RESUMEN
Detection of hepatitis B Virus surface antigen (HBsAg) is an established method for diagnosing both acute and chronic hepatitis B virus (HBV) infection. In addition to enzyme immunoassays (EIAs), rapid diagnostic tests (RDTs) are available for the detection of HBsAg in resource-poor settings. However, the available RDTs have inadequate sensitivity and therefore are not suitable for diagnosis of patients with low levels of HBsAg and for blood screening. To provide a high-sensitivity RDT, we developed a lateral flow immunoassay (LFIA) for HBsAg utilizing upconverting nanoparticle (UCNP) reporter. The UCNP-LFIA can use whole blood, serum, or plasma and the results can be read in 30 min using a reader device. When compared with a commercial conventional visually read LFIA, the developed UCNP-LFIA had a Limit of Detection (LoD) of 0.1 IU HBsAg/ml in spiked serum, whereas the LoD of the conventional LFIA was 3.2 IU HBsAg/ml. The developed UCNP-LFIA fulfills the WHO criterion for blood screening (LoD ≤ 0.13 IU HBsAg/ml) in terms of LoD. The UCNP-LFIA and conventional LFIA were evaluated with well-characterized sample panels. The UCNP-LFIA detected 20/24 HBsAg-positive samples within the HBsAg Performance Panel and 8/10 samples within the Mixed Titer Performance Panel, whereas the conventional LFIA detected 8/24 and 4/10 samples in these panels, respectively. The performance of the assays was further evaluated with HBsAg-positive (n = 108) and HBsAg-negative (n = 315) patient samples. In comparison with a central laboratory test, UCNP-LFIA showed 95.4% (95% CI: 89.5-98.5%) sensitivity whereas sensitivity of the conventional LFIA was 87.7% (95%CI: 79.9-93.3%).
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Anticuerpos Inmovilizados/química , Antígenos de Superficie de la Hepatitis B/análisis , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B/diagnóstico , Inmunoensayo/métodos , Nanopartículas/química , Diseño de Equipo , Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/sangre , Humanos , Inmunoensayo/instrumentación , Límite de Detección , Mediciones Luminiscentes/instrumentación , Mediciones Luminiscentes/métodosRESUMEN
Rapid diagnostic tests (RDTs) are often used for the detection of anti-human immunodeficiency virus (HIV) antibodies in remote locations in low- and middle-income countries (LMIC) with low or limited access to central laboratories. The typical format of an RDT is a lateral flow assay (LFA) with visual interpretation prone to subjectivity. This risk of misinterpretation can be overcome with luminescent upconverting nanoparticle reporters (UCNPs) measured with a miniaturized easy-to-use reader instrument. An LFA with UCNPs for anti-HIV-1/2 antibodies was developed and the assay performance was evaluated extensively with challenging patient sample panels. Sensitivity (n = 145) of the UCNP-LFA was 96.6% (95% CI: 92.1-98.8%) and specificity (n = 309) was 98.7% (95% CI: 96.7-99.7%). Another set of samples (n = 200) was used for a comparison between the UCNP-LFA and a conventional visual RDT. In this comparison, the sensitivities for HIV-1 were 96.4% (95% CI: 89.8-99.3%) and 97.6% (95% CI: 91.6-99.7%), for the UCNP-LFA and conventional RDT, respectively. The specificity was 100% (95% CI: 96.4-100%) for both assays. The developed UCNP-LFA demonstrates the applicability of UCNPs for the detection of anti-HIV antibodies. The signal measurement is done by a reader instrument, which may facilitate automated result interpretation, archiving and transfer of data from de-centralized locations.
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VIH-1 , Nanopartículas , Anticuerpos , Humanos , Inmunoensayo , Pruebas Inmunológicas , Sensibilidad y EspecificidadRESUMEN
OBJECTIVES: To devise a novel, simple chest x-ray (CXR) scoring system which would help in prognosticating the disease severity and ability to predict comorbidities and in-hospital mortality. METHODS: We included a total of 343 consecutive hospitalised patients with COVID-19 in this study. The chest x-rays of these patients were scored retrospectively by three radiologists independently. We divided CXR in to six zones (right upper, mid & lower and left, upper mid & lower zones). We scored each zone as- 0, 1 or 2 as follows- if that zone was clear (0) Ground glass opacity (1) or Consolidation (2). A total of score from 0 to 12 could be obtained. RESULTS: A CXR score cut off ≥3 independently predicted mortality. Along with a relatively higher NPV ≥80%, it reinforced the importance of CXR score is a screening tool to triage patients according to risk of mortality. CONCLUSIONS: We propose that Pennine score is a simple tool which can be adapted by various countries, experiencing a large surge in number of patients, to decide which patient would need a tertiary Hospital referral/admission as opposed to patients that can be managed locally or at basic/primary care hospitals.
