Rol antigénico del sitio de N-glicosilación 144 en la hemaglutinina del virus influenza A H1N1

Tesis (Magíster en Biotecnología)Durante la adaptación del virus Influenza A (VIA) en el humano, el cambio antigénico es un proceso clave. Este integra mutaciones primordialmente en la glicoproteína hemaglutinina (HA), la cual es la proteína más inmunogénica del virus y reconoce el receptor en las células del hospedero. Un tipo de mutaciones que utiliza el VIA para evadir los anticuerpos preexistentes es la adición de sitios de N-glicosilación, principalmente en HA. Previamente hemos demostrado que la glicosilación en la posición 144 de la HA H1N1 induce una respuesta policlonal capaz de neutralizar otras variantes de H1N1 N-glicosiladas. A su vez, este sitio de N-glicosilación enmascara de manera efectiva los sitios antigénicos en HA. Estos datos sugieren que el tipo de N-glicosilación en la posición 144 modula las propiedades antigénicas de HA redireccionando la respuesta inmune humoral. Para evaluar las características de este oligosacárido, expresamos de manera soluble la HA del virus H1N1 A/Netherlands/602/09 con N-glicosilaciones adicionales en los sitios 144, 142, 172 y 144-172. Además, produjimos las mismas variantes de HA como virus recombinantes en presencia o ausencia del inhibidor de α-manosidasa I kifunensina, lo que generó N-glicosilaciones del tipo simple o complejas, respectivamente. Mediante Western Blot, observamos que la HA 144 tenía un peso molecular mayor que HA 142 y HA 172, sugiriendo que esta posición permite el ensamble de oligosacáridos más largos en comparación con otros sitios cercanos. Además, observamos que el oligosacárido en la posición 144 afecta negativamente la avidez del VIA por su receptor, pero al reducir la longitud de este (tipo simple) su avidez aumenta significativamente. También se evaluó la relevancia de la posición y tipo de N-glicosilaciones en la cabeza de HA con respecto a su modulación de la respuesta humoral. Se inmunizaron ratones y se determinó que las N-glicosilaciones de tipo complejo producen más anticuerpos contra HA que las de tipo simple. El conjunto de sitios de N-glicosilación 144-172 en HA evaden la respuesta humoral de manera más eficaz que solo el sitio 144 y a su vez inducen anticuerpos que inhiben eficientemente a las otras variantes. Estos resultados proporcionan nuevos conocimientos sobre la relevancia de la N-glicosilación en el sitio 144 HA del VIA y su potencial uso para el desarrollo de vacunas más eficientes.During the adaptation of the Influenza A virus (IAV) in humans, antigenic drift is a key process. This integrates mutations primarily in the hemagglutinin (HA) glycoprotein which is the most immunogenic protein of the virus and recognizes the receptor in the host cells. A type of mutations that IAV uses to evade pre-existing antibodies is the addition of N-glycosylation sites, mainly in HA. We have previously shown that glycosylation at position 144 of HA H1N1 induces a polyclonal response capable of neutralizing other N-glycosylated H1N1 variants. In turn, this N-glycosylation site effectively masks the antigenic sites in HA. These data suggest that the type of N-glycosylation at position 144 modulates the antigenic properties of HA redirecting the humoral immune response. To evaluate the characteristics of this oligosaccharide, we express soluble HAs of the H1N1 virus A/Netherlands/602/09 with additional N-glycosylations sites at positions 144, 142, 172 and 144-172. In addition, we produced the same HA variants as recombinant viruses in the presence or absence of the α-mannosidase I inhibitor kifunensine, which generated N-glycosylations of the simple or complex type, respectively. Through Western Blot, we observed that HA 144 had a molecular weight higher than HA 142 and HA 172, suggesting that this position allows the assembly of longer oligosaccharides in comparison with other nearby sites. In addition, we observed that the oligosaccharide in position 144 negatively affects the avidity of IAV for its receptor, but by reducing the length of this (simple type) its avidity increases significantly. The relevance of the position and type of N-glycosylations in the HA head was also evaluated with respect to its modulation of the humoral response. Mice were immunized, and it was determined that the complex-type N-glycosylations produce more antibodies against HA than the simple type. The set of N-glycosylation sites 144-172 in HA evade the humoral response more effectively than only site 144 and in turn induce antibodies that efficiently inhibit the other variants. These results provide new insights into the relevance of N-glycosylation at the 144 HA site of IAV and its potential use for the development of more efficient vaccines

‘Neighbourhood effects’ on children’s educational achievement in Chile: the effects of inequality and polarization

This article studies the effects of the neighborhood in which a school is located on children’s mathematics achievement in Chile. It uses data taken from a sample of 127,020 sixth grade students measured by the National Education Quality Measurement System [Sistema Nacional de Medicio´n de la Calidad de la Educacio´ n]. The incorporation of a measurement of socioeconomic polarization of the geographic environment, which is innovative in urban studies, allows us to qualify some critical aspects suggested in the academic discussion. A lagged dependent variable model is used, controlling for the score obtained by the same students in fourth grade. Using multilevel linear regressions, the results show positive effects related to participation in neighborhood organizations. One critical finding is that socio-economic polarization has a negative and significant impact on the educational achievement of sixth graders. The conclusions highlight the repercussions associated with acute inequalities in the neighborhoods, and speak to the importance of accessing dimensions which are more closely linked to cities’ social structure.Centre for Social Conflict and Cohesion Studies (CONICYT/FONDAP/15130009

A Canadian Survey of Research on HIV-1 Latency—Where Are We Now and Where Are We Heading?

Worldwide, almost 40 million people are currently living with HIV-1. The implementation of cART inhibits HIV-1 replication and reduces viremia but fails to eliminate HIV-1 from latently infected cells. These cells are considered viral reservoirs from which HIV-1 rebounds if cART is interrupted. Several efforts have been made to identify these cells and their niches. There has been little success in diminishing the pool of latently infected cells, underscoring the urgency to continue efforts to fully understand how HIV-1 establishes and maintains a latent state. Reactivating HIV-1 expression in these cells using latency-reversing agents (LRAs) has been successful, but only in vitro. This review aims to provide a broad view of HIV-1 latency, highlighting Canadian contributions toward these aims. We will summarize the research efforts conducted in Canadian labs to understand the establishment of latently infected cells and how this informs curative strategies, by reviewing how HIV latency is established, which cells are latently infected, what methodologies have been developed to characterize them, how new compounds are discovered and evaluated as potential LRAs, and what clinical trials aim to reverse latency in people living with HIV (PLWH)