RESUMEN
The interactions between antigen and adjuvant were among the most significant factors influencing the immunogenicity of vaccines, especially for unstable antigens like inactivated foot and mouth disease virus (iFMDV). Here we propose a novel antigen delivery pattern based on the coordination interaction between transition metal ions Zn2+ chelated to chitosan nanoparticles and iFMDV, which is known to be rich in histidine. The zinc chelated chitosan particles (CP-PEI-Zn) were prepared by cross-linking chitosan particles (CP) with sodium tripolyphosphate (TPP), modifying with metal chelator polyethylenimine (PEI), and subsequent chelating of Zn2+. The coordination interaction was confirmed by analyzing the adsorption and desorption behavior of iFMDV on CP-PEI-Zn by high-performance size exclusion chromatography (HPSEC), while the CP-PEI without chelating Zn2+ loads iFMDV mainly through electrostatic interactions. The iFMDV loaded on CP-PEI-Zn showed better thermal stability than that on CP-PEI, as revealed by a slightly higher transition temperature (Tm) related to iFMDV dissociation. After subcutaneous immunization in female Balb/C mice, antigens loaded on CP-PEI and CP-PEI-Zn all induced higher specific antibody titers, better activation of B lymphocytes, and more effector-memory T cells proliferation than the free antigen and iFMDV adjuvanted with ISA 206 emulsion did. Moreover, CP-PEI-Zn showed superior efficacy to CP-PEI in promoting the proliferation of effector-memory T cells and secretion of cytokines, indicating a more potent cellular immune response. In summary, the CP-PEI-Zn stabilized the iFMDV after loading and promoted both humoral and cellular immune responses, thus reflecting its potential to be a promising adjuvant for the iFMDV vaccine and other unstable viral antigens.
Asunto(s)
Antígenos Virales/química , Antígenos Virales/inmunología , Virus de la Fiebre Aftosa/inmunología , Inmunidad Celular/inmunología , Vacunas de Productos Inactivados/inmunología , Zinc/química , Adyuvantes Inmunológicos/farmacología , Animales , Línea Celular , Cricetinae , Citocinas/inmunología , Sistemas de Liberación de Medicamentos/métodos , Femenino , Fiebre Aftosa/inmunología , Inmunidad Humoral/inmunología , Memoria Inmunológica/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Linfocitos T/inmunología , Vacunas Virales/inmunologíaRESUMEN
In the rapidly evolving field of nanotechnology, plant virus nanoparticles (pVNPs) are emerging as powerful tools in diverse applications ranging from biomedicine to materials science. The proteinaceous structure of plant viruses allows the capsid structure to be modified by genetic engineering and/or chemical conjugation with nanoscale precision. This means that pVNPs can be engineered to display peptides and proteins on their external surface, including immunodominant peptides derived from pathogens allowing pVNPs to be used for active immunization. In this context, pVNPs are safer than VNPs derived from mammalian viruses because there is no risk of infection or reversion to pathogenicity. Furthermore, pVNPs can be produced rapidly and inexpensively in natural host plants or heterologous production platforms. In this review, we discuss the use of pVNPs for the delivery of peptide antigens to the host immune in pre-clinical studies with the final aim of promoting systemic immunity against the corresponding pathogens. Furthermore, we described the versatility of plant viruses, with innate immunostimulatory properties, in providing a huge natural resource of carriers that can be used to develop the next generation of sustainable vaccines.
