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Vaccination is one of the most effective prophylactic public health interventions for the prevention of infectious diseases such as coronavirus disease (COVID-19). Considering the ongoing need for new COVID-19 vaccines, it is crucial to modify our approach and incorporate more conserved regions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to effectively address emerging viral variants. The nucleocapsid protein is a structural protein of SARS-CoV-2 that is involved in replication and immune responses. Furthermore, this protein offers significant advantages owing to the minimal accumulation of mutations over time and the inclusion of key T-cell epitopes critical for SARS-CoV-2 immunity. A novel strategy that may be suitable for the new generation of vaccines against COVID-19 is to use a combination of antigens, including the spike and nucleocapsid proteins, to elicit robust humoral and potent cellular immune responses, along with long-lasting immunity. The strategic use of multiple antigens aims to enhance vaccine efficacy and broaden protection against viruses, including their variants. The immune response against the nucleocapsid protein from other coronavirus is long-lasting, and it can persist up to 11 years post-infection. Thus, the incorporation of nucleocapsids (N) into vaccine design adds an important dimension to vaccination efforts and holds promise for bolstering the ability to combat COVID-19 effectively. In this review, we summarize the preclinical studies that evaluated the use of the nucleocapsid protein as antigen. This study discusses the use of nucleocapsid alone and its combination with spike protein or other proteins of SARS-CoV-2.
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Vacunas contra la COVID-19 , COVID-19 , Proteínas de la Nucleocápside de Coronavirus , SARS-CoV-2 , Humanos , Vacunas contra la COVID-19/inmunología , SARS-CoV-2/inmunología , COVID-19/prevención & control , COVID-19/inmunología , Proteínas de la Nucleocápside de Coronavirus/inmunología , Proteínas de la Nucleocápside de Coronavirus/genética , Inmunogenicidad Vacunal , Animales , Fosfoproteínas/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Epítopos de Linfocito T/inmunología , Anticuerpos Antivirales/inmunología , Proteínas de la Nucleocápside/inmunologíaRESUMEN
In mammals, the placenta is a connection between a mother and a new developing organism. This tissue has a protective function against some microorganisms, transports nutrients, and exchanges gases and excretory substances between the mother and the fetus. Placental tissue is mainly composed of chorionic villi functional units called trophoblasts (cytotrophoblasts, the syncytiotrophoblast, and extravillous trophoblasts). However, some viruses have developed mechanisms that help them invade the placenta, causing various conditions such as necrosis, poor perfusion, and membrane rupture which, in turn, can impact the development of the fetus and put the mother's health at risk. In this study, we collected the most relevant information about viral infection during pregnancy which can affect both the mother and the fetus, leading to an increase in the probability of vertical transmission. Knowing these mechanisms could be relevant for new research in the maternal-fetal context and may provide options for new therapeutic targets and biomarkers in fetal prognosis.
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Viruses have a wide repertoire of molecular strategies that focus on their replication or the facilitation of different stages of the viral cycle. One of these strategies is mediated by the activity of viroporins, which are multifunctional viral proteins that, upon oligomerization, exhibit ion channel properties with mild ion selectivity. Viroporins facilitate multiple processes, such as the regulation of immune response and inflammasome activation through the induction of pore formation in various cell organelle membranes to facilitate the escape of ions and the alteration of intracellular homeostasis. Viroporins target diverse membranes (such as the cellular membrane), endoplasmic reticulum, and mitochondria. Cumulative data regarding the importance of mitochondria function in multiple processes, such as cellular metabolism, energy production, calcium homeostasis, apoptosis, and mitophagy, have been reported. The direct or indirect interaction of viroporins with mitochondria and how this interaction affects the functioning of mitochondrial cells in the innate immunity of host cells against viruses remains unclear. A better understanding of the viroporin-mitochondria interactions will provide insights into their role in affecting host immune signaling through the mitochondria. Thus, in this review, we mainly focus on descriptions of viroporins and studies that have provided insights into the role of viroporins in hijacked mitochondria.
