Desarrollo de anticuerpos contra virus Andes (Hantaviridae) a partir de partículas semejantes a virus (VLPs) y caracterización de su actividad neutralizante y mecanismo de acción
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Fecha
2020
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es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
Los orthohantavirus son patógenos transmitidos por roedores y se encuentran
distribuidos en gran parte del mundo. En Chile, el virus Andes (ANDV) puede producir
síndrome cardiopulmonar por hantavirus (SCPH) con tasas de mortalidad de 30 a 40%.
Sin embargo, a la fecha no existen antivirales efectivos contra el SCPH ensayados en
estudios clínicos. ANDV infecta principalmente células endoteliales de pulmón, proceso
mediado por las glicoproteínas de envoltura, Gn y Ge, encargadas de la unión a
receptores y la posterior fusión de membranas viral y celular. Moléculas que se unan a
estas glicoproteínas podrían inhibir la entrada del virus a células.
Se ha establecido en nuestro laboratorio que mediante la transfección del gen precursor
de las glicoproteínas (GPC) en células de mamífero es posible obtener VLPs (vírus-líke
partícles), las cuales son semejantes en su conformación al virus nativo.
El objetivo de la presente tesis es desarrollar anticuerpos contra ANDV, usando VLPs
como inmunógeno y caracterizar sus propiedades de unión a Gn/Gc y el mecanismo de
inhibición de la infección viral.
El método de obtención de VLPs fue optimizado y escalado para posibilitar su uso como
inmunógeno. Además se usaron VLPs con espículas de Gn/Gc estabilizadas, diseñados
en colaboración con el Dr. Félix Rey y el Dr. Pablo Guardado-Calvo, en las cuales se
introdujeron sustituciones de residuos por cisteína para permitir la formación de puentes
disulfuro. Las VLPs nativas y estabilizadas se usaron para inmunizar ratones BALB/c para
la obtención de anticuerpos monoclonales murinos (mAbs) y para inmunizar alpacas para
la obtención de sueros policlonales. Se evaluó la reactividad de los los anticuerpos a
glicoproteínas de ANDV mediante ELISA, western blot y citometría de flujo y se
determinó la neutralización contra el virus en ensayos de infección in vítro. El mecanismo
de neutralización de los mAbs se determinó mediante ensayos de fusión con virus
infeccioso y con células transfectadas con GPC de ANDV.
Los mAbs reaccionaron específicamente contra la glicoproteína Ge de ANDV y
presentaron reactividad cruzada contra los ortohantavirus Sin Nombre (SNV), Puumala
(PUUV) y Hantaan (HNTV). Los mAbs presentaron actividad neutralizante parcial en
ensayos de neutralización con virus infeccioso. En el caso del clon 7 A6/C7 arreglos con
péptidos permitieron identificar su epitopo que está ubicado en el dominio I y que se
encuentra oculto dentro de las espículas en su conformación previa a la fusión, dado que este anticuerpo reacciona con VLPs solo en su estructura abierta (50-60º C) o en la
conformación post-fusión de Ge. Ensayos de fusión in vitro demostraron que este
anticuerpo inhibe específicamente la fusión de membranas de manera parcial; similar a
lo observado con péptidos inhibitorios de Ge previamente descritos.
Por otro lado, sueros policlonales de alpaca obtenidos con VLPs de ANDV se prepararon
en colaboración con el Dr. Alejandro Rojas y Ronald Jara, los cuales neutralizaron por
completo la infección de virus ANDV en ensayos de infección in vitro hasta diluciones 10-
5, demostrando que las alpacas son una plataforma biológica atractiva para el desarrollo
de moléculas que inhiban la infección del virus. Se logró aislar clones de nanobodies que
serán a futuro caracterizados por su actividad neutralizante.
Finalmente, al utilizar las VLPs de ANDV estabilizadas mediante mutaciones sitio
dirigidas para la inmunización de ratones BALB/c se logró aumentar el título de
anticuerpos neutralizantes de los sueros murinos comparado con la inmunización con
VLPs nativos. Estos VLPs estabilizados corresponden, por ende, a inmunógenos
mejorados.