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COVID-19/diagnóstico por imagen , Radiografía Torácica , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , COVID-19/diagnóstico , COVID-19/mortalidad , Comorbilidad , Femenino , Mortalidad Hospitalaria , Humanos , Tiempo de Internación , Masculino , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Pronóstico , Radiografía Torácica/métodos , Radiografía Torácica/estadística & datos numéricos , Estudios Retrospectivos , Factores de Riesgo , Índice de Severidad de la EnfermedadRESUMEN
Plasmodium falciparum malaria is widespread in the tropical and subtropical regions of the world. There is ongoing effort to eliminate malaria from endemic regions, and sensitive point-of-care (POC) diagnostic tests are required to support this effort. However, current POC tests are not sufficiently sensitive to detect P. falciparum in asymptomatic individuals. After extensive optimization, we have developed a highly sensitive and robust POC test for the detection of P. falciparum infection. The test is based on upconverting nanophosphor-based lateral flow (UCNP-LF) immunoassay. The developed UCNP-LF test was validated using whole blood reference panels containing samples at different parasite densities covering eight strains of P. falciparum from different geographical areas. The limit of detection was compared to a WHO-prequalified rapid diagnostic test (RDT). The UCNP-LF achieved a detection limit of 0.2-2 parasites/µL, depending on the strain, which is 50- to 250-fold improvement in analytical sensitivity over the conventional RDTs. The developed UCNP-LF is highly stable even at 40 °C for at least 5 months. The extensively optimized UCNP-LF assay is as simple as the conventional malaria RDTs and requires 5 µL of whole blood as sample. Results can be read after 20 min from sample addition, with a simple photoluminescence reader. In the absence of a reader device at the testing site, the strips after running the test can be transported and read at a central location with access to a reader. We have found that the test and control line signals are stable for at least 10 months after running the test. The UCNP-LF has potential for diagnostic testing of both symptomatic and asymptomatic individuals.
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Inmunoensayo , Malaria Falciparum/diagnóstico , Pruebas en el Punto de Atención , HumanosAsunto(s)
Vacunas contra el Dengue , Virus del Dengue/inmunología , Dengue , Adolescente , Niño , HumanosRESUMEN
BACKGROUND: Dengue is a viral disease spread to humans by mosquitoes. Notably, there are four serotypes of dengue viruses (DENV) that places ~40 % of the global population at risk of infection. However, lack of a suitable drug or a preventive vaccine exacerbates the matter further. Envelope domain-III (EDIII) antigen of dengue virus (DENV) has garnered much attention as a promising vaccine candidate for dengue, in addition to its use as a diagnostic intermediate. Hence developing a method for efficient production of high quality recombinant EDIII is important for research and industrial purpose. RESULTS: In this work, a Pichia pastoris system was optimized for the secretory over-expression of DENV serotype-3 EDIII under the control of methanol inducible AOX1 promoter. Temperature alone had a significant impact upon the amount of secretory EDIII, with 2.5-fold increase upon reducing the induction temperature from 30 to 20 °C. However surprisingly, supplementation of culture media with Casamino acids (CA), further augmented secretory EDIII titer, with a concomitant drop of intracellular EDIII levels at both temperatures. Though, reduction in intracellular retention of EDIII was more prominent at 20 °C than 30 °C. This suggests that CA supplementation facilitates overexpressing P. pastoris cells to secrete more EDIII by reducing the proportion retained intracellularly. Moreover, a bell-shaped correlation was observed between CA concentration and secretory EDIII titer. The maximum EDIII expression level of 187 mg/L was achieved under shake flask conditions with induction at 20 °C in the presence of 1 % CA. The overall increase in EDIII titer was ~9-fold compared to un-optimized conditions. Notably, mouse immune-sera, generated using this purified EDIII antigen, efficiently neutralized the DENV. CONCLUSIONS: The strategy described herein could enable fulfilling the mounting demand for recombinant EDIII as well as lay direction to future studies on secretory expression of recombinant proteins in P. pastoris with CA as a media supplement.