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Proteínas Viroporinas , Virus , Proteínas Viroporinas/metabolismo , Proteínas Virales/metabolismo , Canales Iónicos/metabolismo , Inmunidad InnataRESUMEN
Despite all successful efforts to develop a COVID-19 vaccine, the need to evaluate alternative antigens to produce next-generation vaccines is imperative to target emerging variants. Thus, the second generation of COVID-19 vaccines employ more than one antigen from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to induce an effective and lasting immune response. Here, we analyzed the combination of two SARS-CoV-2 viral antigens that could elicit a more durable immune response in both T- and B-cells. The nucleocapsid (N) protein, Spike protein S1 domain, and receptor binding domain (RBD) of the SARS-CoV-2 spike surface glycoproteins were expressed and purified in a mammalian expression system, taking into consideration the posttranscriptional modifications and structural characteristics. The immunogenicity of these combined proteins was evaluated in a murine model. Immunization combining S1 or RBD with the N protein induced higher levels of IgG antibodies, increased the percentage of neutralization, and elevated the production of cytokines TNF-α, IFN-γ, and IL-2 compared to the administration of a single antigen. Furthermore, sera from immunized mice recognized alpha and beta variants of SARS-CoV-2, which supports ongoing clinical results on partial protection in vaccinated populations, despite mutations. This study identifies potential antigens for second-generation COVID-19 vaccines.
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Zika virus (ZIKV) infection is a major public health threat, making the study of its biology a matter of great importance. By analyzing the viral-host protein interactions, new drug targets may be proposed. In this work, we showed that human cytoplasmic dynein-1 (Dyn) interacts with the envelope protein (E) of ZIKV. Biochemical evidence indicates that the E protein and the dimerization domain of the heavy chain of Dyn binds directly without dynactin or any cargo adaptor. Analysis of this interactions in infected Vero cells by proximity ligation assay suggest that the E-Dyn interaction is dynamic and finely tuned along the replication cycle. Altogether, our results suggest new steps in the replication cycle of the ZIKV for virion transport and indicate a suitable molecular target to modulate infection by ZIKV.
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Infección por el Virus Zika , Virus Zika , Chlorocebus aethiops , Humanos , Animales , Dineínas Citoplasmáticas , Células Vero , Transporte BiológicoRESUMEN
The COVID-19 pandemic has caused major disturbances to human health and economy on a global scale. Although vaccination campaigns and important advances in treatments have been developed, an early diagnosis is still crucial. While PCR is the golden standard for diagnosing SARS-CoV-2 infection, rapid and low-cost techniques such as ATR-FTIR followed by multivariate analyses, where dimensions are reduced for obtaining valuable information from highly complex data sets, have been investigated. Most dimensionality reduction techniques attempt to discriminate and create new combinations of attributes prior to the classification stage; thus, the user needs to optimize a wealth of parameters before reaching reliable and valid outcomes. In this work, we developed a method for evaluating SARS-CoV-2 infection and COVID-19 disease severity on infrared spectra of sera, based on a rather simple feature selection technique (correlation-based feature subset selection). Dengue infection was also evaluated for assessing whether selectivity toward a different virus was possible with the same algorithm, although independent models were built for both viruses. High sensitivity (94.55%) and high specificity (98.44%) were obtained for assessing SARS-CoV-2 infection with our model; for severe COVID-19 disease classification, sensitivity is 70.97% and specificity is 94.95%; for mild disease classification, sensitivity is 33.33% and specificity is 94.64%; and for dengue infection assessment, sensitivity is 84.27% and specificity is 94.64%.
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Dengue and Zika viruses cocirculate annually in endemic areas of Mexico, causing outbreaks of different magnitude and severity every year, suggesting a continuous selection of Flavivirus variants with variable phenotypes of transmissibility and virulence. To evaluate if Flavivirus variants with different phenotypes cocirculate during outbreaks, we isolated dengue and Zika viruses from blood samples of febrile patients from Oaxaca City during the 2016 and 2019 epidemic years. We compared their replication kinetics in human cells, susceptibility to type I interferon antiviral response, and the accumulation of subgenomic RNA on infected cells. We observed correlations between type I interferon susceptibility and subgenomic RNA accumulation, with high hematocrit percentage and thrombocytopenia. Our results suggest that Flaviviruses that cocirculate in Oaxaca, Mexico, have variable sensitivity to the antiviral activity of type I interferons, and this phenotypic trait correlates with the severity of the disease.
