Polyanionic dendritic nanoparticles against human immunodeficiency virus Type 1 and herpes simplex virus Type 2: searching for microbicides and new therapeutic approaches

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Departamento de Biología Facultad de Biología

Universidad Autónoma de Madrid

TESIS DOCTORAL

POLYANIONIC DENDRITIC NANOPARTICLES AGAINST HUMAN IMMUNODEFICIENCY VIRUS

TYPE 1 AND HERPES SIMPLEX VIRUS TYPE 2:

SEARCHING FOR MICROBICIDES AND NEW THERAPEUTIC APPROACHES

Memoria presentada por el graduado en Bioquímica Carlos Guerrero Beltrán

Directores de Tesis

Dra. Mª Ángeles Muñoz Fernández Dr. Jose Luis Jiménez Fuentes

Lugar de realización

Laboratorio de Inmuno-Biología Molecular Plataforma de Laboratorio. IiSGM

Hospital General Universitario Gregorio Marañón

Madrid, 2018

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This Thesis has been conducted at Laboratorio de Inmuno-Biología Molecular, Sección de Inmunología and Spanish HIV HGM BioBank and Plataforma de Laboratorio at Hospital General Universitario Gregorio Marañón de Madrid under the direction of Dra.

Mª Ángeles Muñoz-Fernández and Dr. Jose Luis Jiménez Fuentes. This Thesis was supported by the RD12/0017/0037 and RD16/0025/0019 projects as part of the Acción Estratégica en Salud, Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica (2008-2011 and 2013-2016, respectively) and cofinanced by Instituto de Salud Carlos III (Subdirección General de Evaluación) and Fondo Europeo de Desarrollo Regional (FEDER), Instituto de Salud Carlos III (grant numbers PI13/02016, PI14/00882, PI16/01863 and PI17/01115); RETIC (PT13/0010/0028;

RETIC PT17/0015/0042); Comunidad de Madrid (B2017/BMD-3703), European EPIICAL Project and the “Programa de Investigación de la Consejería de Sanidad de la Comunidad de Madrid”. CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, the Consolider Program, and CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.

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La Dra. Mª Ángeles Muñoz Fernández, Jefa de Sección del Servicio de Inmunología, Jefa del Laboratorio de Inmuno-Biología Molecular y Directora del Biobanco VIH del Hospital General Universitario Gregorio Marañón y el Dr. Jose Luis Jiménez Fuentes, Investigador Senior de la Plataforma de Laboratorio del Hospital General Universitario Gregorio Marañón (IiSGM)

CERTIFICAN QUE,

El trabajo de investigación y la redacción de la Tesis Doctoral titulada: “Polyanionic Dendritic Nanoparticles Against Human Immunodeficiency Virus Type 1 and Herpes Simplex Virus Type 2: Searching for microbicides and new therapeutic approaches” han sido realizadas por Carlos Guerrero Beltrán bajo nuestra dirección. Revisado el trabajo, consideramos éste como satisfactorio y autorizamos su presentación y defensa para optar al grado de Doctor por la Universidad Autónoma de Madrid.

Y para que quede constancia de ello, firmamos el presente documento.

Madrid, 30 de Septiembre de 2018

Fdo. Dra. Mª Ángeles Muñoz Fernández Fdo. Dr. Jose Luis Jiménez Fuentes

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ACKNOWLEDGEMENTS

Hace tres años comenzó una aventura cuyo final veía muy difícil y lejano. No ha sido un camino ni mucho menos fácil, lleno de incertidumbres pero también repleto de aprendizaje que me ha hecho crecer como persona. Por supuesto no lo habría logrado sin todas las personas que de una forma u otra han puesto su granito de arena para que haya podido llegar hasta aquí.

En primer lugar, me gustaría dar las gracias a las personas que me dieron la oportunidad de estar en el laboratorio, y han hecho este proyecto posible. Muchas gracias a mi directora, María Ángeles por la oportunidad que me diste de empezar el camino, por tu constancia, confianza en todo momento y sobre todo, tu fuerza que me ha empujado hasta el final. He aprendido muchísimas cosas de ti, pero quiero resaltar tu capacidad para llevarnos a todos hacia delante con tu esfuerzo diario, eres el motor que hace que todo vaya siempre en la buena dirección. Muchas gracias a mi co-director, Jose Luis, por estar siempre ahí para lo que haga falta. Tus consejos durante estos años y tu capacidad de solucionar cualquier problema han sido un apoyo inmejorable. Sé que vas a echar de menos mi acento granaíno, mi tupé y mis alpargatas veraniegas que tanto te gustan jaja. Muchas gracias a los dos por vuestra labor de directores de mi tesis, por guiarme en este trabajo, y aconsejarme durante todo el camino.

Vamos con la gente del laboratorio, muchas gracias Nacho (pequeño), porque desde el primer momento has sido mi apoyo número uno dentro y fuera del hospital. Somos “Pili y Mili” y la complicidad que tenemos se refleja hasta en la cabina, con esos temazos que solíamos componer y que envidiaría el mismísimo Sabina. No sé qué habría hecho sin ti. Todo el mundo sabe que la β-galactosidasa la hice yo, y así quedará para los anales de la historia, tontito jaja. Siento decirte que no tienes ni idea de gramática (te LU digo yo) ni de Clash Royale, así que espero que en la ciencia te vaya un poquito mejor, porque si no, estás jodido moreno.

Muchas gracias Rafa por haberme enseñado mil cosas en el laboratorio. Vas a llegar a donde te propongas, no pierdas nunca esa confianza que tienes en ti mismo y que te caracteriza. Se me hacían cortas esas horas en el gimnasio, aunque los dos sabemos que hubiesen sido necesarias muchas más. Nunca dejes de escalar y persigue aquello que más quieres. Muchas gracias a ti Alba, simplemente por ser como eres, tu energía y positivismo nos contagian día a día. Gracias por estar ahí en los días en los que todo

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parece negro y hace falta alguien con quien poder desahogarse. A pesar de todas las dificultades, espero que siempre mires hacía delante y llegues a donde te propongas.

Nunca dejes de bailar y sobre todo nunca cambies tú. Sabes que se quedan pendientes (como siempre) las clases de bachata jaja. Sin ti, estos agradecimientos no se habrían escrito. Una de las personas que en muy poco tiempo se ha convertido en un apoyo enorme eres tú, Laura (Albacete). Sabes que me encanta picarte, pero también sabes que eres la única que me sigue el ritmo. Muchas gracias por esas cervezas después del trabajo que hacían todo mucho más llevadero. Eso sí, tengo que decirte que la feria de Albacete tampoco es para tanto, sólo merece la pena las navajas y los quesos. Bueno, y tú también. Gracias por tus consejos y apoyo en todo el tiempo que has estado aquí.

