Desarrollo de un prototipo de vacuna contra el virus Andes basado en la tecnología de ARN mensajero.
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Date
2026
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Universidad de Concepción
Abstract
El virus Andes es el principal agente etiológico del Síndrome Cardiopulmonar por Hantavirus en Chile, una enfermedad infecciosa grave que puede progresar hacia falla cardiorrespiratoria y presentar alta letalidad. Además, este virus mantiene relevancia epidemiológica por su condición endémica en Sudamérica y por la evidencia de transmisión persona a persona. A pesar de ello, no existen vacunas aprobadas para uso humano ni tratamientos antivirales específicos frente a este virus. Esta brecha sanitaria refuerza la necesidad de desarrollar nuevas estrategias vacunales dirigidas a blancos antigénicos relevantes, como las glicoproteínas Gn/Gc, que participan en la entrada viral y representan blancos importantes para la inducción de anticuerpos neutralizantes. Este trabajo tuvo como objetivo desarrollar y evaluar un prototipo vacunal basado en ARN mensajero encapsulado en nanopartículas lipídicas, codificante de Gn/Gc del virus Andes. El ARNm se produjo mediante transcripción in vitro, se incorporó una caperuza 5’ tipo Cap-1 y se formuló en nanopartículas lipídicas empleando el método de nanoprecipitación. El proceso de síntesis del transcrito presentó rendimientos consistentes, recuperación eficiente después del proceso de adición de caperuza, e integridad compatible con su uso en la formulación. La nanoformulación obtenida mostró una alta eficiencia de encapsulación, tamaño nanométrico homogéneo, baja dispersión y potencial zeta ligeramente negativo. Además, el prototipo mantuvo viabilidad celular por encima del umbral predefinido y no mostró reducción relevante a la concentración de trabajo en las líneas HeLa y HFF, y mostró actividad biológica in vitro, evidenciada por la expresión detectable de la glicoproteína Gc en células HEK-293A y por una expresión relativa superior al comparador interno bajo las condiciones del ensayo. La prueba de concepto en hámster sirio integró el monitoreo de peso corporal y temperatura como indicadores fisiológicos, junto con la evaluación de inmunogenicidad humoral y celular. El seguimiento aportó una lectura inicial de la tolerancia de las formulaciones durante el ensayo, mientras que la evaluación inmunológica mostró que las dosis de 10 μg y 20 μg indujeron anticuerpos totales, actividad neutralizante y una respuesta celular detectable compatible con orientación Th1 frente a Gn/Gc. Sin embargo, la respuesta humoral disminuyó hacia el final del seguimiento, lo que indicó la necesidad de optimizar variables asociadas con la magnitud y persistencia de la respuesta. Además, la dosis de 20 μg no mostró una ventaja inmunológica clara sobre 10 μg, así que esta última se definió como la alternativa más adecuada para la formulación inicial. Estos resultados demostraron la factibilidad técnica e inmunológica del desarrollo de un prototipo vacunal de ARNm frente al virus Andes, desde la producción del ácido nucleico hasta su formulación, caracterización y evaluación funcional. El presente trabajo aporta una base experimental para optimizar el diseño molecular, la formulación y el esquema de inmunización de candidatos vacunales de ARNm frente al virus Andes, y establece un punto de partida para futuros estudios orientados a evaluar su capacidad protectora.