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Aminoácidos/metabolismo , Virus del Dengue/genética , Pichia/genética , Proteínas Recombinantes/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Aminoácidos/química , Animales , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
BACKGROUND: Four antigenically distinct serotypes (1-4) of dengue viruses (DENVs) cause dengue disease. Antibodies to any one DENV serotype have the potential to predispose an individual to more severe disease upon infection with a different DENV serotype. A dengue vaccine must elicit homotypic neutralizing antibodies to all four DENV serotypes to avoid the risk of such antibody-dependent enhancement in the vaccine recipient. This is a formidable challenge as evident from the lack of protective efficacy against DENV-2 by a tetravalent live attenuated dengue vaccine that has completed phase III trials recently. These trial data underscore the need to explore non-replicating subunit vaccine alternatives. Recently, using the methylotrophic yeast Pichia pastoris, we showed that DENV-2 and DENV-3 envelope (E) glycoproteins, expressed in absence of prM, implicated in causing severe dengue disease, self-assemble into virus-like particles (VLPs), which elicit predominantly virus-neutralizing antibodies and confer significant protection against lethal DENV challenge in an animal model. The current study extends this work to a third DENV serotype. RESULTS: We cloned and expressed DENV-1 E antigen in P. pastoris, and purified it to near homogeneity. Recombinant DENV-1 E underwent post-translational processing, namely, signal peptide cleavage and glycosylation. Purified DENV-1 E self-assembled into stable VLPs, based on electron microscopy and dynamic light scattering analysis. Epitope mapping with monoclonal antibodies revealed that the VLPs retained the overall antigenic integrity of the virion particles despite the absence of prM. Subtle changes accompanied the efficient display of E domain III (EDIII), which contains type-specific neutralizing epitopes. These VLPs were immunogenic, eliciting predominantly homotypic EDIII-directed DENV-1-specific neutralizing antibodies. CONCLUSIONS: This work demonstrates the inherent potential of P. pastoris-expressed DENV-1 E glycoprotein to self-assemble into VLPs eliciting predominantly homotypic neutralizing antibodies. This work justifies an investigation of the last remaining serotype, namely, DENV-4, to assess if it also shares the desirable vaccine potential manifested by the remaining three DENV serotypes. Such efforts could make it possible to envisage the development of a tetravalent dengue vaccine based on VLPs of P. pastoris-expressed E glycoproteins of the four DENV serotypes.
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Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/inmunología , Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Pichia/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Ratones , Pichia/genéticaRESUMEN
BACKGROUND: Dengue is today the most significant of arboviral diseases. Novel tools are necessary to effectively address the problem of dengue. Virus-like particles (VLP) offer a versatile nanoscale platform for developing tools with potential biomedical applications. From the perspective of a potentially useful dengue-specific tool, the dengue virus envelope protein domain III (EDIII), endowed with serotype-specificity, host receptor recognition and the capacity to elicit virus-neutralizing antibodies, is an attractive candidate. METHODS: We have developed a strategy to co-express and co-purify Hepatitis B virus surface (S) antigen in two forms: independently and as a fusion with EDIII. We characterized these physically and functionally. RESULTS: The two forms of the S antigen associate into VLPs. The ability of these to display EDIII in a functionally accessible manner is dependent upon the relative levels of the two forms of the S antigen. Mosaic VLPs containing the fused and un-fused components in 1:4 ratio displayed maximal functional competence. CONCLUSIONS: VLPs armed with EDIII may be potentially useful in diagnostic, therapeutic and prophylactic applications.