Muchas gracias Susana por tu apoyo, conocimiento y visión crítica, sobre todo en este tramo final de la tesis. Tú ya sabes porqué... Chusa, jamás se me olvidará eso de “sabéis que por las buenas ruedo, pero por las malas…”, y es cierto. Ha habido momentos que me has dado caña y mucha, pero en el fondo sé que siempre lo has hecho porque era lo más justo. Muchas gracias por todo lo que me has enseñado y por tus consejos en los momentos más complicados. Santi, ¡qué grande eres! Alegras los días simplemente con tus visitas y tu forma de ser. Eres el “relaciones públicas” del grupo, como pude comprobar en el congreso de GESIDA. El ambiente del labo no sería lo mismo sin ti, sin tu quiniela y las cañas, pero no muchas, que te pones cariñoso.

Muchas gracias a Nacho (Grande) y Raquel, por hacer más llevadero el día a día, sobre todo de estos últimos meses, dadle mucha caña a esos herpes de mi parte jaja. Lola, que sepas que no olvidaré tus consejos femeninos jaja. Muchas gracias Irene, no sólo por salvarme la vida con los papeleos, sino también por todos esos momentos (buenos y malos) fuera del labo, acompañados por zumos de cebada. No me puedo olvidar de la última incorporación al labo, JR, en este poco tiempo ha sido suficiente para ver lo gran persona y científico que eres. Muchas gracias por tu ayuda desinteresada.

Muchas gracias a toda la gente del Biobanco. Coral, fuiste la primera que me vio meter las manos en una cabina y siempre te estaré agradecido por tu cariño y paciencia. Isa, fuiste el mejor descubrimiento del congreso GESIDA de Vigo, me alegro de haber conocido la gran persona que eres. Paula, muchas gracias por tu apoyo y consejo todos estos años. Jorge, no se me olvidarán esos ratos de cañas y tus conocimientos sobre artes marciales y otras artes oscuras.

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Muchas gracias al resto de gente del hospital, Rafa Correa, Esther, Mabela, Luis y Miguel porque cada uno en su medida, siempre habéis aportado algo positivo; y en especial a Marjorie, porque siempre que he tenido dudas me las has respondido con la mejor de las sonrisas. Eres un modelo a seguir como científica y como persona.

No me puedo olvidar de toda la gente que ha pasado por el laboratorio y que han dejado su huella a su manera. Laura, Raúl, Pili, Jacobo (mi bro), Verónica, David, Irene, Dina y Mica. Silvia, gracias por tu buenrollismo en el labo, sabes que siempre seré tu yogurín favorito y tu mi princes.

No podía olvidarme de mis amigos de la universidad que son para toda la vida: Puerma, Pablo, Carmen, Paula, Juanpe, Sandra, Rafa, Maite, etc. Hemos compartido mil momentos desde que nos conocimos, que recuerdos más bonitos. Sólo vosotros podéis decir que me habéis visto beber aquarius en el botellón jaja. ¡Cómo hemos cambiado desde entonces! Me habéis visto desde mis inicios en la bioquímica hasta llegar aquí, y espero que sigamos compartiendo millones de momentos más, nos queda mucho por recorrer y espero que sea juntos. Mención especial a Puerma y a sus dotes en el diseño gráfico, ya tú sabes jaja.

A mi grupo de andaluces en Madrid, Luis, Oti, Violeta, Raquel, Emi, mi tocayo, etc.

aparecisteis en mi vida justo en el momento en que más lo necesitaba, y aunque no os lo creáis, me hicisteis sentir un poquito más cerca de mi tierra. Sois de lo mejorcito que me llevo de Madrid, sabéis que en Granada tenéis vuestra casa.

Muchas gracias a mis amigos de toda la vida, Largo, Manu, Utrilla, Dani y Jaime, porque veros los findes en Granada ha sido la mejor medicina para desconectar. Y en especial a mi Javi, el de toda la vida, gente como tú queda muy poca.

Puedo decir que sólo existe una persona a caballo entre mis mejores amigos y familia, y esa es mi prima Laura. Mil gracias por ser mi confidente. Has sido mi válvula de desahogo. Gracias a toda mi familia, abuelos, tíos y primos. En especial a mi tito Paco, por tener siempre palabras de apoyo, ánimo y darme la confianza para que aflorasen mis sentimientos. A mi tata, por tener esa magia, por hacerme soñar y tener esa confianza en mí. A mi prima María, porque en nuestros peores momentos supimos apoyarnos mutuamente. A mi primo Santi, gracias por estar ahí y simplemente ser como eres. A mi primo Jesús, solo los dos sabemos lo duro que estar lejos de la familia. A mi tito Jesús y a mi tita Paqui, eres mi segunda madre, gracias por darme siempre un punto de vista

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distinto. Todo lo que me has aportado estos años de tesis no se puede describir, me has dado el equilibrio necesario en los momentos en los que me hundía. Has estado a mi lado desde que era un bebé (sí, me cambiabas los pañales jaja) y quiero que eso no cambie nunca. Sabes que te quiero un montón.

No puedo olvidarme de aquellas personas que ya no están, pero que siempre nos cuidan y seguro que están orgullosas de mí estén donde estén. Tita Dori, gracias por todo tu apoyo, cariño y esa fuerza que nos aportabas, incluso cuando tú no la tenías. A mi abuelo Paco, no ha habido un día que no me acuerde de ti, y a pesar de todo el tiempo que ha pasado te siento muy cerca. Ojalá estuvieras aquí para ver lo que he conseguido.

Finalmente, quiero dar las gracias a los tres pilares fundamentales de mi vida por aguantarme día a día: mi hermano, y los dos mejores padres del mundo. Pablo, mi enana, mi hermano, que haría yo sin ti. Muchas gracias por ser como eres, a pesar de que eres un malafollá, también eres totalmente auténtico y vacilón. Me has enseñado muchas cosas, así que más que un hermano mayor, me siento un amigo con el que sabes que podrás contar siempre. Te quiero un montón, aunque tú nunca me lo digas, eh crack. Papá, muchas gracias por apoyarme en todo lo que hago, incluida esta etapa en Madrid. Por esas charlas telefónicas casi diarias y por esas ganas constantes de que vuelva a casa, que me hacen sentir tan especial. Me has enseñado el significado de las palabras esfuerzo y sacrificio cada día. Solo tú eres capaz de construir cosas grandes de la nada. Te quiero. Mamá, todo lo que diga de ti se queda corto. Es imposible reflejar el inmenso cariño, y admiración que me demuestras cada día. Me has dado la fuerza necesaria para continuar en el camino. Me has apoyado incluso en aquellas decisiones que tú sabías que no eran las mejores y nadie más lo hacía. Me has arropado en la distancia y lo has dado todo por mí. Algo que jamás podre agradecerte. Te quiero.