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Dengue , Flavivirus , Interferón Tipo I , Infección por el Virus Zika , Virus Zika , Antivirales , Flavivirus/genética , Humanos , México/epidemiología , ARN Viral/genética , Índice de Severidad de la Enfermedad , Replicación Viral , Virus Zika/genéticaRESUMEN
Severe coronavirus disease 2019 (COVID-19) is characterized by lung injury, cytokine storm, and increased neutrophil-to-lymphocyte ratio (NLR). Current therapies focus on reducing viral replication and inflammatory responses, but no specific treatment exists to prevent the development of severe COVID-19 in infected individuals. Angiotensin-converting enzyme-2 (ACE2) is the receptor for SARS-CoV-2, the virus causing COVID-19, but it is also critical for maintaining the correct functionality of lung epithelium and endothelium. Coronaviruses induce activation of a disintegrin and metalloprotease 17 (ADAM17) and shedding of ACE2 from the cell surface resulting in exacerbated inflammatory responses. Thus, we hypothesized that ADAM17 inhibition ameliorates COVID-19-related lung inflammation. We employed a preclinical mouse model using intratracheal instillation of a combination of polyinosinic:polycytidylic acid (poly(I:C)) and the receptor-binding domain of the SARS-CoV-2 spike protein (RBD-S) to mimic lung damage associated with COVID-19. Histologic analysis of inflamed mice confirmed the expected signs of lung injury including edema, fibrosis, vascular congestion, and leukocyte infiltration. Moreover, inflamed mice also showed an increased NLR as observed in critically ill COVID-19 patients. Administration of the ADAM17/MMP inhibitors apratastat and TMI-1 significantly improved lung histology and prevented leukocyte infiltration. Reduced leukocyte recruitment could be explained by reduced production of proinflammatory cytokines and lower levels of the endothelial adhesion molecules ICAM-1 and VCAM-1. Additionally, the NLR was significantly reduced by ADAM17/MMP inhibition. Thus, we propose inhibition of ADAM17/MMP as a novel promising treatment strategy in SARS-CoV-2-infected individuals to prevent the progression toward severe COVID-19.
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Tratamiento Farmacológico de COVID-19 , Lesión Pulmonar , Proteína ADAM17 , Enzima Convertidora de Angiotensina 2 , Animales , Modelos Animales de Enfermedad , Humanos , Lesión Pulmonar/etiología , Lesión Pulmonar/prevención & control , Metaloproteinasas de la Matriz , Ratones , SARS-CoV-2 , Glicoproteína de la Espiga del CoronavirusRESUMEN
The coronavirus disease 2019 (COVID-19) pandemic has reached an unprecedented level. There is a strong demand for diagnostic and serological supplies worldwide, making it necessary for countries to establish their own technologies to produce high-quality biomolecules. The two main viral antigens used for the diagnostics for severe acute respiratory syndrome coronavirus (SARS-CoV-2) are the structural proteins spike (S) protein and nucleocapsid (N) protein. The spike protein of SARS-CoV-2 is cleaved into S1 and S2, in which the S1 subunit has the receptor-binding domain (RBD), which induces the production of neutralizing antibodies, whereas nucleocapsid is an ideal target for viral antigen-based detection. In this study, we designed plasmids, pcDNA3.1/S1 and pcDNA3.1/N, and optimized their expression of the recombinant S1 and N proteins from SARS-CoV-2 in a mammalian system. The RBD was used as a control. The antigens were successfully purified from Expi293 cells, with high yields of the S1, N, and RBD proteins. The immunogenic abilities of these proteins were demonstrated in a mouse model. Further, enzyme-linked immunosorbent assays with human serum samples showed that the SARS-CoV-2 antigens are a suitable alternative for serological assays to identify patients infected with COVID-19.