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Virus del Dengue/fisiología , Dengue/diagnóstico , Dengue/virología , Nanopartículas/química , Animales , Antígenos Virales/aislamiento & purificación , Antígenos Virales/ultraestructura , Extractos Celulares , Chlorocebus aethiops , Pichia/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/aislamiento & purificación , Especificidad de la Especie , Células Vero , Proteínas del Envoltorio Viral , Proteínas Virales/química , Proteínas Virales/aislamiento & purificación , Virión/metabolismoRESUMEN
While most vaccines aim to develop a solid humoral and neutralizing antibody response against the pathogen, an effective vaccine candidate should be able to stimulate both the B-cell mediated humoral immunity, and T-cell mediated cellular immunity. The focus of vaccinology is rapidly gaining to generate T cell responses, which can mediate pathogen clearance and help B cells leading to protective antibody responses. Here we evaluate the cellular immune response of the pre-clinical tetravalent dengue subunit vaccine candidate, DSV4, in mice. While we have shown previously that DSV4 induces type-specific neutralizing antibody responses in mice, in this study, we show that the vaccine candidate DSV4 well induces dengue-specific T- cell responses evaluated by their ability to produce IFN-γ. In addition to IFN-γ secretion by both CD4+ and CD8+ T-cells in immunized mice, we observed that DSV4 also induces a higher frequency and cytokine functions of follicular CD4+ helper T-cells (TFH). These cytokines lead to an efficient germinal center reaction and potent B cell antibody response. Apart from TFH response, DSV4 stimulated Type 1 T helper cells (TH1) which is characteristic of a viral infection leading to secretion of pro-inflammatory cytokines and phagocyte-dependent protective immune responses. Our study highlights that DSV4 can mediate both arms of adaptive immunity-humoral and cell-mediated immunity in mice. By elucidating vaccine-specific T cell response, our work has implications in showing DSV4 as an effective, type-specific and safe dengue vaccine candidate.
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Anticuerpos Antivirales , Dengue , Ratones , Animales , Vacunas Combinadas , Inmunidad Celular , Anticuerpos Neutralizantes , Citocinas , Dengue/prevención & controlRESUMEN
Dengue virus (DENV) infection has increased worldwide, with over 400 million infections annually, and has become a serious public health concern. Several drug candidates, new and repurposed, have failed to meet the primary efficacy endpoints. We have recently shown that Aqueous Extract of the stem of Cocculus hirsutus (AQCH) was effective in vitro and in vivo against DENV and was safe in humans. We now report that an active ingredient of AQCH, Sinococuline, protects against the antibody-mediated secondary-DENV infection in the AG129 mouse model. DENV infection markers were assessed, viz. serum viremia and vital organs pathologies-viral load, proinflammatory cytokines and intestinal vascular leakage. The treatment with Sinococuline at 2.0 mg/kg/day; BID (twice a day), was the most effective in protecting the severely DENV-infected AG129 mice. Also, this dose effectively reduced serum viremia and tissue-viral load and inhibited the elevated expression levels of proinflammatory cytokines (TNF-α and IL-6) in several vital organs. Based on these findings, it could be explored further for pre-clinical and clinical developments for the treatment of dengue.
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Cocculus , Virus del Dengue , Morfinanos , Animales , Humanos , Ratones , Cocculus/química , Citocinas/metabolismo , Virus del Dengue/efectos de los fármacos , Modelos Animales de Enfermedad , Viremia/tratamiento farmacológico , Morfinanos/farmacologíaRESUMEN
Japanese encephalitis virus (JEV) is the causal agent behind Japanese encephalitis (JE), a potentially severe brain infection that spreads through mosquito bites. JE is predominant over the Asia-Pacific Region and has the potential to spread globally with a higher rate of morbidity and mortality. Efforts have been made to identify and select various target molecules essential in JEV's progression, but until now, no licensed anti-JEV drug has been available. From a prophylactic point of view, a few licensed JE vaccines are available, but various factors, viz., the high cost and different side effects imposed by them, has narrowed their global use. With an average occurrence of >67,000 cases of JE annually, there is an urgent need to find a suitable antiviral drug to treat patients at the acute phase, as presently only supportive care is available to mitigate infection. This systematic review highlights the current status of efforts put in to develop antivirals against JE and the available vaccines, along with their effectiveness. It also summarizes epidemiology, structure, pathogenesis, and potential drug targets that can be explored to develop a new range of anti-JEV drugs to combat JEV infection globally.