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SUMMARY

In spite of the significant efforts to develop effective vaccines against human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 2 (HSV-2), they still remain elusive and microbicides have emerged as a feasible preventive strategy against both viruses. Up to date, several topically applied compounds have failed and the search of new candidates is urgently needed. Polyanionic dendritic nanoparticles, such as dendrons showed unique chemical properties that could be effective against both HIV-1 and HSV-2. We performed a screening of six polyanionic carbosilane dendrons with fatty acid at the core. Results concluded that only the third generation dendrons with hexanoic or palmitic acid at the core exerted a great broad-spectrum antiviral activity, as well as a suitable efficacy against HIV-1 even if the mucosal disruption occurs as consequence of HSV-2 infection. Third generation dendrons retained their antiviral properties at different pHs and remained capable of blocking HIV-1 and HSV-2 infections at early stages of viral cycles. Unfortunately, the study of their vaginal in vivo toxicity in BALB/c mice revealed that they were not safe.

Previous studies in our laboratory revealed the potent activity of G1-S4, G2-S16 and G3-S16 polyanionic carbosilane dendrimers against HIV-1 and HSV-2 by themselves and in combination with several antiretrovirals, such as tenofovir (TFV) or maraviroc (MVC). We further go deep into these studies using dapivirine (DPV), a leading antiretroviral candidate showing good results on HIV-1 prevention in phase III human clinical trials. The combination of dendrimers, acting at the very first stages of infection together with DPV, which interferes HIV-1 reverse transcriptase (RT), resulted in more than 95% of R5-HIV-1_NLAD8 inhibition with synergistic or additive profiles. We also proved that dendrimer/DPV combination inhibited the HIV-1 infection without altering the potent activity of dendrimers to prevent other viral infections, such as HSV-2.

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Interestingly enough, dendrimers and DPV did not modify the growth of several bacteria strains in vitro. The vaginal microbiome comprised a wide variety of bacterial species and alterations in this ecosystem could lead to higher risk to acquire sexually transmitted infections (STIs), such as HSV-2 and HIV-1. We selected the G2-S16 dendrimer as the leading candidate to further evaluate its effect on vaginal microbiome using BALB/c mice in the context of HSV-2 infection. We characterized the composition of mice vaginal microbiome by a metagenomic approach and reported a significant shift in that microbiome after HSV-2 infection. We proved that G2-S16 prevented this alteration, both in the presence or absence of HSV-2, supporting further assays up to a possible clinical trial.

New therapeutic applications of dendrimers are being studied. In this context, the study of G1-S4, G2-S16 and G3-S16 dendrimers in the X4-HIV-1 tropic cell-cell fusion process, referred to syncytium formation, remains still unknown. Dendrimers prevented the X4-HIV-1 infection, as well as syncytia formation, in a dose-dependent manner. We demonstrated that G2-S16 and G1-S4 reduced significantly syncytia formation in HIV-1 Env-mediated cell-to-cell fusion model. These results were supported by in silico molecular modeling which revealed that G2-S16 dendrimer interfered with gp120-CD4 complex, demonstrating its new and potential use not only as potential microbicides but also as a HIV-1 treatment.

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RESUMEN

A pesar de los esfuerzos realizados para desarrollar una vacuna efectiva frente al virus de la inmunodeficiencia humana tipo 1 (VIH-1) y el virus herpes simple tipo 2 (VHS-2), a día de hoy no se dispone de ninguna vacuna eficaz. Este hecho implica que los microbicidas hayan emergido como una de las principales estrategias preventivas. Sin embargo, la mayoría de los microbicidas han fallado durante su desarrollo, por lo que es necesario continuar buscando nuevos fármacos o moléculas. En este sentido, las nanopartículas con estructura dendrítica, como los dendrones polianiónicos, poseen una serie de características y propiedades químicas únicas que pueden jugar un papel relevante en la prevención de las infecciones generadas por virus de transmisión sexual, como el VIH-1 y VHS-2. Por ello, analizamos la capacidad antiviral de seis dendrones polianiónicos tipo carbosilano, con ácidos grasos en el punto focal y funcionalizados con grupos sulfonato. Los resultados muestran que únicamente los dendrones de tercera generación poseen una potente actividad frente a distintos aislados virales del VIH-1 y del VHS-2. Estos dendrones de tercera generación, son capaces de prevenir la infección por el VIH-1 incluso cuando se produce una ruptura en la mucosa vaginal como consecuencia de la infección por el VHS-2. Además, estos dendrones mantienen su capacidad antiviral a diferentes pHs. Tras estudiar el mecanismo de acción subyacente, se observó que actúan en las primeras etapas del ciclo viral. A pesar de los datos obtenidos in vitro, los resultados de toxicidad a nivel vaginal realizados in vivo, en ratonas BALB/c, mostraron toxicidad e irritación tras su administración, por lo que no se continuó el estudio.

Nuestro grupo de investigación está trabajando con dendrímeros polianiónicos tipo carbosilano (G1-S4, G2-S16 y G3-S16) como potenciales microbicidas frente al VIH-1 y VHS-2. Los resultados obtenidos demuestran una gran actividad antiviral, de forma

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individual y en combinación con antirretrovirales utilizados en clínica como el tenofovir (TFV) o el maraviroc (MVC). En base a estos resultados, también se estudió la combinación de los dendrímeros carbosilano con dapivirina (DPV), un antirretroviral que actualmente se encuentra en ensayos clínicos de fase III como microbicida. Las combinaciones dendrímero/DPV (G1-S4/DPV, G2-S16/DPV y G3-S16/DPV) presentaron inhibiciones >95% frente al aislado viral R5-VIH-1_NLAD8, mostrando un comportamiento sinérgico o aditivo. Por otro lado, la combinación de la DPV con los distintos dendrímeros no alteró la actividad que estos presentan frente al VHS-2.