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A common hallmark of dengue infections is the dysfunction of the vascular endothelium induced by different biological mechanisms. In this paper, we studied the role of recombinant NS1 proteins representing the four dengue serotypes, and their role in promoting the expression and release of endocan, which is a highly specific biomarker of endothelial cell activation. We evaluated mRNA expression and the levels of endocan protein in vitro following the stimulation of HUVEC and HMEC-1 cell lines with recombinant NS1 proteins. NS1 proteins increase endocan mRNA expression 48 h post-activation in both endothelial cell lines. Endocan mRNA expression levels were higher in HUVEC and HMEC-1 cells stimulated with NS1 proteins than in non-stimulated cells (p < 0.05). A two-fold to three-fold increase in endocan protein release was observed after the stimulation of HUVECs or HMEC-1 cells with NS1 proteins compared with that in non-stimulated cells (p < 0.05). The blockade of Toll-like receptor 4 (TLR-4) signaling on HMEC-1 cells with an antagonistic antibody prevented NS1-dependent endocan production. Dengue-infected patients showed elevated serum endocan levels (≥30 ng/mL) during early dengue infection. High endocan serum levels were associated with laboratory abnormalities, such as lymphopenia and thrombocytopenia, and are associated with the presence of NS1 in the serum.
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The intestinal epithelial barrier (IEB) depends on stable interepithelial protein complexes such as tight junctions (TJ), adherens junctions (AJ), and the actin cytoskeleton. During inflammation, the IEB is compromised due to TJ protein internalization and actin remodeling. An important actin regulator is the actin-related protein 2/3 (Arp2/3) complex, which induces actin branching. Activation of Arp2/3 by nucleation-promoting factors is required for the formation of epithelial monolayers, but little is known about the relevance of Arp2/3 inhibition and endogenous Arp2/3 inhibitory proteins for IEB regulation. We found that the recently identified Arp2/3 inhibitory protein arpin was strongly expressed in intestinal epithelial cells. Arpin expression decreased in response to tumor necrosis factor (TNF)α and interferon (IFN)γ treatment, whereas the expression of gadkin and protein interacting with protein C-kinase α-subunit 1 (PICK1), other Arp2/3 inhibitors, remained unchanged. Of note, arpin coprecipitated with the TJ proteins occludin and claudin-1 and the AJ protein E-cadherin. Arpin depletion altered the architecture of both AJ and TJ, increased actin filament content and actomyosin contractility, and significantly increased epithelial permeability, demonstrating that arpin is indeed required for maintaining IEB integrity. During experimental colitis in mice, arpin expression was also decreased. Analyzing colon tissues from ulcerative colitis patients by Western blot, we found different arpin levels with overall no significant changes. However, in acutely inflamed areas, arpin was significantly reduced compared to non-inflamed areas. Importantly, patients receiving mesalazine had significantly higher arpin levels than untreated patients. As arpin depletion (theoretically meaning more active Arp2/3) increased permeability, we wanted to know whether Arp2/3 inhibition would show the opposite. Indeed, the specific Arp2/3 inhibitor CK666 ameliorated TNFα/IFNγ-induced permeability in established Caco-2 monolayers by preventing TJ disruption. CK666 treatment also attenuated colitis development, colon tissue damage, TJ disruption, and permeability in dextran sulphate sodium (DSS)-treated mice. Our results demonstrate that loss of arpin triggers IEB dysfunction during inflammation and that low arpin levels can be considered a novel hallmark of acute inflammation.