En conclusión, se demostró que las VLPs de ANDV permiten la generación de anticuerpos
neutralizantes, tanto en ratones BALB/c como en alpacas. Además, mediante la caracterización de los anticuerpos monoclonales obtenidos se identificó la existencia de
epitopos neutralizantes ocultos en las espiculas de prefusion y epitopos conservados
entre diferentes ortohantavirus lo cual sugiere que es posible el desarrollo de anticuerpos
con reactividad cruzada. Finalmente, el uso de VLPs estabilizadas demostraron ser un
inmunógeno mejorado para la obtención de anticuerpos neutralizantes o para el diseño
de vacunas de próxima generación.
Orthohantaviruses are pathogens transmitted by rodents and are distributed throughout the world. In Chile, the Andes virus (ANDV) can produce hantavirus pulmonary syndrome (HPS) with mortality rates ranging from 30 to 40%. However, to date there are no effective antivirals against HPS tested in clinical trials. ANDV mainly infects lung endothelial cells, a process mediated by envelope glycoproteins, Gn and Ge, responsible for receptor binding and subsequent fusion of viral and cellular membranes. Molecules that bind to these glycoproteins could inhibit the entry of the virus into cells. It has been established in our laboratory that by transfecting the glycoprotein precursor gene (GPC) in mammalian cells it is possible to obtain VLPs, which are similar in their conformation to the native virus. The objective of the present thesis is to develop antibodies against ANDV, using VLPs as immunogens and to characterize their Gn / Ge binding properties and the mechanism of inhibition of viral infection. The method for VLPs production was optimized and scaled to enable their use as an immunogen. In addition, VLPs with stabilized Gn / Ge spicules, designed in collaboration with Dr. Félix Rey and Dr. Pablo Guardado-Calvo, were used, in which residue substitutions by cysteine were introduced to allow the formation of disulfide bridges. The native and stabilized VLPs were used to immunize BALB/c mice to obtain murine monoclonal antibodies (mAbs) and to immunize alpacas to obtain polyclonal serum. The reactivity of the mAbs to ANDV glycoproteins was evaluated by ELISA, western blot and flow cytometry and neutralization against the virus was determined in in vitro infection assays. The neutralization mechanism was determined by fusion assays with infectious virus and with ANDV GPC transfected cells. The mAbs reacted specifically against the Ge glycoprotein of ANDV and showed cross reactivity against the orthohantavirus Sin Nombre (SNV), Puumala (PUUV) and Hantaan (HNTV). mAbs showed partial neutralizing activity in neutralization assays with infectious virus. In the case of clone 7A6/C7, peptide arrangements allowed us to identify that part of its epitope is located in domain I and that it is hidden within the spicules in its pre fusion conformation, coinciding with the result that this antibody reacts with VLPs only in their open structure (50-60 ºC) or in the post-fusion conformation of Ge. In vitro fusion assays demonstrated that this antibody specifically inhibits membrane fusion partially; similar to that observed with Ge inhibitory peptide previously described. On the other hand, polyclonal alpaca sera obtained with ANDV VLPs were prepared in collaboration with Dr. Alejandro Rojas and Ronald Jara, which completely neutralized ANDV virus infection using in vitro infection assays up to 10-s dilutions, demonstrating that alpacas are an attractive biological platform for the development of molecules that inhibit the virus infection. Furthermore, It was possible to isolate clones of nanobodies that will be characterized in the future by their neutralizing activity. Finally, by using ANDV VLPs stabilized by site-directed mutations for the immunization of BALB/c mice, it was possible to increase the titer of neutralizing antibodies in murine sera compared to immunization with native VLPs. These stabilized VLPs therefore correspond to improved immunogens. In conclusion, it was demonstrated that ANDV VLPs allow the generation of neutralizing antibodies, both in BALB/c mice and alpacas. Furthermore, by characterizing the monoclonal antibodies obtained, the existence of hidden neutralizing epitopes in the prefusion spicules and conserved epitopes between different orthohantaviruses was identified, suggesting that the development of antibodies with cross-reactivity is possible. Finally, the use of stabilized VLPs proved to be an improved immunogen for obtaining neutralizing antibodies or for the design of next-generation vaccines.