Además, ninguno de los dendrímeros ni la DPV inhibieron el crecimiento de distintas cepas bacterianas in vitro. Este punto es de gran relevancia, ya que el microbioma vaginal dispone de una enorme variabilidad. Las alteraciones en dicho ecosistema pueden incrementar el riesgo de adquirir infecciones de transmisión sexual, por lo que es de vital importancia que los nuevos candidatos a microbicidas vaginales mantengan intacto dicho microambiente. Por ello, evaluamos el efecto in vivo del dendrímero G2- S16 sobre el microbioma vaginal de ratonas BALB/c en el contexto de la infección por el VHS-2. Mediante estudios de metagenómica, se caracterizó el microbioma vaginal y se determinó que tras la infección por el VHS-2 se producía una desregulación significativa. El dendrímero G2-S16 además de prevenir la infección por el VHS-2 evitó la alteración del microbioma, tanto en presencia como en ausencia del VHS-2.

Como previamente hemos demostrado, los dendrímeros son nanomoléculas con una gran versatilidad ya sea en su modo de acción o en su actividad antiviral. Por este motivo, analizamos el efecto de los dendrímeros G1-S4, G2-S16 y G3-S16 en el proceso de fusión celular o formación de sincitios inducido por el aislado viral X4-VIH- 1. Los resultados mostraron que los dendrímeros prevenían tanto la infección por el X4- VIH-1 como la formación de sincitios, de manera dosis dependiente. Además, los

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dendrímeros G1-S4 y G2-S16 reducían la formación de sincitios significativamente en un modelo de fusión celular. Los resultados in silico de modelaje molecular revelaron que el dendrímero G2-S16 interfería con el complejo gp120-CD4, induciendo la formación de un número incorrecto de uniones. Este estudio supone una primera aproximación para poder llegar a utilizar los dendrímeros no solo como microbicidas, sino también como posibles tratamientos frente a la infección por el VIH-1.

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VALUES...70 8.3 INHIBITORY PROFILE OF DENDRIMERS/DPV COMBINATIONS AGAINST R5-HIV-1...74 8.4 DENDRIMERS RETAINED THEIR ANTI-HSV-2 ACTIVITY IN PRESENCE OF DAPIVIRINE...79 8.5 SUSCEPTIBILITY OF DENDRIMERS AND DPV ON BACTERIA...80 9. ROLE OF G2-S16 ON VAGINAL MICROBIOME AND HETEROGENEITY IN HSV-2 INFECTED MICE...83

9.1 VAGINAL MICROBIOME ANALYSES...83 9.2 MICROBIAL COMMUNITY OF HEALTHY MICE VAGINA...84 9.3 VARIABILITY OF MICROBIAL COMPOSITION IN HSV-2 INFECTED

MICE...86 9.4 EFFECT OF G2-S16 DENDRIMER ON VAGINAL MICROBIOME...88 10. POLYANIONIC CARBOSILANE DENDRIMERS IN THE PREVENTION OF X4-HIV-1 AND CELL-TO-CELL FUSION: A NEW THERAPEUTIC APRROACH...90

10.1 EVALUATION OF DENDRIMERS TOXICITY...90 10.2 DENDRIMER´S ABILITY TO INHIBIT X4-HIV-1NL4.3 INFECTION ON MT2 CELL LINE...91 10.3 INHIBITION OF X4-HIV-1NL4.3 SYNCYTIA FORMATION...95 10.4 UNDERLYING MECHANISM OF ACTION OF DENDRIMERS ON

INHIBITION OF SYNCYTIA FORMATION AND CELL-TO-CELL FUSION...98 10.4.1 EFFECT OF DENDRIMERS ON TETRASPANIN PATTERN...98 10.4.2 HIV-1 ENV-MEDIATED CELL-TO-CELL FUSION ASSAY...99

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10.4.3 G2-S16 DENDRIMER MOLECULAR MODELLING...101

DISCUSSION

11. DISCUSSION...107

CONCLUSIONS

12. CONCLUSIONS...122

REFERENCES

13. REFERENCES...126

PUBLICATIONS

14. PUBLICATIONS...143

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1

LIST OF ABBREVIATIONS

3-OS-HS 3-o-Sulfated Heparan Sulfate

ACV Acyclovir

AIDS Acquired Immune Deficiency Syndrome ANOVA Analysis of Variance

ARV Antiretroviral

ATCC American Type Culture Collection cART Combined Antiretroviral Therapy

BV Bacterial Vaginosis

CBMSO Centro de Biología Molecular Severo Ochoa

CCR Chemokine (C-C) motif Receptor. It can be extended to the rest of abbreviations: CCR2, CCR3, CCR5…

CD4 Cluster of Differentiation. It can be extended to the rest of abbreviations: CD3, CD4, CD8…

CEEA-CBMSO Institutional Animal Care and Use Committee-CBMSO

CFU Colony Forming Units

CI Control of Infection

CIX Combination Index

CXCR4 Chemokine (C-X-C motif) Receptor 4 DAPI 4',6-diamidino-2-phenylindole

DMEM Dulbecco’s Modified Eagle’s Medium

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DPV Dapivirine

DPX Distyrene Platiciser Xilene

DRI Dose Reduction Index

dsDNA Double-strand DNA

E. coli Escherichia coli

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2

EBI European Bioinformatics Institute

EC50 Effective Concentration 50%. It can extended to the rest of abbreviations: EC90 and EC95

ELISA Enzyme-Linked ImmunoSorbent Assay

Env Gene encoding the structural Env polyprotein of HIV-1

FA Fatty acid

FBS Fetal Bovine Serum

FfTK Force Field Toolkit plugin

FISABIO Foundation for the Promotion of Health and Biomedical Research

G0, G1, G2, G3 Generation of dendrimers or dendrons

G1d-STE2Hx A first generation dendron synthesized by thiol-ene pathway with hexanoic acid at the focal point and 2 sulfonate groups in the periphery

G1d-STE2Pm A first generation dendron synthesized by thiol-ene pathway with palmitic acid at the focal point and 2 sulfonate groups in the periphery

G1-S4 A first generation dendrimer with 8 sulfate groups in the periphery

G2d-STE4Hx A second generation dendron synthesized by thiol-ene pathway with hexanoic acid at the focal point and 4 sulfonate groups in the periphery

G2d-STE4Pm A second generation dendron synthesized by thiol-ene pathway with palmitic acid at the focal point and 4 sulfonate groups in the periphery

G2-S16 A second generation dendrimer with 16 sulfonate groups in the periphery

G3d-STE8Hx A third generation dendron synthesized by thiol-ene pathway with hexanoic acid at the focal point and 8 sulfonate groups in the periphery