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Dengue is a worldwide expanding threat caused by dengue virus (DENV) infection. To date, no specific treatment or effective vaccine is available. Antibodies produced by plasma cells (PCs) might be involved concomitantly in protection and severe dengue immunopathology. Although a massive appearance of PCs has been reported during acute DENV infection in humans, this response has been poorly characterized. Here, we show the dynamic of PC generation in immune-competent mice cutaneously inoculated with DENV compared with two control experimental groups: mice inoculated with inactivated DENV or with PBS. We found that PC numbers increased significantly in the skin-draining lymph node (DLN), peaking at day 10 and abruptly decreasing by day 14 after DENV inoculation. Class-switched IgG+ PCs appeared from day 7 and dominated the response, while in contrast, the frequency of IgM+ PCs decreased from day 7 onwards. Even though the kinetic of the response was similar between DENV- and iDENV-inoculated mice, the intensity of the response was significantly different. Interestingly, we demonstrated a similar PC response to virus antigens (E and prM) by ELISPOT. In situ characterization showed that PCs were distributed in the medullary cords and in close proximity to germinal centers (GCs), suggesting both an extrafollicular and a GC origin. Proliferating PCs (Ki-67+) were found as early as 3-day postinoculation, and in-depth analysis showed that these PCs were in active phases of cell cycle during the kinetic. Finally, we found a progressive appearance of high-affinity neutralizing DENV-specific IgG further supporting GC involvement. Of note, these antibodies seem to be highly cross-reactive, as a large proportion recognizes Zika virus (ZIKV). The strong PC response to skin-inoculated DENV in this work resembles the findings already described in humans. We consider that this study contributes to the understanding of the in vivo biology of the humoral immune response to DENV in an immunocompetent murine model.
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Virus del Dengue/inmunología , Dengue/inmunología , Células Plasmáticas/inmunología , Animales , Anticuerpos Neutralizantes/análisis , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Antivirales/análisis , Anticuerpos Antivirales/metabolismo , Reacciones Cruzadas , Dengue/patología , Dengue/virología , Modelos Animales de Enfermedad , Centro Germinal/citología , Centro Germinal/inmunología , Centro Germinal/metabolismo , Humanos , Masculino , Ratones , Células Plasmáticas/metabolismo , Piel/inmunología , Piel/patología , Piel/virología , Organismos Libres de Patógenos Específicos , Virus Zika/inmunologíaRESUMEN
Dengue is a global health problem without current specific treatment nor safe vaccines available. While severe dengue is related to pre-existing non-neutralizing dengue virus (DENV) antibodies, the role of T cells in protection or pathology is unclear. Using cutaneous DENV infection in immunocompetent mice we previously showed the generation of PNA+ germinal centers (GCs), now we assessed the activation and proliferation of B and T cells in draining lymph nodes (DLNs). We found a drastic remodelling of DLN compartments from 7 to 14 days post-infection (dpi) with greatly enlarged B cell follicles, occupying almost half of the DLN area compared to ~24% in naïve conditions. Enormous clusters of proliferating (Ki-67+) cells inside B follicles were found 14 dpi, representing ~33% of B cells in DLNs but only ~2% in non-infected mice. Inside GCs, we noticed an important recruitment of tingle body macrophages removing apoptotic cells. In contrast, the percentage of paracortex area and total T cells decreased by 14-16 dpi, compared to controls. Scattered randomly distributed Ki-67+ T cells were found, similar to non-infected mice. CD69 expression by CD4+ and CD8+ T cells was minor, while it was remarkable in B cells, representing 1764.7% of change from basal levels 3 dpi. The apparent lack of T cell responses cannot be attributed to apoptosis since no significant differences were observed compared to non-infected mice. This study shows massive B cell activation and proliferation in DLNs upon DENV infection. In contrast, we found very poor, almost absent CD4+ and CD8+ T cell responses.
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Virus del Dengue , Dengue , Animales , Linfocitos B , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T , RatonesRESUMEN
Dengue is the most prevalent and rapidly transmitted mosquito-borne viral disease of humans. One of the fundamental innate immune responses to viral infections includes the processing and release of pro-inflammatory cytokines such as interleukin (IL-1ß and IL-18) through the activation of inflammasome. Dengue virus stimulates the Nod-like receptor (NLRP3-specific inflammasome), however, the specific mechanism(s) by which dengue virus activates the NLRP3 inflammasome is unknown. In this study, we investigated the activation of the NLRP3 inflammasome in endothelial cells (HMEC-1) following dengue virus infection. Our results showed that dengue infection as well as the NS2A and NS2B protein expression increase the NLRP3 inflammasome activation, and further apoptosis-associated speck-like protein containing caspase recruitment domain (ASC) oligomerization, and IL-1ß secretion through caspase-1 activation. Specifically, we have demonstrated that NS2A and NS2B, two proteins of dengue virus that behave as putative viroporins, were sufficient to stimulate the NLRP3 inflammasome complex in lipopolysaccharide (LPS)-primed endothelial cells. In summary, our observations provide insight into the dengue-induced inflammatory response mechanism and highlight the importance of DENV-2 NS2A and NS2B proteins in activation of the NLRP3 inflammasome during dengue virus infection.
