RESUMEN
Influenza and dengue viruses present a growing global threat to public health. Both viruses depend on the host endoplasmic reticulum (ER) glycoprotein folding pathway. In 2014, Sadat et al. reported two siblings with a rare genetic defect in ER α-glucosidase I (ER Glu I) who showed resistance to viral infections, identifying ER Glu I as a key antiviral target. Here, we show that a single dose of UV-4B (the hydrochloride salt form of N-(9'-methoxynonyl)-1-deoxynojirimycin; MON-DNJ) capable of inhibiting Glu I in vivo is sufficient to prevent death in mice infected with lethal viral doses, even when treatment is started as late as 48 h post infection. The first crystal structure of mammalian ER Glu I will constitute the basis for the development of potent and selective inhibitors. Targeting ER Glu I with UV-4B-derived compounds may alter treatment paradigms for acute viral disease through development of a single-dose therapeutic regime.
Asunto(s)
Dengue/prevención & control , Retículo Endoplásmico/efectos de los fármacos , Inhibidores de Glicósido Hidrolasas/administración & dosificación , Gripe Humana/prevención & control , alfa-Glucosidasas , Animales , Dengue/tratamiento farmacológico , Dengue/enzimología , Virus del Dengue/efectos de los fármacos , Virus del Dengue/enzimología , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/enzimología , Humanos , Gripe Humana/tratamiento farmacológico , Gripe Humana/enzimología , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Estructura Secundaria de Proteína , alfa-Glucosidasas/metabolismoRESUMEN
The recent Ebola virus (EBOV) epidemic in West Africa demonstrates the potential for a significant public health burden caused by filoviral infections. No vaccine or antiviral is currently FDA approved. To expand the vaccine options potentially available, we assessed protection conferred by an EBOV vaccine composed of vesicular stomatitis virus pseudovirions that lack native G glycoprotein (VSVΔG) and bear EBOV glycoprotein (GP). These pseudovirions mediate a single round of infection. Both single-dose and prime/boost vaccination regimens protected mice against lethal challenge with mouse-adapted Ebola virus (ma-EBOV) in a dose-dependent manner. The prime/boost regimen provided significantly better protection than a single dose. As N-linked glycans are thought to shield conserved regions of the EBOV GP receptor-binding domain (RBD), thereby blocking epitopes within the RBD, we also tested whether VSVΔG bearing EBOV GPs that lack GP1 N-linked glycans provided effective immunity against challenge with ma-EBOV or a more distantly related virus, Sudan virus. Using a prime/boost strategy, high doses of GP/VSVΔG partially or fully denuded of N-linked glycans on GP1 protected mice against ma-EBOV challenge, but these mutants were no more effective than wild-type (WT) GP/VSVΔG and did not provide cross protection against Sudan virus. As reported for other EBOV vaccine platforms, the protection conferred correlated with the quantity of EBOV GP-specific Ig produced but not with the production of neutralizing antibodies. Our results show that EBOV GP/VSVΔG pseudovirions serve as a successful vaccination platform in a rodent model of Ebola virus disease and that GP1 N-glycan loss does not influence immunogenicity or vaccination success.IMPORTANCE The West African Ebola virus epidemic was the largest to date, with more than 28,000 people infected. No FDA-approved vaccines are yet available, but in a trial vaccination strategy in West Africa, recombinant, infectious VSV encoding the Ebola virus glycoprotein effectively prevented virus-associated disease. VSVΔG pseudovirion vaccines may prove as efficacious and have better safety, but they have not been tested to date. Thus, we tested the efficacy of VSVΔG pseudovirions bearing Ebola virus glycoprotein as a vaccine platform. We found that wild-type Ebola virus glycoprotein, in the context of this platform, provides robust protection of EBOV-challenged mice. Further, we found that removal of the heavy glycan shield surrounding conserved regions of the glycoprotein does not enhance vaccine efficacy.