G3d-STE8Pm A third generation dendron synthesized by thiol-ene pathway with palmitic acid at the focal point and 8 sulfonate groups in the periphery

G3-S16 A third generation dendrimer with 8 sulfate groups in the periphery

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3 gag Group-specific antigen. Gene encoding the structural gag

polyprotein of HIV-1

gB Glycoprotein exposed in the surface of HSV-2 membrane. It can be extended to the rest of abbreviations: gB, gC, gD, gE, gG, gH, gI, gJ, gK, gL and gM

GFP Green Fluorescent Protein

gp120 Glycoprotein exposed on the surface of the HIV-1

gp160 Precursor of the HIV envelope glycoproteins gp41 and gp120 gp41 Transmembrane envelope glycoprotein of the HIV-1

HEC Hydroxyethyl cellulose

HIV-1 Human Immunodeficiency Virus Type 1

HS Heparan Sulfate

HSV-2 Herpes Simplex Virus type 2 HVEM Herpesvirus Entry Mediator

IgG Immunoglobulin G

IL-2 Interleukin 2. It can be extended to the rest of interleukins

IN Integrase of HIV-1

Isoflurane 2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane

kbp kilobase pair

LTR Long Terminal Repeat

mAB Monoclonal Antibody

MD Molecular Dynamic

MIC Minimal Inhibitory Concentration

MM-GBSA Molecular Mechanics/Generalized Born Surface Area method

mRNA messenger RNA

MTT 3-(4-5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Mw Molecular weight

N9 Nonoxynol-9

NCBI National Center for Biotechnology Information

Nef Gene encoding the accessory Nef protein of HIV

NI Non infected

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4

NIH National Institute of Health

NNRTI Non-Nucleoside Reverse Transcriptase Inhibitor

NP-40 Nonidet P-40

NT Non treated

nt Nucleotide

PAMAM Poly(Amido Amine) dendrimer

PBS Phosphate-buffered saline

PCoA Principal Coordinate Analysis

PDB Protein Data Bank

P-dendrimer Phosphorus dendrimer

PERMANOVA Permutational Multivariate Analysis of Variance

PET Polyethylene Terephthalate

PFA Paraformaldehide

PFU Plaque Forming Units

PLL Poly-L-Lysine dendrimer

pol DNA polymerase. Gene encoding the structural Pol polyprotein of HIV-1

PME Particle-Mesh Ewald method

PPI Poly(Propylene Imine) dendrimer

PR Protease of HIV-1

Pr160GagPol Polyprotein precursor of RT and IN encoded by pol gene Pr55Gag Polyprotein precursor of p17, p24, p7 and p6 proteins encoded

by gag gene

rpm Revolutions per minute

RAL Raltegravir

RL Repeat Long

RMSD Root Means Square Distance

RNA Ribonucleic acid

RPMI Roswell Park Memorial Institute

RS Repeat Short Region

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RT Reverse Transcriptase of HIV-1

S. sciuri Staphylococcus sciuri

SAQ Saquinavir

SASA Solvent Accessible Surface Area

SD Standard Deviation

SLP7013 A fourth generation polylysine-based dendrimer with 32 napthylsulfonate acid groups in the periphery

STI Sexually Transmitted Infection

T/F Transmitter Founder

TDF Tenofovir Disoproxil Fumarate

TFV Tenofovir

TNF Tumor Necrosis Factor

UL Unique Long

UNAIDS United Nations Programme on HIV and AIDS

US Unique Short

V3 Variable 3 region of the HIV-1 gp120

Vif Viral infectivity factor. Gene encoding the accessory Vif protein of HIV-1

VMD Visual Molecular Dynamics

VOICE Vaginal and Oral Interventions to Control the Epidemic

Vpr Viral protein r. Gene encoding the accessory Vpr protein of HIV-1

Vpu Viral protein unique. Gene encoding the accessory Vpu protein of HIV-1

VZV Varicella Zoster Virus

w/v Weight/volume

WHO World Human Organization

X-Gal 5-bromo-4-chloro-3-indoyl-D-galactopyranoside

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6

INTRODUCTION

1. HUMAN IMMUNODEFICIENCY VIRUS TYPE 1

1.1 EPIDEMIOLOGY

The AIDS epidemic was first identified in the early 1980s. Since then, approximately 70 million people have been infected worldwide, resulting in 35 million deaths (1).

Estimations revealed that in 2016, 36.7 million of people were living with HIV-1 and one million died by AIDS-related diseases. The latest data report 1.8 millions of new infections per year (2). In this regard, global efforts to prevent new HIV-1 infections achieved that the annual number of new HIV-1 infections has declined by 16% of 1.8 million since 2010. However, the targeted number agreed upon by the United Nations General Assembly in 2016 which supposed to be lower than 500,000 new infections per year by 2020 are still far away (2).

The global pandemic situation has improved. In example, in sub-Saharan Africa, the region where most people live with HIV (25.6 million) (3) and approximately two third of new infections worldwide are registered (2). This fact occurs due to the higher treatment coverage and better adherence to treatment, as well as to policies implemented in each region, which results in a significant reduction of AIDS-related deaths (4). Declines in AIDS-related deaths over the last decade were achieved in the Caribbean (52% reduction), Europe and North America (45% reduction), Asia and the Pacific (39% reduction) and Western and Central Africa (30% reduction). In Latin America the reduction was just 16%. Worrying increases in AIDS-related mortality have occurred over the past decade in the Middle East and North Africa (48% increase), Eastern Europe and Central Asia (38% increase) (2).

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7 1.2 MORPHOLOGY AND STRUCTURE

HIV-1 belongs to the subfamily Lentivirinae of the family Retroviridae (5). HIV-1 is a single-stranded positive-sense RNA virus. The HIV-1 genome encodes major structural and non-structural proteins common to all replication-competent retroviruses. From the 5'- to 3'-ends of the genome are found the gag, pol and env genes. The gag gene encodes a polyprotein precursor (Pr55Gag), which is cleaved by the viral protease (PR) to generate p6, p7, p17 and p24 proteins. The pol-encoded the enzymes reverse transcriptase (RT) and integrase (IN), whose are cleaved from Pr160GagPol precursor (6). The viral surface is thought to be encoded in the env gene region of the HIV-1 genome. Gp160 is the primary env gene product that will be proteolytically processed into gpl20 and gp41 (7). Gp120 and gp41 are both glycoproteins playing an important role in the interaction with host receptors during the HIV-1 infection.