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Virus del Dengue/inmunología , Dengue/inmunología , Células Endoteliales/fisiología , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas no Estructurales Virales/metabolismo , Proteínas Viroporinas/metabolismo , Proteínas Adaptadoras de Señalización CARD/metabolismo , Caspasa 1/metabolismo , Línea Celular Transformada , Dengue/virología , Virus del Dengue/patogenicidad , Humanos , Inmunidad Innata , Interleucina-1beta/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas Viroporinas/genética , VirulenciaRESUMEN
Zika virus (ZIKV) is a mosquito-borne flavivirus that has caused recent large outbreaks in the Americas. Given its association with severe congenital defects including microcephaly, distinguishing infections caused by ZIKV from those caused by dengue virus (DENV) is of primordial importance. The objectives of this study were to evaluate the recombinant proteins rEIII-ZIKV (Envelope protein domain III) and rNS1ß-leader-ZIKV (non-structural protein 1) for the serological diagnosis of ZIKV in the Mexican population. We also evaluated potential cross-reactivity in commercial enzyme-linked immunosorbent assays (ELISA) based on the ZIKV NS1 and DENV NS1 proteins. rEIII-ZIKV and rNS1ß-leader-ZIKV proteins were tested with sera from 30 PCR-confirmed ZIKV cases, 50 ZIKV-naive, DENV-exposed subjects with no acute febrile disease, (asymptomatic subjects, AS), and 50 ZIKV-naive and DENV naive AS. Commercial ELISA tests were evaluated with sera from 57 ZIKV and 20 DENV PCR-confirmed cases, and 50 ZIKV-naive, DENV-exposed AS. In-house ELISA assays showed that IgM antibody levels against rEIII-ZIKV and rNS1ß-ZIKV were higher in ZIKV naive, DENV-exposed AS than in acutely infected ZIKV individuals. IgG reactivity was highest for rEIII-ZIKV, and indistinguishable between acutely infected ZIKV cases and DENV exposed AS. Positivity for the Euroimmun Zika IgM assay at 7-10 days was considerably higher in DENV-naive ZIKV patients (86%) than in DENV-exposed ZIKV patients (33%), while 39% of the latter had false-negative anti-ZIKV IgG before 7 days of onset. DENV-exposed ZIKV patients presented lower anti-ZIKV IgM and higher IgG responses similar to a secondary dengue response. Forty-four percent of DENV- exposed acute ZIKV patients were DENV IgM positive with the Panbio Dengue assay, and two (15%) of the DENV-naive ZIKV patients presented false DENV IgG conversion. Given the extensive cross-reactivity to both the NS1 and EDIII proteins in current serological methods, the development of sensitive and specific serological tests to distinguish ZIKV from DENV infections is an urgent priority.