RESUMEN
BACKGROUND: Pichia pastoris is an established eukaryotic host for the production of recombinant proteins. Most often, protein production is under the control of the strong methanol-inducible aox1 promoter. However, detailed information about the physiological alterations in P. pastoris accompanying the shift from growth on glycerol to methanol-induced protein production under industrial relevant conditions is missing. Here, we provide an analysis of the physiological response of P. pastoris GS115 to methanol-induced high-level production of the Hepatitis B virus surface antigen (HBsAg). High product titers and the retention of the protein in the endoplasmic reticulum (ER) are supposedly of major impact on the host physiology. For a more detailed understanding of the cellular response to methanol-induced HBsAg production, the time-dependent changes in the yeast proteome and ultrastructural cell morphology were analyzed during the production process. RESULTS: The shift from growth on glycerol to growth and HBsAg production on methanol was accompanied by a drastic change in the yeast proteome. In particular, enzymes from the methanol dissimilation pathway started to dominate the proteome while enzymes from the methanol assimilation pathway, e.g. the transketolase DAS1, increased only moderately. The majority of methanol was metabolized via the energy generating dissimilatory pathway leading to a corresponding increase in mitochondrial size and numbers. The methanol-metabolism related generation of reactive oxygen species induced a pronounced oxidative stress response (e.g. strong increase of the peroxiredoxin PMP20). Moreover, the accumulation of HBsAg in the ER resulted in the induction of the unfolded protein response (e.g. strong increase of the ER-resident disulfide isomerase, PDI) and the ER associated degradation (ERAD) pathway (e.g. increase of two cytosolic chaperones and members of the AAA ATPase superfamily) indicating that potential degradation of HBsAg could proceed via the ERAD pathway and through the proteasome. However, the amount of HBsAg did not show any significant decline during the cultivation revealing its general protection from proteolytic degradation. During the methanol fed-batch phase, induction of vacuolar proteases (e.g. strong increase of APR1) and constitutive autophagic processes were observed. Vacuolar enclosures were mainly found around peroxisomes and not close to HBsAg deposits and, thus, were most likely provoked by peroxisomal components damaged by reactive oxygen species generated by methanol oxidation. CONCLUSIONS: In the methanol fed-batch phase P. pastoris is exposed to dual stress; stress resulting from methanol degradation and stress resulting from the production of the recombinant protein leading to the induction of oxidative stress and unfolded protein response pathways, respectively. Finally, the modest increase of methanol assimilatory enzymes compared to the strong increase of methanol dissimilatory enzymes suggests here a potential to increase methanol incorporation into biomass/product through metabolic enhancement of the methanol assimilatory pathway.
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Antígenos de Superficie de la Hepatitis B/metabolismo , Metanol/metabolismo , Pichia/metabolismo , Autofagia , Degradación Asociada con el Retículo Endoplásmico , Proteínas Fúngicas/metabolismo , Glicerol/metabolismo , Antígenos de Superficie de la Hepatitis B/genética , Chaperonas Moleculares/metabolismo , Estrés Oxidativo , Peroxirredoxinas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Respuesta de Proteína Desplegada , Vacuolas/metabolismoRESUMEN
BACKGROUND: The Human Immunodeficiency Virus type 1 (HIV-1) envelope glycoprotein gp160, useful in detecting anti-HIV-1 antibodies, is difficult to express in heterologous hosts. The major hurdles are its signal sequence, strong hydrophobic regions and heavy glycosylation. While it has not been possible to express full length recombinant (r)-gp160 in E. coli, it can be expressed in insect and mammalian cells, but at relatively higher cost. In this work, we report E. coli-based over-expression of r-gp160 variant and evaluate its performance in diagnostic immunoassays for the detection of anti-HIV-1 antibodies. METHODS: A deletion variant of r-gp160 lacking hydrophobic regions of the parent full length molecule was expressed in E. coli and purified to near homogeneity using single-step Ni(II)-affinity chromatography. Biotinylated and europium(III) chelate-labeled versions of this antigen were used to set up one- and two-step time-resolved fluorometric double antigen sandwich assays. The performance of these assays was evaluated against a collection of well-characterized human sera (n=131), that included an in-house panel and four commercially procured panels. RESULTS: In-frame deletion of three hydrophobic regions, spanning amino acid residues 1-43, 519-538 and 676-706, of full length HIV-1 gp160 resulted in its expression in E. coli. Both the one- and two-step assays manifested high sensitivity unambiguously identifying 75/77 and 77/77 HIV-1 positive sera, respectively. Both assays also identified all 52 HIV-seronegative sera correctly. Between the two assays, the mean signal-to-cutoff value of the two-step assay was an order of magnitude greater than that of the one-step assay. Both assays were highly specific manifesting no cross-reactivity towards antibodies specific to other viruses like hepatitis B, C, and human T cell leukemia viruses. CONCLUSIONS: This study has demonstrated the expression of r-gp160 variant in E. coli, by deletion of hydrophobic regions, and its purification in reasonable yields. This underscores the potential for cost saving in antigen production. Evaluation of this antigen in a double antigen sandwich two-step assay showed it to be a highly sensitive and specific HIV-1 diagnostic reagent. The amenability of this assay to the one-step format suggests its potential utility in developing a rapid point-of-care HIV-1 diagnostic test.