HIV particles are spherical with 80-150 nm of diameter. The structure consists of an envelope, a matrix and a core. The envelope is a lipid bilayer derived from the host cell’s membrane decorated with viral glycoproteins. The matrix shell is formed by several copies of p17 proteins as the inner surface of external lipid bilayer. The inner core architecture is conical with a stick-like shape comprising polymers of p24 protein.

The core contains key enzymes like the viral protease, the reverse transcriptase and the integrase. It also contains several accessory proteins (Nef, Vif and Vpr, Vpu) (8) and two copies of non-covalently linked, unspliced, positive-sense single stranded RNA stabilized by the nucleocapsid protein p7.

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8

Figure 1. HIV-1 particle. (A) Schematic structure of HIV-1 including the gp41 and gp120, as well as the main proteins that play an important role in its replicative cycle (modified from Gonzalo-Gil et al., 2017) (9). (B) Organization of the HIV-1 genome. The long terminal repeats are at both ends of the HIV-1 genome depicted in a three-color-code: U3 (grey), R (black) and U5 (white) (Sertznig et al., 2018) (10).

1.3 VIRAL CYCLE

The HIV-1 replication cycle initiates with the attachment of the viral envelope gp120 to the amino-terminal immunoglobulin domain of CD4 host receptor. Subsequent interactions with CCR5 or CXCR4 co-receptor (11) is essential to induced the fusion of the viral and host cellular membranes (12). In this sense, the V3 loop region of gp120 plays a major role to determine the viral tropism. Gp41 not only anchors the gp120/gp41 complex, but also catalyzes the membrane fusion reaction between viral and host lipid bilayers. These events promote the formation of a pore and the subsequent release of the viral core in the cytoplasm cell (13). The viral RNA is transcribed into double-stranded DNA by the RT enzyme (14). The high frequency of

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9 genetic recombination and the elevated mutation rate of HIV-1 RT result in highly heterogeneous HIV populations (“quasispecies”). Consequently, HIV-1 develops efficient resistance to antiviral drugs and evades the host immune response (15).

Following nuclear import, the integrase catalyzes the insertion of the linear, double- stranded viral DNA in the host cell chromosome (6). The integrated provirus is transcribed for the synthesis of the viral RNA that finally encodes the proteins used to direct virus replication. The transcription process leads to the generation of a large number (>30) of viral RNA (16) in order to complete the synthesis of the full complement of viral proteins. During the assembly step, the Gag precursor polyprotein, Pr55^Gag plays an important role due to the fact that it binds the plasma membrane itself, promotes Gag-Gag interactions, encapsidates the viral RNA genome, goes together with the viral Env glycoproteins and stimulates budding from the cell (17). Finally, the viral protease cleaves the Gag and Pol polyprotein precursors to generate HIV-1 mature infectious particles (Figure 2).

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Figure 2. HIV-1 replication cycle. The first step is the binding of HIV-1 surface glycoproteins to host receptor CD4 and the fusion with the cell membrane. After the uncoating in the cytoplasm, the RT transcribed the viral RNA to DNA, which will be translocated into the nucleus. Viral DNA is integrated in the host genome, transcribed and translated to produce viral RNA and proteins, which are moved to the cell membrane in order to assembly. The release of new viral particles and the maturation by the action of the protease results in the production of new virions. Adapted from Gonzalo-Gil et al., 2017 (9).

1.4 CLINICAL COURSE

Most HIV-1 infections occur by sexual exposure through the genital tract or rectal mucosa. The HIV-1 diffusion across the vaginal mucosa up to target cells occur when the genital mucosa is damaged by physical trauma or co-existing genital infections (18).

Among the HIV-1 quasispecies, only those viruses capable of binding the chemokine receptor CCR5 appear to be mainly at the very first stages of infection, known as R5- tropic viruses (19). At this point, “founder virus” is established in CD4⁺memory T cells

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11 residing in the submucosa expressing the CCR5 co-receptor, which is consistent with the cell tropism of HIV-1 founder virus (20). The virus population expands and disseminates to lymph nodes, resulting in an exponential increase of viral production up to reach a peak and a progressive depletion of CD4⁺ T cells (21).

In late stages of infection, a switch from CCR5-tropic to CXCR4-tropic viruses (22) is associated with a massive loss of CD4⁺ T cells, increasing pathogenicity and disease progression to AIDS (23). A high cell density and migratory T cells are located in the lymph nodes inducing the interaction among HIV-1 infected and non-infected cells.

Subsequently, nuclear fusion occurs causing the formation of giant multinucleated cells known as syncytia (24, 25).

The introduction of combined antiretroviral therapy (cART) improved the survival rate and reduced the number of individuals developing AIDS (26). The control of the viremia up to viral undetectable limits lead to a precipitous decline in AIDS- opportunistic infections in HIV-infected people. Thus, in the post-cART era, the life expectancy of HIV-infected individuals has improve due to better treatments, management and care of disease that lead them to older age (27). Nevertheless, the immune dysregulation persists and is associated with the occurrence of HIV and with comorbidities connected with aging, including cardiovascular disease (4), cancers (28) and liver disease, among others.

Although cART is effective in suppressing HIV-1 replication, it is not curative due to the existence of a latent viral reservoir in resting memory CD4⁺ T cells. The viral genome persists in a DNA form as integrated provirus that is not actively transcribed (29). The viral reservoir is inaccessible either to immune system or cART which needs a lifelong treatment to maintain undetectable viremia (30). Many international efforts are focused on a functional cure that eradicates latently HIV-1-infected cells.

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Figure 3. Time course of untreated HIV infection and cART. (A) The nontreated course of HIV infection is characterized by progressively lost of CD4+ T cells in blood and an increase in the viral load after the acute phase that promotes the development of AIDS. (B) The implementation of cART, significantly decrease the viral load up to undetectable levels followed by recovery of CD4 T cells.

Nevertheless, one of the main challenges is the eradication of viral reservoirs, which are inaccessible to cART. Adapted from Maartens et al., 2014 (31).

2. HERPES SIMPLEX VIRUS TYPE 2 (HSV-2)

2.1 EPIDEMIOLOGY

Epidemiological studies estimated that approximately 500 million people (range: 274–

678 million) (11.3%) world-wide aged 15–49 years are currently infected with HSV-2 and that 19.2 million (range: 13.0–28.6 million) became newly infected per year. The global burden of HSV-2 infection is large, leaving people at increased risk of genital ulcer disease, HIV acquisition and transmission of HSV-2 to partners or neonates (32).