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Anticuerpos Antivirales/sangre , Virus del Dengue/inmunología , Virus Zika/inmunología , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Pruebas SerológicasRESUMEN
Zika virus (ZIKV), an emerging mosquito-borne flavivirus, has quickly spread in many regions around the world where dengue virus (DENV) is endemic. This represents a major health concern, given the high homology between these two viruses, which can result in cross-reactivity. The aim of this study was to determine the cross-reacting antibody response of the IgM and IgG classes against the recombinant envelope protein of ZIKV (rE-ZIKV) in sera from patients with acute-phase infection of different clinical forms of dengue, i.e., dengue fever (DF) and dengue hemorrhagic fever (DHF) (before the arrival of ZIKV in Mexico 2010), as well as acute-phase sera of ZIKV patients, together with the implications in neutralization and antibody-dependent enhancement. Differences in IgM responses were observed in a number of DF and DHF patients whose sera cross-reacted with the rE-ZIK antigen, with 42% recognition between acute-phase DHF and ZIKV but 27% recognition between DF and ZIKV. Regarding IgG antibodies, 71.5% from the DF group showed cross-reactivity to rE-ZIKV in contrast with 50% and only 25% of DHF and ZIKV serum samples, respectively, which specifically recognized the homologous antigen. The DHF group showed more enhancement of ZIKV infection of FCRγ-expressing cells compared to the DF group. Furthermore, the DHF group also showed a higher cross-neutralizing ability than that of DF. This is the first report where DF and DHF serum samples were evaluated for cross-reactivity against Zika protein and ZIKV. Furthermore, DENV serum samples cross-protect against ZIKV through neutralizing antibodies but at the same time mediate antibody-dependent enhancement in the sequential ZIKV infection.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Reacciones Cruzadas/inmunología , Virus del Dengue/inmunología , Dengue/inmunología , Infección por el Virus Zika/inmunología , Virus Zika/inmunología , Adolescente , Adulto , Línea Celular , Niño , Preescolar , Dengue/epidemiología , Virus del Dengue/genética , Femenino , Humanos , Masculino , México/epidemiología , Persona de Mediana Edad , Pruebas de Neutralización , Vigilancia de la Población , Adulto Joven , Virus Zika/genética , Infección por el Virus Zika/epidemiologíaRESUMEN
The envelope (E) protein from Dengue and Zika viruses comprises three functional and structural domains (DI, DII, and DIII). Domain III induces most of the neutralizing antibodies and, as such, is considered as having the highest antigenic potential for the evaluation of population-level surveillance and for detecting past infections in both Dengue and Zika patients. The present study aimed to clone and express recombinant proteins of domain III from Dengue virus serotype 2 and from Zika virus in a prokaryotic system, as well as evaluate their immunogenicity and cross-reactivity. Both antigens were successfully purified and their antigenicity was assessed in mice. The antibodies elicited by domain III of Zika and Dengue virus antigens recognized specifically the native proteins in infected cells. Furthermore, the antigens showed a more specific immunogenic response than that of domain III proteins from Dengue virus. The generated recombinant proteins can be potentially used in subunit vaccines or for surveillance studies.
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Virus del Dengue/genética , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/aislamiento & purificación , Virus Zika/genética , Animales , Anticuerpos Antivirales/inmunología , Reacciones Cruzadas , Dengue/inmunología , Dengue/prevención & control , Dengue/virología , Vacunas contra el Dengue , Virus del Dengue/química , Virus del Dengue/inmunología , Femenino , Expresión Génica , Humanos , Ratones , Ratones Endogámicos BALB C , Dominios Proteicos , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/química , Vacunas Virales/genética , Vacunas Virales/inmunología , Vacunas Virales/aislamiento & purificación , Virus Zika/química , Virus Zika/inmunología , Infección por el Virus Zika/inmunología , Infección por el Virus Zika/prevención & control , Infección por el Virus Zika/virologíaRESUMEN
BACKGROUND: Co-circulation of dengue virus (DENV) and chikungunya virus (CHIKV) is increasing worldwide but information on the viral dynamics and immune response to DENV-CHIKV co-infection, particularly in young infants, is scant. METHODS: Blood samples were collected from 24 patients, aged 2 months to 82 years, during a CHIKV outbreak in Mexico. DENV and CHIKV were identified by RT-PCR; ELISA was used to detect IgM and IgG antibodies. CHIKV PCR products were cloned, sequenced and subjected to BLAST analysis. To address serological findings, HMEC-1 and Vero cells were inoculated with DENV-1, DENV-2 and CHIKV alone and in combination (DENV-2-CHIKV and DENV-1-CHIKV); viral titers were measured at 24, 48 and 72 h. RESULTS: Nine patients (38%) presented co-infection, of who eight were children. None of the patients presented severe illness. Sequence analysis showed that the circulating CHIKV virus belonged to the Asian lineage. Seroconversion to both viruses was only observed in the four patients five years or older, while the five infants under two years of age only seroconverted to CHIKV. Viral titers in the CHIKV mono-infected cells were greater than in the DENV-1 and DENV-2 mono-infected cells. Furthermore, we observed significantly increased CHIKV progeny and reduction of DENV progeny in the co-infected cells. CONCLUSIONS: In our population, DENV-CHIKV co-infection was not associated with increased clinical severity. Our in vitro assay findings strongly suggest that the lack of DENV IgG conversion in the co-infected infants is due to suppression of DENV replication by the Asian lineage CHIKV. The presence of maternal antibody and immature immune responses in the young infants may also play a role.