Asunto(s)
Nanopartículas/química , Nanotecnología/métodos , Nicotiana/genética , Virus de Plantas/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Vigna/genética , Animales , Antígenos Virales/química , Antígenos Virales/genética , Antígenos Virales/inmunología , Cápside/química , Cápside/inmunología , Evaluación Preclínica de Medicamentos , Ingeniería Genética/métodos , Humanos , Inmunización , Inmunogenicidad Vacunal , Ratones , Nanopartículas/administración & dosificación , Péptidos/química , Péptidos/genética , Péptidos/inmunología , Virus de Plantas/genética , Nicotiana/virología , Vacunas de Subunidad , Vacunas de Partículas Similares a Virus/biosíntesis , Vacunas de Partículas Similares a Virus/genética , Vigna/virologíaRESUMEN
Mucosal nasal vaccine development, although ideal to protect from pathogens invading mucosally, is limited by the lack of specific adjuvant. We recently used P1, a conserved region of HIV-1 gp41-envelope glycoprotein, as efficient antigen in a prophylactic HIV-1 mucosal vaccine applied nasally. Herein, P1 immunomodulation properties were assessed on human nasal mucosal models by measuring induction of cytokine and chemokine production, intracellular signaling pathways, mucosal dendritic cell (DC) activation, and T cell proliferation. P1 adjuvant properties were evaluated by quantification of antigen-specific B cell responses against a model antigen in an in vitro immunization model. We now demonstrated that P1 has additional immunological properties. P1 initiates immune responses by inducing nasal epithelial cells to secrete the Th2-cytokine thymic stromal lymphopoietin (TSLP), a described mucosal adjuvant. Secreted TSLP activates, in turn, intracellular calcium flux and PAR-2-associated NFAT signaling pathway regulated by microRNA-4485. Thereafter, P1 induces mucosal dendritic cell maturation, secretion of TSLP in a TSLP-receptor (R)-dependent autocrine loop, but also IL-6, IL-10, IL-8, CCL20, CCL22, and MMP-9, and proliferation of CD4+ T cells. Finally, P1 acts as an adjuvant to stimulate antigen-specific B cell responses in vitro. Overall, P1 is a multi-functional domain with various immuno-modulatory properties. In addition to being a protective vaccine antigen for HIV prevention, P1 acts as adjuvant for other mucosal vaccines able to stimulate humoral and cellular antigen-specific responses.
Asunto(s)
Adyuvantes Inmunológicos , Antígenos Virales , Proteína gp41 de Envoltorio del VIH/química , VIH-1/química , Inmunidad Innata/efectos de los fármacos , Inmunidad Mucosa/efectos de los fármacos , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/farmacología , Antígenos Virales/química , Antígenos Virales/farmacología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Línea Celular Tumoral , Citocinas/inmunología , HumanosRESUMEN
Current challenges in global immunization indicate the demand for new delivery strategies, which could be applied to the development of new vaccines against emerging diseases, as well as to improve safety and efficacy of currently existing vaccine formulations. Here, we report a novel antigen nanocarrier consisting of an oily core and a protamine shell, further stabilized with pegylated surfactants. These nanocarriers, named protamine nanocapsules, were rationally designed to promote the intracellular delivery of antigens to immunocompetent cells and to trigger an efficient and long-lasting immune response. Protamine nanocapsules have nanometric size, positive zeta potential and high association capacity for H1N1 influenza hemagglutinin, a protein that was used here as a model antigen. The new formulation shows an attractive stability profile both, as an aqueous suspension or a freeze-dried powder formulation. In vitro studies showed that protamine nanocapsules were efficiently internalized by macrophages without eliciting significant toxicity. In vivo studies indicate that antigen-loaded nanocapsules trigger immune responses comparable to those achieved with alum, even when using significantly lower antigen doses, thus indicating their adjuvant properties. These promising in vivo data, alongside with their versatility for the loading of different antigens and oily immunomodulators and their excellent stability profile, make these nanocapsules a promising platform for the delivery of antigens. CHEMICAL COMPOUNDS: Protamine sulphate (PubChem SID: 7849283), Sodium Cholate (PubChem CID: 23668194), Miglyol (PubChem CID: 53471835), α tocopherol (PubChem CID: 14985), Tween® 20(PubChem CID: 443314), Tween® 80(PubChem CID: 5281955), TPGS (PubChem CID: 71406).