HSV-2 is the main cause of genital herpes. The prevalence of HSV-2 rises with initiation of sexual activity in adolescence and steadily increases through adulthood.

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13 Women are more susceptible to HSV-2 infection than men (33). Although reasons are still unclear, studies suggest that men could be more resistant to HSV-2 infection because of stronger keratinization of the skin at the external surfaces of the genitalia (33) or even due to the sexual behavior (such as different patterns of sexual mixing) (34, 35). The prevalence of HSV-2 varies significantly depending on the global region, ranging from ~5% for countries such as Spain and up to 70% for Sub-Saharan Africa (33). In Western countries, genital herpes is a relatively common infection with a prevalence rate of 13–25%. Many infections go unrecognized as they may be clinically atypical, short lived and the majority is asymptomatic. This largely explains the constant transmission rate from one partner to another (36).

The seroprevalence of HSV-2 in countries belonging to Asia and Oceania showed percentages ranging from ~5% for New Zealand and Japan, ~10% for Australia and the Philippines and up to ~20% for China (37). In Latin America, relatively few epidemiological studies assessing the seroprevalence of HSV-2 can be found.

Nevertheless, studies suggest nearly 54 million cases for South America and the Caribbean (32).

2.2 MORPHOLOGY AND STRUCTURE

HSV-2 is a double stranded, enveloped DNA virus that belongs to the herpesviridae family (36). It is classified in the subfamily alphaherpesviruses, which include HSV-1, HSV-2 and varicella zoster virus (VZV). These are neurotropic cytolytic viruses with the potential ability to establish asymptomatic latent infections in neurons of the peripheral nervous system (38).

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The linear DNA of HSV consists of two covalent linked segments, the long (L) and short (S). The L component consists of unique sequences (UL) bounded by inverted repeats (R_L and R_L′). The S component is composed of unique sequences (U_S) bounded by inverted repeats (RS and RS′) (Figure 4B) (39).

The size of the genome is about 154 kbp and codified for more than 74 genes. This genetic material is packaged in an icosapentahedral capsid consisting of 162 capsomers (40), which is wrapped in a lipid bilayer (envelope). The tegument consists of a protein layer that surrounds the capsid and is enclosed within the envelope (Figure 4A). The complete structure is known as HSV-2 virion with a diameter of approximately 200 nanometers.

Figure 4. Representation of the HSV-2 particle. (A) Structure of HSV-2 including the main glycoproteins gB, gC, gD and the heterodimer gH-gL, as well as other relevant parts of the virion. (B) Overall organization of the HSV-2 genome. The U_L and U_S portions of the genome are represented as solid lines, and the major repeat elements (TR_L, IR_L, IR_S and TR_S) as open boxes. TR_L, U_L and IR_Lare regarded as comprising the L region, and IR_S, U_S and TR_S as comprising the S region. Dolan et al., 1998 (41).

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15 2.3 VIRAL CYCLE

The HSV-2 entry into host cells is a multistep process that begins with the specific binding of viral envelope glycoproteins to the host-cell surface receptors (40). The glycoprotein C (gC) mediates the initial attachment of the virions to certain cell-surface glycosaminoglycans, most notably heparan sulfate (HS) (42). The gC is not essential for viral entry due to in its absence, the gB mediates the initial attachment (43). The next step is triggered by gD, which is also essential in this process and mediated the binding with three host receptor: nectins, 3-O-sulfated heparin sulfate (3-O-HS) and the herpesvirus entry mediator (HVEM), which belongs to the tumor necrosis factor (TNF) receptor family (44). Subsequent conformational changes in gD recruits and binds to gB and gH/gL heterodimer. The activation of gH/gL heterodimer support the fusogenic activity of gB and membrane fusion (42, 45). As a consequence, the interaction among cell membrane and the vesicular membrane result in the release of the viral capsid into the cytoplasm. The incoming capsids are directed to the nuclear pores where the genomic viral DNA is released in the nucleus. The transcription of the viral genome is carried out by a host RNA polymerase II in a cascade-like process that expresses immediate-early, early, and late viral mRNA. The immediate-early genes encoded proteins that regulate the expression of subsequent ones (46), meanwhile late genes encode structural components of the virion, including capsid and envelope proteins (47).

After the concatemeric viral DNA replication, the genomic material is cleaved to unit lengths of the nucleus in preformed capsids. These structures are translocated to the cytosol through the nuclear membrane, which provides the nucleocapsid with a primary envelope (48). After the translocation, the nucleocapsid acquire more than 15 tegument proteins as well as the lipid envelope, containing >10 viral glycoproteins in the Golgi or trans-Golgi network (49, 50). The result is an enveloped virion within a secretory

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vesicle that is transported to the cell membrane where vesicle and plasma membranes fuse and mature, and then virions are released (51).

Figure 5. HSV-2 replication cycle. Schematic representation of the main steps involved in the HSV-2 replication cycle, including the initial viral attachment, entry and dissociation of tegument, transport of incoming capsids to the nuclear pore, and release of viral DNA in the nucleus where transcription occurs in a cascade-like fashion and viral DNA replication takes place. Late events, such as capsid assembly, secondary envelopment and the release of new virions are also displayed.

2.4 CLINICAL COURSE

HSV typically infects epithelial cells of the skin and mucosa. It is acquired with the direct contact of infected tissue or secretions. Sexual intercourse is the main route of

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17 HSV-2 transmission. It was assumed that genital ulcerations were caused by this virus.

However, HSV-1 is more often in the genitalia lessons due to the decreasing prevalence of orolabial HSV-1 in the young adult population and due to an increase in the frequency of oral sex (36).

Once target cells are infected, the viral cycle is about one day with an estimated production of 10²-10³ virions/infected cell. Viral progeny infects surrounding cells and then enters sensory neuronal axons via free nerve endings in the epidermis. HSV-2 particles are transported retrogradely to neuronal cell bodies in the dorsal root ganglia where HSV-2 establishes latency (52).

During latency, the viral RNA expression is limited and very few or no viral proteins are synthesized. Symptomatic or asymptomatic recurrences of HSV-2 infection are initiated in latently infected neurons when the HSV genome switches from silent to gene expression and subsequent lytic cycle. HSV-2 virions are produced and delivered from axons of sensory ganglions to epithelial cells (skin or mucous membranes) (53) with anterograde transfer. Reactivation can be caused by several factors, including hormonal changes, stress, heat, fever and physical damage to the neuron.

Clinical lessons after primary infection appear only in the 10-25% of infections.