Andes virus is the main etiological agent of Hantavirus Cardiopulmonary Syndrome in Chile, a severe infectious disease that can progress to cardiorespiratory failure and is associated with high lethality. In addition, this virus remains epidemiologically relevant due to its endemicity in South America and the evidence of person-to-person transmission. Despite this, there are no approved vaccines for human use or specific antiviral treatments against this virus. This public health gap reinforces the need to develop new vaccine strategies targeting relevant antigenic targets, such as the Gn/Gc glycoproteins, which participate in viral entry and represent important targets for the induction of neutralizing antibodies. This study aimed to develop and evaluate a vaccine prototype based on messenger RNA encapsulated in lipid nanoparticles, encoding Andes virus Gn/Gc. The mRNA was produced by in vitro transcription, incorporated a 5’ Cap-1 structure, and was formulated into lipid nanoparticles using the nanoprecipitation method. The transcript synthesis process showed consistent yields, efficient recovery after the capping process, and integrity compatible with its use in the formulation. The resulting nanoformulation showed high encapsulation efficiency, homogeneous nanoscale size, low dispersion, and a slightly negative zeta potential. In addition, the prototype maintained cell viability above the predefined threshold and showed no significant reduction at the test concentration in the HeLa and HFF cell lines, and exhibited in vitro biological activity, evidenced by detectable expression of the Gc glycoprotein in HEK-293A cells and by an expression greater than the internal comparator under the test conditions. The proof-of-concept study in Syrian hamsters integrated body weight and temperature monitoring as physiological indicators, together with the evaluation of humoral and cellular immunogenicity. This follow-up provided an initial assessment of the tolerability of the formulations during the assay, while the immunological evaluation showed that the 10 μg and 20 μg doses induced total antibodies, neutralizing activity, and a detectable cellular response compatible with Th1 orientation against Gn/Gc. However, the humoral response decreased toward the end of the follow-up, indicating the need to optimize variables associated with the magnitude and persistence of the response. In addition, the 20 μg dose did not show a clear immunological advantage over 10 μg; therefore, the latter was defined as the most suitable alternative for the initial formulation. These results demonstrated the technical and immunological feasibility of developing an mRNA vaccine prototype against Andes virus, from nucleic acid production to its formulation, characterization, and functional evaluation. This work provides an experimental basis for optimizing the molecular design, formulation, and immunization schedule of mRNA vaccine candidates against Andes virus and establishes a starting point for future studies aimed at evaluating its protective capacity.
Andes virus is the main etiological agent of Hantavirus Cardiopulmonary Syndrome in Chile, a severe infectious disease that can progress to cardiorespiratory failure and is associated with high lethality. In addition, this virus remains epidemiologically relevant due to its endemicity in South America and the evidence of person-to-person transmission. Despite this, there are no approved vaccines for human use or specific antiviral treatments against this virus. This public health gap reinforces the need to develop new vaccine strategies targeting relevant antigenic targets, such as the Gn/Gc glycoproteins, which participate in viral entry and represent important targets for the induction of neutralizing antibodies. This study aimed to develop and evaluate a vaccine prototype based on messenger RNA encapsulated in lipid nanoparticles, encoding Andes virus Gn/Gc. The mRNA was produced by in vitro transcription, incorporated a 5’ Cap-1 structure, and was formulated into lipid nanoparticles using the nanoprecipitation method. The transcript synthesis process showed consistent yields, efficient recovery after the capping process, and integrity compatible with its use in the formulation. The resulting nanoformulation showed high encapsulation efficiency, homogeneous nanoscale size, low dispersion, and a slightly negative zeta potential. In addition, the prototype maintained cell viability above the predefined threshold and showed no significant reduction at the test concentration in the HeLa and HFF cell lines, and exhibited in vitro biological activity, evidenced by detectable expression of the Gc glycoprotein in HEK-293A cells and by an expression greater than the internal comparator under the test conditions. The proof-of-concept study in Syrian hamsters integrated body weight and temperature monitoring as physiological indicators, together with the evaluation of humoral and cellular immunogenicity. This follow-up provided an initial assessment of the tolerability of the formulations during the assay, while the immunological evaluation showed that the 10 μg and 20 μg doses induced total antibodies, neutralizing activity, and a detectable cellular response compatible with Th1 orientation against Gn/Gc. However, the humoral response decreased toward the end of the follow-up, indicating the need to optimize variables associated with the magnitude and persistence of the response. In addition, the 20 μg dose did not show a clear immunological advantage over 10 μg; therefore, the latter was defined as the most suitable alternative for the initial formulation. These results demonstrated the technical and immunological feasibility of developing an mRNA vaccine prototype against Andes virus, from nucleic acid production to its formulation, characterization, and functional evaluation. This work provides an experimental basis for optimizing the molecular design, formulation, and immunization schedule of mRNA vaccine candidates against Andes virus and establishes a starting point for future studies aimed at evaluating its protective capacity.
Description
Tesis presentada para optar al grado de Doctor/a en Biotecnología Molecular.
Keywords
Hantavirus, ARN mensajero, Vacunas, Síndrome pulmonar por hantavirus