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Antígenos Virales , Técnicas de Laboratorio Clínico/métodos , Anticuerpos Anti-VIH/sangre , Proteínas gp160 de Envoltorio del VIH , Infecciones por VIH/diagnóstico , Antígenos Virales/genética , Antígenos Virales/aislamiento & purificación , Cromatografía de Afinidad , Escherichia coli/genética , Expresión Génica , Proteínas gp160 de Envoltorio del VIH/genética , Proteínas gp160 de Envoltorio del VIH/aislamiento & purificación , Humanos , Inmunoensayo/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Sensibilidad y Especificidad , Eliminación de SecuenciaRESUMEN
BACKGROUND: Dengue is a global public health problem for which no drug or vaccine is available. Currently, there is increasing interest in developing non-replicating dengue vaccines based on a discrete antigenic domain of the major structural protein of dengue viruses (DENVs), known as envelope domain III (EDIII). The use of bio-nanoparticles consisting of recombinant viral structural polypeptides, better known as virus-like particles (VLPs), has emerged as a potential platform technology for vaccine development. This work explores the feasibility of developing nanoparticles based on E. coli-expressed recombinant Hepatitis B virus core antigen (HBcAg) designed to display EDIII moiety of DENV on the surface. FINDINGS: We designed a synthetic gene construct encoding HBcAg containing an EDIII insert in its c/e1 loop. The fusion antigen HBcAg-EDIII-2 was expressed in E. coli, purified to near homogeneity using Ni+2 affinity chromatography and demonstrated to assemble into discrete 35-40 nm VLPs by electron microscopy. Competitive ELISA analyses showed that the EDIII-2 moieties of the VLPs are accessible to anti-EDIII-2-specific monoclonal and polyclonal antibodies, suggesting that they are surface-displayed. The VLPs were highly immunogenic eliciting high titer anti-EDIII-2 antibodies that were able to recognize, bind and neutralize infectious DENV based on ELISA, immunofluorescence and virus-neutralization assays. CONCLUSION: This work demonstrates that HBcAg-derived nanoparticles can serve as a useful platform for the display of DENV EDIII. The EDIII-displaying nanoparticles may have potential applications in diagnostics/vaccines for dengue.