Orthohantaviruses are pathogens transmitted by rodents and are distributed throughout the world. In Chile, the Andes virus (ANDV) can produce hantavirus pulmonary syndrome (HPS) with mortality rates ranging from 30 to 40%. However, to date there are no effective antivirals against HPS tested in clinical trials. ANDV mainly infects lung endothelial cells, a process mediated by envelope glycoproteins, Gn and Ge, responsible for receptor binding and subsequent fusion of viral and cellular membranes. Molecules that bind to these glycoproteins could inhibit the entry of the virus into cells. It has been established in our laboratory that by transfecting the glycoprotein precursor gene (GPC) in mammalian cells it is possible to obtain VLPs, which are similar in their conformation to the native virus. The objective of the present thesis is to develop antibodies against ANDV, using VLPs as immunogens and to characterize their Gn / Ge binding properties and the mechanism of inhibition of viral infection. The method for VLPs production was optimized and scaled to enable their use as an immunogen. In addition, VLPs with stabilized Gn / Ge spicules, designed in collaboration with Dr. Félix Rey and Dr. Pablo Guardado-Calvo, were used, in which residue substitutions by cysteine were introduced to allow the formation of disulfide bridges. The native and stabilized VLPs were used to immunize BALB/c mice to obtain murine monoclonal antibodies (mAbs) and to immunize alpacas to obtain polyclonal serum. The reactivity of the mAbs to ANDV glycoproteins was evaluated by ELISA, western blot and flow cytometry and neutralization against the virus was determined in in vitro infection assays. The neutralization mechanism was determined by fusion assays with infectious virus and with ANDV GPC transfected cells. The mAbs reacted specifically against the Ge glycoprotein of ANDV and showed cross reactivity against the orthohantavirus Sin Nombre (SNV), Puumala (PUUV) and Hantaan (HNTV). mAbs showed partial neutralizing activity in neutralization assays with infectious virus. In the case of clone 7A6/C7, peptide arrangements allowed us to identify that part of its epitope is located in domain I and that it is hidden within the spicules in its pre fusion conformation, coinciding with the result that this antibody reacts with VLPs only in their open structure (50-60 ºC) or in the post-fusion conformation of Ge. In vitro fusion assays demonstrated that this antibody specifically inhibits membrane fusion partially; similar to that observed with Ge inhibitory peptide previously described. On the other hand, polyclonal alpaca sera obtained with ANDV VLPs were prepared in collaboration with Dr. Alejandro Rojas and Ronald Jara, which completely neutralized ANDV virus infection using in vitro infection assays up to 10-s dilutions, demonstrating that alpacas are an attractive biological platform for the development of molecules that inhibit the virus infection. Furthermore, It was possible to isolate clones of nanobodies that will be characterized in the future by their neutralizing activity. Finally, by using ANDV VLPs stabilized by site-directed mutations for the immunization of BALB/c mice, it was possible to increase the titer of neutralizing antibodies in murine sera compared to immunization with native VLPs. These stabilized VLPs therefore correspond to improved immunogens. In conclusion, it was demonstrated that ANDV VLPs allow the generation of neutralizing antibodies, both in BALB/c mice and alpacas. Furthermore, by characterizing the monoclonal antibodies obtained, the existence of hidden neutralizing epitopes in the prefusion spicules and conserved epitopes between different orthohantaviruses was identified, suggesting that the development of antibodies with cross-reactivity is possible. Finally, the use of stabilized VLPs proved to be an improved immunogen for obtaining neutralizing antibodies or for the design of next-generation vaccines.
Notas
Tesis (Doctor en Biotecnología)
La presente tesis fue realizada gracias a las siguientes fuentes de financiamiento: Beca de Doctorado Nacional Conicyt NM Proyecto FONDECYT 1181799 Proyecto Basal AFB 170004 Proyecto FONDEF CA12l 10367
La presente tesis fue realizada gracias a las siguientes fuentes de financiamiento: Beca de Doctorado Nacional Conicyt NM Proyecto FONDECYT 1181799 Proyecto Basal AFB 170004 Proyecto FONDEF CA12l 10367
Palabras clave
Anticuerpos, Virus Andes, Análisis