Asunto(s)
Antígenos Virales/administración & dosificación , Portadores de Fármacos/administración & dosificación , Glicoproteínas Hemaglutininas del Virus de la Influenza/administración & dosificación , Nanocápsulas/administración & dosificación , Protaminas/administración & dosificación , Animales , Antígenos Virales/química , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos/química , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Inmunización , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Nanocápsulas/química , Protaminas/química , Células RAW 264.7RESUMEN
Hepatitis C virus (HCV) infection is still a serious global health burden. Despite improved therapeutic options, a preventative vaccine would be desirable especially in undeveloped countries. Traditionally, highly conserved epitopes are targets for antibody-based prophylactic vaccines. In HCV-infected patients, however, neutralizing antibodies are primarily directed against hypervariable region I (HVRI) in the envelope protein E2. HVRI is the most variable region of HCV, and this heterogeneity contributes to viral persistence and has thus far prevented the development of an effective HVRI-based vaccine. The primary goal of an antibody-based HCV vaccine should therefore be the induction of cross-reactive HVRI antibodies. In this study we approached this problem by presenting selected cross-reactive HVRI variants in a highly symmetric repeated array on capsid-like particles (CLPs). SplitCore CLPs, a novel particulate antigen presentation system derived from the HBV core protein, were used to deliberately manipulate the orientation of HVRI and therefore enable the presentation of conserved parts of HVRI. These HVRI-CLPs induced high titers of cross-reactive antibodies, including neutralizing antibodies. The combination of only four HVRI CLPs was sufficient to induce antibodies cross-reactive with 81 of 326 (24.8%) naturally occurring HVRI peptides. Most importantly, HVRI CLPs with AS03 as an adjuvant induced antibodies with a 10-fold increase in neutralizing capability. These antibodies were able to neutralize infectious HCVcc isolates and 4 of 19 (21%) patient-derived HCVpp isolates. Taken together, these results demonstrate that the induction of at least partially cross-neutralizing antibodies is possible. This approach might be useful for the development of a prophylactic HCV vaccine and should also be adaptable to other highly variable viruses.
Asunto(s)
Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Hepacivirus/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis C/prevención & control , Vacunas contra Hepatitis Viral/inmunología , Proteínas Virales/inmunología , Animales , Presentación de Antígeno , Antígenos Virales/química , Antígenos Virales/genética , Antígenos Virales/inmunología , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Protección Cruzada , Combinación de Medicamentos , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Expresión Génica , Virus de la Hepatitis B/genética , Hepatitis C/inmunología , Hepatitis C/virología , Humanos , Inmunidad Humoral , Inyecciones Intramusculares , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos C57BL , Polisorbatos/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Escualeno/administración & dosificación , Vacunas contra Hepatitis Viral/administración & dosificación , Vacunas contra Hepatitis Viral/genética , Proteínas Virales/química , Proteínas Virales/genética , alfa-Tocoferol/administración & dosificaciónRESUMEN
The vaccine safety surveillance system effectively detected a very rare adverse event, narcolepsy, in subjects receiving AS03-adjuvanted A(H1N1) pandemic vaccine made using the European inactivation/purification protocol. The reports of increased cases of narcolepsy in non-vaccinated subjects infected with wild A(H1N1) pandemic influenza virus suggest a role for the viral antigen(s) in disease development. However, additional investigations are needed to better understand what factor(s) in wild influenza infection trigger(s) narcolepsy in susceptible hosts. An estimated 31 million doses of European AS03-adjuvanted A(H1N1) pandemic vaccine were used in more than 47 countries. The Canadian AS03-adjuvanted A(H1N1) pandemic vaccine was used with high coverage in Canada where an estimated 12 million doses were administered. As no similar narcolepsy association has been reported to date with the AS03-adjuvanted A(H1N1) pandemic vaccine made using the Canadian inactivation/purification protocol, this suggests that the AS03 adjuvant alone may not be responsible for the narcolepsy association. To date, no narcolepsy association has been reported with the MF59®-adjuvanted A(H1N1) pandemic vaccine. This review article provides a brief background on narcolepsy, outlines the different types of vaccine preparations including the ones for influenza, reviews the accumulated evidence for the safety of adjuvants, and explores the association between autoimmune diseases and natural infections. It concludes by assimilating the historical observations and recent clinical studies to formulate a feasible hypothesis on why vaccine-associated narcolepsy may not be solely linked to the AS03 adjuvant but more likely be linked to how the specific influenza antigen component of the European AS03-adjuvanted pandemic vaccine was prepared. Careful and long-term epidemiological studies of subjects who developed narcolepsy in association with AS03-adjuvanted A(H1N1) pandemic vaccine prepared with the European inactivation/purification protocol are needed.