RESUMEN
Previous efforts to identify cross-neutralizing antibodies to the receptor-binding site (RBS) of ebolavirus glycoproteins have been unsuccessful, largely because the RBS is occluded on the viral surface. We report a monoclonal antibody (FVM04) that targets a uniquely exposed epitope within the RBS; cross-neutralizes Ebola (EBOV), Sudan (SUDV), and, to a lesser extent, Bundibugyo viruses; and shows protection against EBOV and SUDV in mice and guinea pigs. The antibody cocktail ZMapp™ is remarkably effective against EBOV (Zaire) but does not cross-neutralize other ebolaviruses. By replacing one of the ZMapp™ components with FVM04, we retained the anti-EBOV efficacy while extending the breadth of protection to SUDV, thereby generating a cross-protective antibody cocktail. In addition, we report several mutations at the base of the ebolavirus glycoprotein that enhance the binding of FVM04 and other cross-reactive antibodies. These findings have important implications for pan-ebolavirus vaccine development and defining broadly protective antibody cocktails.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Ebolavirus/fisiología , Epítopos/inmunología , Glicoproteínas/metabolismo , Fiebre Hemorrágica Ebola/inmunología , Receptores Virales/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/ultraestructura , Anticuerpos Neutralizantes , Anticuerpos Antivirales/química , Sitios de Unión , Modelos Animales de Enfermedad , Femenino , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/ultraestructura , Cobayas , Células HEK293 , Humanos , Cinética , Ratones Endogámicos BALB C , Modelos Moleculares , Mutación/genética , Coloración Negativa , Pruebas de Neutralización , Resultado del TratamientoRESUMEN
UNLABELLED: Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models. IMPORTANCE: Filoviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus. Since the nature of future outbreaks cannot be predicted, there is an urgent need for therapeutics with broad protective efficacy against multiple filoviruses. Here we describe a set of monoclonal antibodies cross-reactive with multiple filovirus species. These antibodies target novel conserved epitopes within the envelope glycoprotein and exhibit protective efficacy in mice. We further present novel concepts for combination of cross-reactive antibodies against multiple epitopes that show enhanced efficacy compared to monotherapy and provide complete protection in mice. These findings set the stage for further evaluation of these antibodies in nonhuman primates and development of effective pan-filovirus immunotherapeutics for use in future outbreaks.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Epítopos/inmunología , Filoviridae/inmunología , Glicoproteínas/inmunología , Fiebre Hemorrágica Ebola/prevención & control , Proteínas Virales/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/aislamiento & purificación , Anticuerpos Antivirales/uso terapéutico , Reacciones Cruzadas , Modelos Animales de Enfermedad , Femenino , Inmunización Pasiva , Macaca , Ratones Endogámicos BALB C , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
The filoviruses, which include the marburg- and ebolaviruses, have caused multiple outbreaks among humans this decade. Antibodies against the filovirus surface glycoprotein (GP) have been shown to provide life-saving therapy in nonhuman primates, but such antibodies are generally virus-specific. Many monoclonal antibodies (mAbs) have been described against Ebola virus. In contrast, relatively few have been described against Marburg virus. Here we present ten mAbs elicited by immunization of mice using recombinant mucin-deleted GPs from different Marburg virus (MARV) strains. Surprisingly, two of the mAbs raised against MARV GP also cross-react with the mucin-deleted GP cores of all tested ebolaviruses (Ebola, Sudan, Bundibugyo, Reston), but these epitopes are masked differently by the mucin-like domains themselves. The most efficacious mAbs in this panel were found to recognize a novel "wing" feature on the GP2 subunit that is unique to Marburg and does not exist in Ebola. Two of these anti-wing antibodies confer 90 and 100% protection, respectively, one hour post-exposure in mice challenged with MARV.
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Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Fiebre Hemorrágica Ebola/inmunología , Inmunización , Enfermedad del Virus de Marburg/prevención & control , Marburgvirus/inmunología , Animales , Anticuerpos Antivirales/inmunología , Reacciones Cruzadas/inmunología , Ebolavirus/inmunología , Femenino , Masculino , Enfermedad del Virus de Marburg/inmunología , Ratones Endogámicos BALB CRESUMEN
Infectious disease has only recently been recognized as a major threat to the survival of Endangered chimpanzees and Critically Endangered gorillas in the wild. One potentially powerful tool, vaccination, has not been deployed in fighting this disease threat, in good part because of fears about vaccine safety. Here we report on what is, to our knowledge, the first trial in which captive chimpanzees were used to test a vaccine intended for use on wild apes rather than humans. We tested a virus-like particle vaccine against Ebola virus, a leading source of death in wild gorillas and chimpanzees. The vaccine was safe and immunogenic. Captive trials of other vaccines and of methods for vaccine delivery hold great potential as weapons in the fight against wild ape extinction.