Nevertheless, systemic complications (recurrent meningitis, hepatitis, and pneumonitis) could take place, particularly among immunosuppressed individuals (54). In erythema or ulcer formation the viral replication and infectivity is high and the peak viral titer occurs in the first 24 h after the lesion. The standard treatment regimens for initial primary HSV include acyclovir (ACV), valacyclovir and famciclovir (55) and a shorter course of antivirals commenced early in the lesion development for recurrent episodic (56).

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Figure 6. Pathogenesis of HSV-2. HSV-2 enters through breaks in the skin or mucosa where it replicates. Remarkably, only 10–25% of new infections are symptomatic. Virions enter to sensory neuronal axons via free nerve endings in the epidermis through retrograde transport where the virus remains in latency. Periodically, the virus reactivates from the latent state and travels by anterograde transport to the skin where it could exert clinical manifestations. Shedding from mucosal surfaces leads to transmission to other sexual partners or even from mother-to-child (Adapted from Gupta et al., 2007) (35).

3. HSV-2 AND HIV-1 COINFECTION

Epidemiological studies have suggested that HSV-2 infection increases the likelihood of HIV-1 infection by 3-fold to 4-fold (57-59). In fact, HIV-1 in co-infected HSV-2/HIV-1 individuals is frequently acquired from genital herpes lesions (60). The resulting coinfection is involved in synergistic relationships, leading in a vicious circle of mutual facilitations. The genital ulcerations caused by HSV-2 primary infection disrupt the epithelial surface, that provokes the HIV-1 to break through the submucosa where it

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19 encounters mucosal CD4⁺ T cells and macrophages, infecting target cells and disseminating throughout the lymphoid tissue (58, 61, 62).

The HSV-2 upregulates the expression of the CCR5 in immune cells, an essential co- receptor for HIV-1 entry in macrophages and activated CD4⁺ T cells. Due to the fact that acute HIV-1 infection has been attributed to R5-tropic viruses, it could be one of the possible mechanisms by which HSV-2 increases acquiring HIV-1 infection (63).

Another possible mechanism could take place through indirect support HIV-1 replication by the release of pro-inflammatory cytokines and chemokines from HSV-2 infected cells (62). The prevention of new HSV-2 infections could indirectly reduce the number of new HIV-1 infections.

Figure 7. HSV-2 and HIV-1 relationship at the site of infection. 1. The HSV-2 infection causes lesions in the mucosa that facilitates the infection of HIV-1. 2. HSV-2 infected cells produce the secretion of pro- inflammatory IL-6, TNF-α or IL-1β cytokines in response to viral infection. 3. This fact induces the recruitment of immune cells to the site of infection, such as dendritic cells and T cells, which are the main HIV-1 targets. (Modified from Suazo et al., 2015) (33).

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4. NANOTECHNOLOGY

Nanotechnology is defined as the science involved in the design, synthesis, characterization and application of materials and devices in the nanometer scale. (64).

One nanometer is one-billionth part of a meter or about the width of 6 carbon atoms (65). Atoms are <1 nm, whereas many molecules including several proteins have a size of 1 nm and even more (66). It represents a multidisciplinary scientific field undergoing a technological revolution in the last years (67). Nano-sized particles possess remarkable self-ordering and assembly behaviors under the control of forces quite different from macro objects (68). This fact causes the genesis of several materials, such as nanocrystals or nanoparticles with unique properties and applications in diverse areas of research.

The potential applications of nanotechnology are too vast due to the possibility to create new products with new features (68). Many examples of these applications are found in the field of cosmetics, electronics, energy production, food industry and optic.

Nevertheless, one of the areas where nanotechnology plays an important role is the biomedicine (64).

4.1 NANOMEDICINE

Nanomedicine represents one of the most studied applications of nanotechnology. It could be define as the monitoring, repairing, construction, and control of human biological systems at the molecular level by using engineered nanostructures.

The field of nanotechnology has been developed very rapidly over the past decade lending great promises to medical applications in drug delivery, therapeutics, and biological imaging (67). The fact that nanomaterials are similar in size to biological

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21 structures coupled with the easy synthesis, high biocompatibility and customizability supports their potential use in prevention, diagnosis and treatment of several pathologies (69).

Nanoparticles have various shapes, different three-dimensional architecture and chemical compositions. The most common types used in nanomedicine are: polymers, liposomes, dendrimers, micelles, carbon nanotubes, quantum dots and dendrons (Figure 8). In particular, dendrons and dendrimers have emerged as potential candidates for drug delivery carriers or antiviral drugs, among other applications. This Thesis has been focused on the use of these nanoparticles.

Figure 8. Examples of nanoparticles used in nanomedicine. Adapted from Mc Carthy et al., 2015 (70).

4.2 DENDRONS

Dendrons are synthetic nano-sized structures with a tree-like shape emerging from a focal point. They could be defined as wedge-shaped dendrimer sections with various

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end-groups in the periphery and a single reactive function at the focal point. The small size of these nanoparticles allows them to have a high flexibility, thus the ability to interact with several biological targets is enormous (71, 72). One of the most interesting aspects of dendrons is their functionality since it is possible to graft dendrons to a surface, to another dendron to design high density structures, or even to other macromolecules (Figure 9A).

The synthesis of these nanoparticles is relatively easy with a high structural control resulting in monodisperse products. Their ability to carry out two distinct functional groups in the same molecule conferred multiple options in the biomedical field (73) and more specifically in the search of powerful microbicides to STI worldwide.

Figure 9. Schematic representation of the architecture of dendrons and dendrimers. (A) Typical structure of a dendron and (B) architecture of a dendrimer, showing the three main parts: core, branching units or internal layers and surface groups.

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23 4.3 DENDRIMERS

The word “dendrimer” derived from the Greek words dendri- (tree-branch-like) and meros- (part of), was used about 20 years by Donald A. Tomalia (74). Dendrimers are nano-sized molecules with high branched three-dimensional structure characterized by a high degree of structural control. The architecture of dendrimers is divided into three main components: i) the core, the inner part of the structure where the branching units raised; ii) interior layers with repeated units that will define the generation of the dendrimer and iii) functional groups, which represent the outer shell or periphery (Figure 9B). It is widely defined that the character of the core affects the shape of the dendrimer. The interior layers affect the host–guest properties (75). In addition, the surface of the molecule is polymerized with several functional groups (76). According to the characteristics and charge of these groups, dendrimers can be divided into cationic or anionic with distinct nanomedicine applications (Figure 10).

Figure 10. Applications of dendrimers in the biomedicine field.