Asunto(s)
Antígenos Virales/química , Autoinmunidad , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/efectos adversos , Gripe Humana/prevención & control , Narcolepsia/inducido químicamente , Pandemias/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/química , Anticuerpos Antivirales/sangre , Antígenos Virales/inmunología , Canadá/epidemiología , Combinación de Medicamentos , Europa (Continente)/epidemiología , Humanos , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Vacunas contra la Influenza/biosíntesis , Vacunas contra la Influenza/inmunología , Gripe Humana/epidemiología , Gripe Humana/inmunología , Narcolepsia/fisiopatología , Polisorbatos/administración & dosificación , Polisorbatos/química , Escualeno/administración & dosificación , Escualeno/química , Escualeno/inmunología , Vacunación/efectos adversos , alfa-Tocoferol/administración & dosificación , alfa-Tocoferol/química , alfa-Tocoferol/inmunologíaRESUMEN
The aqueous extract of Peltophorum pterocarpum (Fabaceae) wood exhibited potent inhibitory effects against EpsteinBarr virus early antigen (EBV-EA) activation induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells and against melanogenesis in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16 melanoma cells, as well as potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging activity. Two phenolic acid derivatives, bergenin (1) and gallic acid (2), were isolated from the ethyl acetate (AcOEt)-soluble fraction obtained from the extract. Compound 1 exhibited potent inhibitory effect against EBV-EA activation and against skin tumor promotion in an in vivo two-stage mouse skin carcinogenesis test based on 7,12-dimethylbenz[a]anthracene (DMBA) as initiator, and with TPA as promoter. Both compounds 1 and 2 exhibited melanogenesis-inhibitory activities in α-MSH-stimulated B16 melanoma cells, and, in addition, compound 2 showed strong DPPH radical-scavenging activity.
Asunto(s)
Anticarcinógenos/química , Benzopiranos/química , Fabaceae/química , Depuradores de Radicales Libres/química , Ácido Gálico/química , Animales , Anticarcinógenos/farmacología , Anticarcinógenos/uso terapéutico , Anticarcinógenos/toxicidad , Antígenos Virales/química , Antígenos Virales/metabolismo , Apoptosis/efectos de los fármacos , Benzopiranos/farmacología , Benzopiranos/uso terapéutico , Línea Celular Tumoral , Fabaceae/metabolismo , Depuradores de Radicales Libres/aislamiento & purificación , Ácido Gálico/farmacología , Ácido Gálico/uso terapéutico , Células HL-60 , Humanos , Melaninas/antagonistas & inhibidores , Melaninas/metabolismo , Ratones , Extractos Vegetales/química , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/prevención & controlRESUMEN
Inactivation of intact influenza viruses using formaldehyde or ß-propiolactone (BPL) is essential for vaccine production and safety. The extent of chemical modifications of such reagents on viral proteins needs to be extensively investigated to better control the reactions and quality of vaccines. We have evaluated the effect of BPL inactivation on two candidate re-assortant vaccines (NIBRG-121xp and NYMC-X181A) derived from A/California/07/2009 pandemic influenza viruses using high-resolution FT-ICR MS-based proteomic approaches. We report here an ultra performance LC MS/MS method for determining full-length protein sequences of hemagglutinin and neuraminidase through protein delipidation, various enzymatic digestions, and subsequent mass spectrometric analyses of the proteolytic peptides. We also demonstrate the ability to reliably identify hundreds of unique sites modified by propiolactone on the surface of glycoprotein antigens. The location of these modifications correlated with changes to protein folding, conformation, and stability, but demonstrated no effect on protein disulfide linkages. In some cases, these modifications resulted in suppression of protein function, an effect that correlated with the degree of change of the modified amino acids' side chain length and polarity.