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Control de Enfermedades Transmisibles , Vacunas contra el Virus del Ébola/uso terapéutico , Fiebre Hemorrágica Ebola/prevención & control , Pan troglodytes/inmunología , Vacunación , Animales , Animales Salvajes , Enfermedades Transmisibles/inmunología , Islas de CpG , Modelos Animales de Enfermedad , Especies en Peligro de Extinción , Femenino , Inmunoglobulina G/inmunología , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
Two common methods of quantifying filovirus infectivity, a plaque assay and 50% cell culture infectious dose (TCID50) endpoint dilution assay, were compared. The two assays were performed side by side using the same virus stock sample to determine the correlation between the results of the two assays. The TCID50 assay appeared to be more sensitive but slightly more variable, and there was a tenfold difference in the numerical results of these methods of enumeration. The advantages and disadvantages of both assays are discussed. Both methods are useful and practicable in filovirus research, and this comparison will be hugely beneficial to the filovirus research community as it seeks to become more united. Further work in this area should be performed to ensure consistency in filovirus research.
Asunto(s)
Filoviridae/fisiología , Viabilidad Microbiana , Carga Viral/métodos , Ensayo de Placa Viral/métodos , Animales , Chlorocebus aethiops , Filoviridae/crecimiento & desarrollo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Células Vero , Cultivo de Virus/métodosRESUMEN
The filovirus plaque assay serves as the assay of choice to measure infectious virus in a cell culture, blood, or homogenized tissue sample. It has been in use for more than 30 years and is the generally accepted assay used to titrate virus in samples from animals treated with a potential antiviral therapeutic or vaccine. As these animal studies are required for the development of vaccines and therapeutics under the FDA Animal Rule, it is essential to have a standardized assay to compare their efficacies against the various filoviruses. Here, we present an evaluation of the conditions under which the filovirus plaque assay performs best for the Ebola virus Kikwit variant and the Angola variant of Marburg virus. The indicator cell type and source, inoculum volumes, length of incubation and general features of filovirus biology as visualized in the assay are addressed in terms of the impact on the sample viral titer calculations. These optimization studies have resulted in a plaque assay protocol which can be used for preclinical studies, and as a standardized protocol for use across institutions, to aid in data comparison. This protocol will be validated for use in GLP studies supporting advanced development of filovirus therapeutics and vaccines.
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Ebolavirus/aislamiento & purificación , Marburgvirus/aislamiento & purificación , Carga Viral/métodos , Carga Viral/normas , Ensayo de Placa Viral/métodos , Ensayo de Placa Viral/normas , Animales , Chlorocebus aethiops , Ebolavirus/crecimiento & desarrollo , Marburgvirus/crecimiento & desarrollo , Células VeroRESUMEN
Viruses of the family Filoviridae represent significant health risks as emerging infectious diseases as well as potentially engineered biothreats. While many research efforts have been published offering possibilities toward the mitigation of filoviral infection, there remain no sanctioned therapeutic or vaccine strategies. Current progress in the development of filovirus therapeutics and vaccines is outlined herein with respect to their current level of testing, evaluation, and proximity toward human implementation, specifically with regard to human clinical trials, nonhuman primate studies, small animal studies, and in vitro development. Contemporary methods of supportive care and previous treatment approaches for human patients are also discussed.
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Infecciones por Filoviridae/terapia , Filoviridae/inmunología , Profilaxis Posexposición/métodos , Vacunas Virales/inmunología , Animales , Ensayos Clínicos como Asunto , Humanos , Inmunoterapia/métodos , PrimatesRESUMEN
Filovirus infections can cause a severe and often fatal disease in humans and nonhuman primates, including great apes. Here, three anti-Ebola virus mouse/human chimeric mAbs (c13C6, h-13F6, and c6D8) were produced in Chinese hamster ovary and in whole plant (Nicotiana benthamiana) cells. In pilot experiments testing a mixture of the three mAbs (MB-003), we found that MB-003 produced in both manufacturing systems protected rhesus macaques from lethal challenge when administered 1 h postinfection. In a pivotal follow-up experiment, we found significant protection (P < 0.05) when MB-003 treatment began 24 or 48 h postinfection (four of six survived vs. zero of two controls). In all experiments, surviving animals that received MB-003 experienced little to no viremia and had few, if any, of the clinical symptoms observed in the controls. The results represent successful postexposure in vivo efficacy by a mAb mixture and suggest that this immunoprotectant should be further pursued as a postexposure and potential therapeutic for Ebola virus exposure.