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Vacunas contra el Dengue/inmunología , Virus del Dengue/inmunología , Dengue/prevención & control , Antígenos del Núcleo de la Hepatitis B/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Proteínas del Envoltorio Viral/inmunología , Animales , Anticuerpos Antivirales/inmunología , Dengue/inmunología , Vacunas contra el Dengue/genética , Vacunas contra el Dengue/aislamiento & purificación , Virus del Dengue/genética , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/genética , Expresión Génica , Antígenos del Núcleo de la Hepatitis B/genética , Antígenos del Núcleo de la Hepatitis B/aislamiento & purificación , Humanos , Ratones , Ratones Endogámicos BALB C , Vacunación , Vacunas de Partículas Similares a Virus/genética , Vacunas de Partículas Similares a Virus/aislamiento & purificación , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/aislamiento & purificaciónRESUMEN
BACKGROUND: A protective immune response against Hepatitis B infection can be obtained through the administration of a single viral polypeptide, the Hepatitis B surface antigen (HBsAg). Thus, the Hepatitis B vaccine is generated through the utilization of recombinant DNA technology, preferentially by using yeast-based expression systems. However, the polypeptide needs to assemble into spherical particles, so-called virus-like particles (VLPs), to elicit the required protective immune response. So far, no clear evidence has been presented showing whether HBsAg assembles in vivo inside the yeast cell into VLPs or later in vitro during down-stream processing and purification. RESULTS: High level production of HBsAg was carried out with recombinant Pichia pastoris using the methanol inducible AOX1 expression system. The recombinant vaccine was isolated in form of VLPs after several down-stream steps from detergent-treated cell lysates. Search for the intracellular localization of the antigen using electron microscopic studies in combination with immunogold labeling revealed the presence of HBsAg in an extended endoplasmic reticulum where it was found to assemble into defined multi-layered, lamellar structures. The distance between two layers was determined as ~6 nm indicating that these lamellas represent monolayers of well-ordered HBsAg subunits. We did not find any evidence for the presence of VLPs within the endoplasmic reticulum or other parts of the yeast cell. CONCLUSIONS: It is concluded that high level production and intrinsic slow HBsAg VLP assembly kinetics are leading to retention and accumulation of the antigen in the endoplasmic reticulum where it assembles at least partly into defined lamellar structures. Further transport of HBsAg to the Golgi apparatus is impaired thus leading to secretory pathway disfunction and the formation of an extended endoplasmic reticulum which bulges into irregular cloud-shaped formations. As VLPs were not found within the cells it is concluded that the VLP assembly process must take place during down-stream processing after detergent-mediated disassembly of HBsAg lamellas and subsequent reassembly of HBsAg into spherical VLPs.
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Antígenos de Superficie de la Hepatitis B/biosíntesis , Pichia/metabolismo , Vacunas de Partículas Similares a Virus/biosíntesis , Aldehído Oxidasa/genética , Aldehído Oxidasa/metabolismo , Retículo Endoplásmico/metabolismo , Antígenos de Superficie de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/inmunología , Inmunohistoquímica , Metanol/química , Metanol/farmacología , Microscopía Electrónica de Transmisión , Vacunas Sintéticas/biosíntesis , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas de Partículas Similares a Virus/genética , Vacunas de Partículas Similares a Virus/inmunologíaRESUMEN
BACKGROUND: Flavivirus cross-reactive antibodies in human sera interfere with the definitive identification of dengue virus (DENV) infections especially in areas with multiple co-circulating flaviviruses. Use of DENV envelope domain-III (EDIII) can partially resolve the problem. This study has examined the effect of (i) incorporating the EDIIIs of four DENV serotypes into a single chimeric antigen, and (ii) immobilizing the antigen through specific interaction on the sensitivity and specificity of anti-DENV antibody detection. METHODS: A sera panel (n = 164) was assembled and characterized using commercial kits for infection by DENV and a host of other pathogens. Anti-DENV antibodies of both IgM and IgG classes in this panel were detected in indirect ELISAs using a mixture of monovalent EDIIIs, a chimeric EDIII-based tetravalent antigen, EDIII-T, and a biotinylated version of the latter as coating antigens. The sensitivity and specificity of these assays were compared to those obtained using the PanBio Dengue IgG/IgM ELISAs. RESULTS: The performance of dengue IgG and IgM indirect ELISAs, using either a physical mixture of four EDIIIs or the single chimeric EDIII-T antigen, were comparable. Coating of a biotinylated version of the tetravalent antigen on streptavidin plates enhanced sensitivity without compromising specificity. CONCLUSIONS: The incorporation of the EDIIIs of the four DENV serotypes into a single chimeric antigen did not adversely affect assay outcome in indirect ELISAs. Oriented, rather than random, immobilization of the tetravalent antigen enhanced sensitivity of detection of anti-DENV antibodies with retention of 100% specificity.