Asunto(s)
Vacunas contra la Influenza/química , Neuraminidasa/química , Propiolactona/química , Proteínas de Unión al ARN/química , Proteínas del Núcleo Viral/química , Proteínas Virales/química , Inactivación de Virus , Secuencia de Aminoácidos , Antígenos Virales/química , Cisteína/química , Hemaglutininas/química , Proteínas de la Nucleocápside , Polisacáridos/química , Espectrometría de Masas en TándemRESUMEN
Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH2-terminal ß-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the ß-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced TH1 and TH2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble (purified F1mut-V mixed with alhydrogel) and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of Y. pestis CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines.
Asunto(s)
Antígenos Bacterianos/metabolismo , Antígenos Virales/metabolismo , Bacteriófago T4/inmunología , Cápside/inmunología , Peste/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Yersinia pestis/virología , Animales , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Antígenos Virales/química , Antígenos Virales/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bacteriófago T4/química , Bacteriófago T4/metabolismo , Cápside/química , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Femenino , Ratones , Ratones Endogámicos BALB C , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Tamaño de la Partícula , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Peste/microbiología , Peste/prevención & control , Peste/virología , Vacuna contra la Peste/química , Vacuna contra la Peste/inmunología , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Dominios y Motivos de Interacción de Proteínas , Distribución Aleatoria , Ratas , Ratas Endogámicas BN , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Vacunas de Partículas Similares a Virus/química , Yersinia pestis/inmunologíaRESUMEN
Vaccination is the most effective means for preventing influenza-associated morbidity and mortality. Since the influenza virus mutates frequently, the virus strains for new vaccine production should be changed according to predicted epidemic strains. The extracellular domain of matrix protein 2 (M2e) is 24 amino acids long, which is highly conserved and therefore a good target for the development of a universal vaccine which may protect against a much wider range of influenza A virus strains. However its low antigenicity and immunogenicity, which are related to its small size, poses a big challenge for vaccine development. Multiple antigen peptide system (MAP) is based on an inert core molecule of radially branching lysine dendrites onto which a number of peptide antigens are anchored. Tuftsin is an immuno-stimulant molecule peptide. Here we developed a novel peptide vaccine by connecting a tuftsin to a branched, four-copy M2e. Not only did this increase the molecular mass, but also potentiate the immunogenicity. Two branched peptides, (M2e)4-tuftsin and (M2e)4-G4(tuftsin was replaced with four glycines), and a M2e monomer were synthesized using standard solid-phase methods. In vitro and in vivo studies were performed to compare their antigenicity and immunogenicity. Experiments in BALB/c mice demonstrated that the branched M2e could induce stronger humoral and cellular immune responses than the M2e monomer, and (M2e)4-tuftsin induced stronger humoral and cellular immune response than (M2e)4-G4. After lethal challenge with influenza virus PR8 strain, up to 80% of the animals in the (M2e)4-tuftsin vaccinated group still survived, in contrast to 44% in the (M2e)4-G4 group and 30% in the M2e monomer group. The combination of branched polypeptides and tuftsin in vaccine design is presented here for the first time, and the results show that the new construct is a promising candidate for a universal vaccine against the influenza A virus.
Asunto(s)
Antígenos Virales/inmunología , Vacunas contra la Influenza/inmunología , Tuftsina/inmunología , Proteínas de la Matriz Viral/inmunología , Animales , Anticuerpos Antivirales/sangre , Antígenos Virales/química , Modelos Animales de Enfermedad , Femenino , Vacunas contra la Influenza/administración & dosificación , Leucocitos Mononucleares/inmunología , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/prevención & control , Análisis de Supervivencia , Tuftsina/química , Proteínas de la Matriz Viral/químicaRESUMEN
A gene encoding VP7, the outer capsid protein of simian rotavirus SA11, was fused to the carboxyl terminus of the cholera toxin B subunit gene. A plant expression vector containing the fusion gene under control of the mannopine synthase P2 promoter was introduced into Solanum tuberosum cells by Agrobacterium tumefaciens-mediated transformation. The CTB::VP7 fusion gene was detected in the genomic DNA of transformed potato leaf cells by polymerase chain reaction (PCR) amplification methods. Immunoblot analysis of transformed potato tuber tissue extracts showed that synthesis and assembly of the CTB::VP7 fusion protein into oligomers of pentameric size occurred in the transformed plant cells. The binding of CTB::VP7 fusion protein pentamers to sialo-sugar containing GM1 ganglioside receptors on the intestinal epithelial cell membrane was quantified by enzyme-linked immunosorbent assay (ELISA). The ELISA results showed that the CTB::VP7 fusion protein made up approx 0.01% of the total soluble tuber protein. Synthesis and assembly of CTB::VP7 monomers into biologically active pentamers in transformed potato tubers demonstrates the feasibility of using edible plants as a mucosal vaccine for the production and delivery system for rotavirus capsid protein antigens.