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Anticuerpos Monoclonales/uso terapéutico , Fiebre Hemorrágica Ebola/prevención & control , Planticuerpos/uso terapéutico , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Células CHO , Cricetinae , Cricetulus , Macaca mulatta , Planticuerpos/aislamiento & purificaciónRESUMEN
We investigated the role of ICAM-3 in DC-SIGN-mediated human immunodeficiency virus (HIV) infection of CD4(+) T cells. Our results demonstrate that ICAM-3 does not appear to play a role in DC-SIGN-mediated infection of CD4(+) T cells as virus is transmitted equally to ICAM-3(+) or ICAM-3(-) Jurkat T cells. However, HIV-1 replication is enhanced in ICAM-3(-) cells, suggesting that ICAM-3 may limit HIV-1 replication. Similar results were obtained when SIV replication was examined in ICAM-3(+) and ICAM-3(-) CEMx174 cells. Furthermore, while ICAM-3 has been proposed to play a co-stimulatory role in T cell activation, DC-SIGN expression on antigen presenting cells did not enhance antigen-dependent activation of T cells. Together, these data indicate that while ICAM-3 may influence HIV-1 replication, it does so independent of DC-SIGN-mediated virus transmission or activation of CD4(+) T cells.
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Antígenos CD/fisiología , Moléculas de Adhesión Celular/fisiología , VIH-1/fisiología , Lectinas Tipo C/fisiología , Receptores de Superficie Celular/fisiología , Animales , Antígenos CD/genética , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Moléculas de Adhesión Celular/deficiencia , Moléculas de Adhesión Celular/genética , Línea Celular , VIH-1/inmunología , VIH-1/patogenicidad , Humanos , Células Jurkat , Lectinas Tipo C/genética , Activación de Linfocitos , Receptores de Superficie Celular/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Virus de la Inmunodeficiencia de los Simios/fisiología , Solubilidad , Replicación Viral/fisiologíaRESUMEN
The C-type lectin DC-SIGN mediates the capture and transfer of simian immunodeficiency virus (SIV) from macaque dendritic cells (DCs) to permissive T-cells. To further identify the determinants in macaque DC-SIGN required for capture and transfer of virus, we created mutants containing deletions or point mutations in the extracellular domains, and tested their ability to capture and transmit SIV. We found that SIV bound to the carbohydrate recognition domain (CRD) of macaque DC-SIGN via the envelope protein. In addition, deleting the C-terminal half of the CRD, or mutating amino acids within this region that contact Ca(2+) or mannose, disrupted virion capture activity. However, an N-terminal CRD deletion mutant was capable of binding SIV, indicating that this region was not necessary for binding. Finally, deletion of the neck domain also reduced the capacity for macaque DC-SIGN to capture SIV. Interestingly, ICAM-3, the cellular ligand for DC-SIGN, did not bind to any of the DC-SIGN mutants, including mutants with amino acid changes in the N-terminal region of the CRD. These data suggest that the binding sites for SIV and ICAM-3 may be distinct but overlapping. Together, the data demonstrate the importance of both the neck and the CRD of macaque DC-SIGN for efficient capture of SIV and binding to ICAM-3.
