, will require support by international philanthropic organizations and governments of the countries more severely stricken by these viruses.

RESUMO

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.

Assuntos

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 C

RESUMO

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.

Assuntos

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 Espécie

RESUMO

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.