Asunto(s)
Antígenos Virales/biosíntesis , Proteínas de la Cápside/biosíntesis , Toxina del Cólera/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Antígenos Virales/química , Antígenos Virales/genética , Biotecnología , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Toxina del Cólera/química , Toxina del Cólera/genética , Ensayo de Inmunoadsorción Enzimática , Hojas de la Planta , Plantas Modificadas Genéticamente , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Transformación GenéticaRESUMEN
The rotavirus capsid is made up of three concentric protein layers. The outer layer, consisting of VP7 and VP4, is lost during virus entry into the host cell. Rotavirus field isolates can be adapted to high-titre growth in tissue culture by treatment with trypsin and by supplementing the culture medium with trypsin, which cleaves VP4 into two fragments, VP8* and VP5*. It is known that protease inhibitors reduce the replication of rotavirus in vitro and in vivo and also diminish disease symptoms in a mouse model. To clarify the molecular basis of these observations, a series of assays were conducted on purified rotavirus particles grown in the presence of trypsin. Results of HPLC and mass spectrometry followed by N-terminal sequencing showed that viral particles contain molecules of trypsin. When associated with triple-layer particles (TLPs), trypsin is inactive and not accessible to protease inhibitors, such as aprotinin. When the outer layer is solubilized by calcium-chelating agents, VP5*, VP8* and VP7 are released and the associated trypsin is activated, allowing cleavage of the viral capsid proteins, as well as other exogenous proteins. It is shown that addition of trypsin inhibitors significantly reduces synthesis of viral mRNA and viral proteins in cells and has a major inhibitory effect if present when virus enters the cell. These data indicate that incorporation of trypsin into rotavirus particles may enhance its infectivity.
Asunto(s)
Antígenos Virales/metabolismo , Proteínas de la Cápside/metabolismo , Rotavirus/química , Tripsina/metabolismo , Antígenos Virales/química , Proteínas de la Cápside/química , Cromatografía Líquida de Alta Presión , Modelos Biológicos , ViriónRESUMEN
Severe acute respiratory syndrome (SARS) is a serious health threat and its early diagnosis is important for infection control and potential treatment of the disease. Diagnostic tools require rapid and accurate methods, of which a capture ELISA method may be useful. Toward this goal, we have prepared and characterized soluble full-length nucleocapsid proteins (N protein) from SARS and 229E human coronaviruses. N proteins form oligomers, mostly as dimers at low concentration. These two N proteins degrade rapidly upon storage and the major degraded N protein is the C-terminal fragment of amino acid (aa) 169-422. Taken together with other data, we suggest that N protein is a two-domain protein, with the N-terminal aa 50-150 as the RNA-binding domain and the C-terminal aa 169-422 as the dimerization domain. Polyclonal antibodies against the SARS N protein have been produced and the strong binding sites of the anti-nucleocapsid protein (NP) antibodies produced were mapped to aa 1-20, aa 150-170 and aa 390-410. These sites are generally consistent with those mapped by sera obtained from SARS patients. The SARS anti-NP antibody was able to clearly detect SARS virus grown in Vero E6 cells and did not cross-react with the NP from the human coronavirus 229E. We have predicted several antigenic sites (15-20 amino acids) of S, M and N proteins and produced antibodies against those peptides, some of which could be recognized by sera obtained from SARS patients. Antibodies against the NP peptides could detect the cognate N protein clearly. Further refinement of these antibodies, particularly large-scale production of monoclonal antibodies, could lead to the development of useful diagnostic kits for diseases associated with SARS and other human coronaviruses.