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Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/fisiología , Lectinas Tipo C/química , Lectinas Tipo C/fisiología , Receptores de Superficie Celular/química , Receptores de Superficie Celular/fisiología , Virus de la Inmunodeficiencia de los Simios/fisiología , Animales , Antígenos CD/fisiología , Sitios de Unión , Línea Celular , Humanos , Macaca , Mutagénesis Sitio-Dirigida , Subunidades de Proteína , Síndrome de Inmunodeficiencia Adquirida del Simio/transmisión , Relación Estructura-ActividadRESUMEN
Day 3 thymectomy (D3Tx) leads to a paucity of CD4(+)CD25(+) suppressor T cells, a loss of peripheral tolerance, and the development of organ-specific autoimmune disease in adult mice. Importantly, D3Tx does not lead to autoimmune disease in all mouse strains, indicating that this process is genetically controlled. Previously, we reported linkage of D3Tx-induced autoimmune ovarian dysgenesis (AOD) and its intermediate phenotypes, antiovarian autoantibody responsiveness, oophoritis, and atrophy, to five quantitative trait loci (QTL), designated Aod1 through Aod5. We also showed interaction between these QTL and H2 as well as Gasa2, a QTL controlling susceptibility to D3Tx-induced autoimmune gastritis. To physically map Aod1, interval-specific bidirectional recombinant congenic strains of mice were generated and studied for susceptibility to D3Tx-induced AOD. Congenic mapping studies revealed that Aod1 controls susceptibility to oophoritis and comprises two linked QTL with opposing allelic effects. Aod1a resides between D16Mit211 (23.3 cM) and D16Mit51 (66.75 cM) on chromosome 16. Aod1b maps proximal of Aod1a between D16Mit89 (20.9 cM) and D16Mit211 (23.3 cM) and includes the candidate genes stefin A1, A2, and A3 (Stfa1-Stfa3), inhibitors of cathepsin S, a cysteine protease required for autoantigen presentation, and the development of autoimmune disease of the salivary and lacrimal glands following D3Tx. cDNA sequencing revealed the existence of structural polymorphisms for both Stfa1 and Stfa2. Given the roles of cathepsins in Ag processing and presentation, Stfa1 and Stfa2 alleles have the potential to control susceptibility to autoimmune disease at the level of both CD4(+)CD25(+) suppressor and CD4(+)CD25(-) effector T cells.
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Alelos , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Predisposición Genética a la Enfermedad , Ooforitis/genética , Ooforitis/inmunología , Sitios de Carácter Cuantitativo/inmunología , Timectomía , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Presentación de Antígeno/genética , Autoantígenos/inmunología , Autoantígenos/metabolismo , Catepsinas/antagonistas & inhibidores , Catepsinas/fisiología , Cistatina A , Cistatinas/genética , Cistatinas/aislamiento & purificación , Inhibidores de Cisteína Proteinasa/genética , Inhibidores de Cisteína Proteinasa/aislamiento & purificación , Femenino , Ligamiento Genético/inmunología , Masculino , Ratones , Ratones Congénicos , Ratones Endogámicos A , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mapeo Físico de CromosomaRESUMEN
Dendritic cells (DCs) are among the first cells encountered by human and simian immunodeficiency virus (HIV and SIV) following mucosal infection. Because these cells efficiently capture and transmit virus to T cells, they may play a major role in mediating HIV and SIV infection. Recently, a C-type lectin protein present on DCs, DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN), was shown to efficiently bind and present HIV and SIV to CD4(+), coreceptor-positive cells in trans. However, the significance of DC-SIGN for virus transmission and pathogenesis in vivo remains unclear. Because SIV infection of macaques may represent the best model to study the importance of DC-SIGN in HIV infection, we cloned and characterized pig-tailed macaque DC-SIGN and generated monoclonal antibodies (MAbs) against it. We demonstrate that, like human DC-SIGN, pig-tailed macaque DC-SIGN (ptDC-SIGN) is expressed on DCs and macrophages but not on monocytes, T cells, or B cells. Moderate levels of ptDC-SIGN expression were detected on the surface of DCs, and low-level expression was found on macrophages. Additionally, we show that ptDC-SIGN efficiently binds and transmits replication-competent SIVmne variants to CD4(+), coreceptor-positive cells. Moreover, transmission of virus between pig-tailed macaque DCs and CD4(+) T cells is largely ptDC-SIGN dependent. Interestingly, MAbs directed against ptDC-SIGN vary in the capacity to block transmission of different SIVmne variants. These data demonstrate that ptDC-SIGN plays a central role in transmitting virus from macaque DCs to T cells, and they suggest that SIVmne variants may differ in their interactions with ptDC-SIGN. Thus, SIVmne infection of pig-tailed macaques may provide an opportunity to investigate the significance of DC-SIGN in primate lentiviral infections.