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Fiebre Chikungunya/epidemiología , Coinfección/epidemiología , Dengue/epidemiología , Replicación Viral , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Anticuerpos Antivirales/sangre , Fiebre Chikungunya/sangre , Fiebre Chikungunya/virología , Virus Chikungunya/genética , Virus Chikungunya/inmunología , Virus Chikungunya/aislamiento & purificación , Niño , Preescolar , Chlorocebus aethiops , Coinfección/sangre , Coinfección/virología , Dengue/sangre , Dengue/virología , Virus del Dengue/genética , Virus del Dengue/inmunología , Virus del Dengue/aislamiento & purificación , Brotes de Enfermedades , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Lactante , Masculino , México/epidemiología , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Pruebas Serológicas , Células Vero , Adulto JovenRESUMEN
The HPV-16 E6/E7 bicistronic immature transcript produces 4 mature RNAs: the unspliced HPV-16 E6/E7pre-mRNA product and 3 alternatively spliced mRNAs. The 3 spliced mRNAs encode short forms of the E6 oncoprotein, namely E6*I, E6*II and E6^E7. In this study we showed that transfection of C-33A cells with monocistronic constructs of these cDNAs fused to GFP, produced different effects on apoptosis, after the treatment with cisplatin. Transfection of C-33A cells with the full-length E6-GFP oncoprotein resulted in a 50% decrease in cell death, while the transfection with the E6*I-GFP construct showed only a 25% of diminution of cell death, compared to the control cells. Transfection with the E6^E7-GFP or E7-GFP construct had no effect on the number of the apoptotic cells, compared with control cells. Conversely, transfection with the E6*II construct resulted in higher cell death than the control cells. Taken together, these results suggested that E6*I or E6*II, the short forms of HPV-16 E6, displayed opposite effects on cisplatin-induced apoptosis, when transfected in C-33A cells.
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Empalme Alternativo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Cisplatino/farmacología , Papillomavirus Humano 16/genética , Proteínas Oncogénicas Virales/genética , Proteínas Represoras/genética , Secuencia de Aminoácidos , Apoptosis/genética , Línea Celular Tumoral , Cuello del Útero/efectos de los fármacos , Cuello del Útero/patología , Cuello del Útero/virología , Femenino , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Papillomavirus Humano 16/metabolismo , Humanos , Proteínas Oncogénicas Virales/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
The envelope (E) protein from DENV, contain three functional and structural domains (DI, DII and DIII). Some studies suggest that neutralizing antibodies during natural DENV infection are predominantly against DI and DII, in contrast, low proportion of the antibodies were against DIII. Thus it is necessary to establish the proportion of human antibodies against DENV E protein that bind to DI and DII during the normal course of infection; as an indicator of the quality of the antibody response and to further design new vaccine candidates for DENV. The aim of this study was to express recombinant proteins harboring a 240-aminoacid fragment of the E protein from DI and DII of DENV serotypes 2 and 3 in a eukaryotic S2 system. Further, we evaluate the antibodies against these antigens in samples from patients in acute phase of DF or DHF and compare it with the response of samples from healthy individuals from the same endemic areas and samples from healthy individuals from a non-endemic area (EA and NEA, respectively). These results suggest that the presence of antibodies against rEDI/DII might be used to identify patients at risk for severe disease.