Asunto(s)
Coronavirus Humano 229E/metabolismo , Proteínas de la Nucleocápside/química , Proteómica/métodos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Anticuerpos Antivirales/química , Antígenos/química , Antígenos Virales/química , Sitios de Unión , Chlorocebus aethiops , Cromatografía en Gel , Dicroismo Circular , Clonación Molecular , Proteínas de la Nucleocápside de Coronavirus , Reactivos de Enlaces Cruzados/farmacología , ADN/química , ADN Complementario/metabolismo , Dimerización , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Humanos , Microscopía Fluorescente , Datos de Secuencia Molecular , Nucleocápside/química , Sistemas de Lectura Abierta , Péptidos/química , Análisis por Matrices de Proteínas/métodos , Unión Proteica , Estructura Terciaria de Proteína , ARN/química , Conejos , Homología de Secuencia de Aminoácido , Síndrome Respiratorio Agudo Grave/diagnóstico , Células VeroRESUMEN
Various peptide-based approaches to simultaneous induction of multiple cytotoxic T lymphocyte (CTL) responses were evaluated as part of ongoing efforts to develop immunotherapeutic vaccines for use in humans. To this end, HLA (human histocompatibility leukocyte antigen)-A2-restricted epitopes from several specific viral proteins were tested in an HLA-A2 transgenic mouse model system, which mimics human CTL responses to these viral proteins. Multiple CTL responses were elicited by immunization with either peptides emulsified in incomplete Freund's adjuvant (IFA), or lipidated peptides administered in phosphate buffered saline (PBS). In the case of lipidated peptides, induction of CTL responses was crucially dependent on the presence of helper T lymphocyte (HTL) epitopes, and most efficient in the case of lipidated covalently linked HTL-CTL epitope constructs. CTL could also be induced by immunization with lipidated HTL epitopes simply mixed with CTL epitopes and formulated in PBS. However, this approach was highly dependent on the particular lipidated HTL/CTL combination utilized, and was marginally effective for simultaneous priming of multiple CTL responses. By contrast, all HTL/CTL combinations were potent immunogens when delivered as lipidated, covalently linked molecules. This was the most effective of the approaches analysed in terms of multi-epitope priming, as demonstrated by the induction of simultaneous CTL responses to a pool of five different epitopes.
Asunto(s)
Antígenos Virales/inmunología , Epítopos/inmunología , Antígeno HLA-A2/inmunología , Hepacivirus/inmunología , Virus de la Hepatitis B/inmunología , Ácido Palmítico/química , Fragmentos de Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Vacunas Sintéticas/inmunología , Vacunas contra Hepatitis Viral/inmunología , Adyuvantes Inmunológicos , Secuencia de Aminoácidos , Animales , Antígenos Virales/química , Epítopos/química , Estudios de Factibilidad , Antígeno HLA-A2/genética , Vacunas contra Hepatitis B/química , Vacunas contra Hepatitis B/inmunología , Humanos , Inmunización , Ratones , Ratones Endogámicos A , Ratones Transgénicos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Cloruro de Sodio , Vacunas Sintéticas/química , Vacunas contra Hepatitis Viral/químicaRESUMEN
We investigated the requirement for calnexin in the biogenesis of MHC class I molecules. Mutant human cells lacking calnexin were infected with recombinant vaccinia viruses encoding mouse MHC class I molecules, Kd, Kb, Kk, Dd, Db, and Ld. Flow cytometry indicated that each of the six MHC class I allomorphs was transported to the cell surface at similar rates in calnexin-deficient cells and transfectants expressing calnexin. For Kb and Kd, the calnexin-independent biogenesis occurred regardless of whether the MHC class I molecules contained human or mouse beta 2-microglobulin. Also addressed was the effect of calnexin on the surface expression of Kb molecules bearing the immunodominant peptide from ovalbumin (OVA257-264). This was detected with a recently described monoclonal antibody specific for the Kb/peptide complex. Calnexin expression had no significant effect on the formation of Kb/peptide complexes generated from full-length OVA, cytosolic OVA257-264, or endoplasmic reticulum-targeted OVA257-264, which was expressed in the presence of the herpes simplex virus ICP47 protein to ensure detection of TAP-independent peptide-MHC class I complexes. Complementary results were obtained with TAP-independent formation of Kd/ peptide complexes. These findings indicate that calnexin is not required for the efficient assembly of MHC class I molecules with TAP-dependent or independent peptides.