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Moléculas de Adhesión Celular/fisiología , Células Dendríticas/inmunología , Células Dendríticas/virología , Lectinas Tipo C/fisiología , Receptores de Superficie Celular/fisiología , Virus de la Inmunodeficiencia de los Simios/inmunología , Virus de la Inmunodeficiencia de los Simios/patogenicidad , Animales , Anticuerpos Monoclonales/farmacología , Sitios de Unión , Linfocitos T CD4-Positivos/virología , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/genética , Línea Celular , VIH-1/inmunología , VIH-1/patogenicidad , Humanos , Lectinas Tipo C/antagonistas & inhibidores , Lectinas Tipo C/química , Lectinas Tipo C/genética , Macaca nemestrina , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Eliminación de Secuencia , Síndrome de Inmunodeficiencia Adquirida del Simio/etiologíaRESUMEN
Day 3 thymectomy (D3Tx) results in a loss of peripheral tolerance mediated by CD4(+)CD25(+) T cells and the development of autoimmune ovarian dysgenesis (AOD) in A/J and (C57BL/6J x A/J)F(1) (B6AF(1)) hybrids but not in C57BL/6J mice. Quantitative trait loci (QTL) linkage analysis using a B6AF(1) x C57BL/6J backcross population verified Aod1 and Aod2 that were previously mapped as qualitative traits. Additionally, three new QTL intervals, Aod3, Aod4, and Aod5, on chromosomes 1, 2, and 7, respectively, influencing specific subphenotypes of AOD were identified. QTL linkage analysis using the A x B and B x A recombinant inbred lines verified Aod3 and confirmed linkage to H2. Aod5 colocalized with Mater, an ovarian-specific autoantigen recognized by anti-ovarian autoantibodies in the sera of D3Tx mice. Sequence analysis of Mater identified allelic, strain-specific splice variants between A/J and C57BL/6J mice making it an attractive candidate gene for Aod5. Interaction analysis revealed significant epistatic effects between Aod1-5 and Gasa2, a locus associated with susceptibility to D3Tx-induced autoimmune gastritis, as well as with H2. These results indicate that the QTL controlling D3Tx-induced autoimmune phenomenon are both organ specific and more generalized in their effects with respect to the genesis and activity of the immunoregulatory mechanisms maintaining peripheral tolerance.
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Antígenos , Mapeo Cromosómico , Predisposición Genética a la Enfermedad , Disgenesia Gonadal/genética , Tolerancia Inmunológica , Enfermedades del Ovario/genética , Carácter Cuantitativo Heredable , Secuencia de Aminoácidos , Animales , Atrofia , Autoantígenos , Proteínas del Huevo/química , Femenino , Ligamiento Genético , Disgenesia Gonadal/inmunología , Masculino , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Enfermedades del Ovario/inmunología , Ovario/inmunología , Proteínas Tirosina Fosfatasas/inmunología , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores , TimectomíaRESUMEN
The replicative, cytopathic, and antigenic properties of simian immunodeficiency virus (SIV) variants influence its replication efficiency in vivo. To further define the viral properties and determinants that may be important for high-level replication in vivo and progression to AIDS, we compared a minimally pathogenic SIVmne molecular clone with two highly pathogenic variants cloned from late stages of infection. Both variants had evolved greater infectivity than the parental clone due to mutations in nef. Interestingly, a pol determinant in one of the highly pathogenic variants also contributed to its increased infectivity. Furthermore, because replication in vivo may also be influenced by the ability of a virus to evade the cellular immune response of the host, we examined whether the variants were more capable of downregulating surface expression of class I major histocompatibility complex (MHC). Decreased MHC class I expression was not observed in cells infected with any of the viruses. Furthermore, the Nef proteins of the highly pathogenic variants only slightly reduced surface MHC class I expression in transfected cells, although they efficiently downregulated CD4. Together, these data demonstrate that mutations which can enhance viral infectivity, as well as CD4 downregulation, may be important for efficient replication of SIV in the host. However, Nef-mediated reduction of MHC class I expression does not appear to be critical for the increased in vivo replicative ability of highly pathogenic late variants.