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Anticuerpos Antivirales/sangre , Antígenos Virales , Técnicas de Laboratorio Clínico/métodos , Virus del Dengue/inmunología , Dengue/diagnóstico , Proteínas del Envoltorio Viral , Virología/métodos , Antígenos Virales/genética , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Proteínas Recombinantes de Fusión/genética , Sensibilidad y Especificidad , Proteínas del Envoltorio Viral/genéticaRESUMEN
Median arcuate ligament syndrome or celiac artery compression syndrome is one of the abdominal vascular compression syndromes due to compression of proximal celiac artery by the median arcuate ligament. The median arcuate ligament unites diaphragmatic crura on either side at the level of aortic hiatus. The ligament has a low insertion causing compression of the celiac artery resulting in clinical symptoms of postprandial pain and weight loss. It is a rare syndrome, detected incidentally on routine Computed Tomography abdomen and pelvis studies. We present a rare case of a 35-year-old female who presented with abdominal pain. She was evaluated by Computed Tomography scan of the abdomen and pelvis. Ultrasound Doppler of mesenteric vasculature helped detect celiac artery stenosis. A referral to the vascular surgery department was made; however, the patient was managed conservatively.
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White matter hyperintensities (WMHs) lacunar infarcts and cerebral microbleeds are well-established features associated with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Increasing case reports and series recounts a wide array of neurological manifestations of COVID-19 including acute cerebrovascular disease, encephalopathy, encephalitis and demyelination. Recently association between COVID-19 and CADASIL has been identified. We describe an unusual case of CADASIL diagnosed as a possible post-infectious manifestation of COVID-19 patient with imaging features closely resembling post-infectious encephalomyelitis.
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Dengue is a serious public health concern worldwide, with â¼3 billion people at risk of contracting dengue virus (DENV) infections, with some suffering severe consequences of disease and leading to death. Currently, there is no broad use vaccine or drug available for the prevention or treatment of dengue, which leaves only anti-mosquito strategies to combat the dengue menace. The present study is an extension of our earlier study aimed at determining the in vitro and in vivo protective effects of a plant-derived phytopharmaceutical drug for the treatment of dengue. In our previous report, we had identified a methanolic extract of aerial parts of Cissampelos pareira to exhibit in vitro and in vivo anti-dengue activity against all the four DENV serotypes. The dried aerial parts of C. pareira supplied by local vendors were often found to be mixed with aerial parts of another plant of the same Menispermaceae family, Cocculus hirsutus, which shares common homology with C. pareira. In the current study, we have found C. hirsutus to have more potent anti-dengue activity as compared with C. pareira. The stem part of C. hirsutus was found to be more potent (â¼25 times) than the aerial part (stem and leaf) irrespective of the extraction solvent used, viz., denatured spirit, hydro-alcohol (50:50), and aqueous. Moreover, the anti-dengue activity of stem extract in all the solvents was comparable. Hence, an aqueous extract of the stem of C. hirsutus (AQCH) was selected due to greater regulatory compliance. Five chemical markers, viz., Sinococuline, 20-Hydroxyecdysone, Makisterone-A, Magnoflorine, and Coniferyl alcohol, were identified in fingerprinting analysis. In a test of primary dengue infection in the AG129 mice model, AQCH extract at 25 mg/kg body weight exhibited protection when administered four and three times a day. The AQCH was also protective in the secondary DENV-infected AG129 mice model at 25 mg/kg/dose when administered four and three times a day. Additionally, the AQCH extract reduced serum viremia and small intestinal pathologies, viz., viral load, pro-inflammatory cytokines, and vascular leakage. Based on these findings, we have undertaken the potential preclinical development of C. hirsutus-based phytopharmaceutical, which could be studied further for its clinical development for treating dengue.
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In vivo gene delivery using human adenovirus serotype 5 (AdV5) vectors is being explored for vaccination purposes. The presence of anti-AdV5 antibodies in human serum arising from natural exposure to AdV5 can interfere potentially with and compromise the efficacy of rAdV5-based vaccine vectors. In this report, a collection of 114 sera from healthy adult Indian blood donors was analyzed for the presence of anti-AdV5 antibodies, using an AdV5 vector encoding the green fluorescent protein (GFP) to monitor the presence of anti-AdV5 neutralizing antibodies in human sera based on their ability to block virus entry into HeLa cells which express the Coxsackievirus-and-Adenovirus Receptor (CAR). In this assay all samples tested were positive for anti-AdV5 antibodies, with titers varying over a very wide range. It was also observed that these antibodies facilitated the uptake of the reporter AdV5 vector into the monocytic cell line U937 which does not express CAR, but expresses Fc receptors (FcRs) instead. These observations have implications for rAdV5-based vaccine development. J. Med. Virol. 82:407-414, 2010. (c) 2010 Wiley-Liss, Inc.