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Antígenos H-2/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia B, Miembro 2 , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Antígenos Virales/química , Antígenos Virales/metabolismo , Transporte Biológico , Calnexina , Compartimento Celular , Células Cultivadas , Humanos , Sustancias Macromoleculares , Ratones , Ovalbúmina/inmunología , Péptidos/inmunología , Péptidos/metabolismo , Unión Proteica , Microglobulina beta-2/metabolismoRESUMEN
The immunogenetic principle of creation of artificial antigens and vaccines possessing the abnormal immunogenecity and having capability to induce the high immune response in weakly reacting individuals have been suggested and proved by experiment. The vaccines of a new generation against the grippe pathogene, salmonellosis have been developed on the basis of this principle. In theory the methods of receiving allergovaccines for allergy disease therapy have been worked out and based.
Asunto(s)
Adyuvantes Inmunológicos , Antígenos/inmunología , Polímeros , Vacunas Sintéticas , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/uso terapéutico , Alérgenos/química , Alérgenos/inmunología , Animales , Antígenos/química , Antígenos Bacterianos/química , Antígenos Bacterianos/inmunología , Antígenos Virales/química , Antígenos Virales/inmunología , Humanos , Inmunidad Celular/inmunología , Polen/inmunología , Polímeros/química , Vacunas Sintéticas/uso terapéuticoRESUMEN
Monoclonal antibodies (MoAbs) against potato virus A (PVA) were examined in their reactivity with PVA and its denatured capsid protein (PVA-CP) bound to the nitrocellulose membrane. Five MoAbs reacted with native PVA, three of them also with PVA-CP. One MoAb gave no reaction in dot-blot test. In polyacrylamide electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE) PVA-CP migrated as two major bands. In immunoblot analysis, two MoAbs reacted only with the slower band, one only with the faster one. We presume that those bands do not correspond to the intact CP but they do to truncated N- and C-terminal CP molecules, respectively, and that the corresponding epitopes reacting with MoAbs are localized near to both termini of CP molecules. After mild trypsinolysis of PVA particles no MoAb reacted with resulting "core" CP.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Virus de Plantas/inmunología , Solanum tuberosum/virología , Antígenos Virales/química , Antígenos Virales/inmunología , Cápside/química , Cápside/inmunología , ImmunoblottingRESUMEN
Two major limitations to the use of peptides as synthetic vaccines are their poor immunogenicity and low antigenic cross-reactivity with the epitopes of virus particles. Recently it has been shown that retro-inverso peptides corresponding to an immunodominant epitope of foot-and-mouth disease virus (FMDV) are able to mimic the structure and antigenic activity of natural L-peptides [1]. A series of L- and retro-inverso peptides of the loop 141-159 of the VP1 protein of FMDV has been synthesized. Antibodies to these peptides were produced by injecting rabbits with peptides covalently coupled to small unilamellar liposomes containing monophosphoryl lipid A as adjuvant. The retro-inverso peptides led to higher serum antibody titres which appeared earlier after the start of immunization and lasted longer than those found with L-peptides. Antibodies to retro-inverso peptides cross-reacted strongly with L-peptides and with virus particles, while guinea pig antisera to VP1 protein and virions cross-reacted strongly with the retro-inverso peptides. In view of their increased stability compared to natural L-peptides, retro-inverso peptidomimetics have considerable potential as synthetic viral vaccines.