Mecanismos inmunoreguladores implicados en la disminución de respuesta vacunal en distintos escenarios marcados por la inmunosenescencia: sujetos ancianos y sujetos con infección por VIH

1 MECANISMOS INMUNOREGULADORES IMPLICADOS EN LA DISMINUCIÓN DE RESPUESTA VACUNAL EN DISTINTOS ESCENARIOS MARCADOS POR LA INMUNOSENESCENCIA: SUJETOS ANCIANOS Y SUJETOS CON INFECCIÓN POR VIH Tesis Doctoral INÉS HERRERO FERNÁNDEZ Instituto de Biomedicina de Sevilla (IBiS) Hospital Universitario Virgen del Rocío Dpto. de Bioquímica Médica y Biología Molecular e Inmunología Universidad de Sevilla Directores D.ª YOLANDA MARÍA PACHECO LÓPEZ D. MANUEL LEAL NOVAL Tutor D. ANTONIO CARRILLO VICO 2 Universidad de Sevilla Facultad de Medicina Dpto. de Bioquímica Médica y Biología Molecular e Inmunología Doctorado en Biología Molecular y Biomedicina D.ª Yolanda María Pacheco López, Investigadora estabilizada del Programa Nicolás Monardes del Sistema Andaluz de Salud. D. Manuel Leal Noval, Profesor asociado del Departamento de Medicina de la Universidad de Sevilla. D. Antonio Carrillo Vico, Profesor Titular del Departamento de Bioquímica Médica, Biología Molecular e Inmunología. CERTIFICAN: Que el trabajo titulado Mecanismos inmunoreguladores implicados en la disminución de respuesta vacunal en distintos escenarios marcados por la inmunosenescencia: sujetos ancianos y sujetos con infección por VIH, presentado por la Graduada en Biología D.ª Inés Herrero Fernández, ha sido realizado bajo nuestra dirección y asesoramiento en el Laboratorio de Inmunovirología del Instituto de Biomedicina de Sevilla (IBiS). Concluido el trabajo experimental y bibliográfico, autorizamos la presentación y la defensa de esta Tesis Doctoral, para que sea juzgada por el tribunal correspondiente. Fdo. Dra. Yolanda María Pachecho López Fdo. Dr. Manuel Leal Noval Fdo. Dr. Antonio Carrillo Vico 3 ABREVIATURAS APC: Células presentadoras de antígenos CCR5: Receptor de quimiocinas CCR5 CD4: Linfocitos T CD4 CD8: Linfocitos T CD8 CMV: Citomegalovirus DC: Células dendríticas ENOS: Eventos no definitorios de SIDA mDC: Células dendríticas mieloides MVC: Maraviroc OMS: Organización Mundial de la Salud RTE: Células recientemente emigradas del timo SIDA: Síndrome de Iinmunodeficiencia adquirida TARc: Tratamiento antirretroviral combinado TCR: Receptores de células T Treg: Células T reguladoras usPCR: Proteína C reactiva ultrasensible VHB: Virus de la Hepatitis B VHC: Virus de la Hepatitis C VIH: Virus de la Inmunodeficiencia Humana 4 ÍNDICE 1. Resumen ........................................................................................................................ 5 2. Introducción ................................................................................................................... 9 3. Objetivos ...................................................................................................................... 16 4. Material, Métodos y Resultados ................................................................................. 18 4.1. Herrero-Fernández I, Rosado-Sánchez I, Álvarez-Ríos AI, Galvá MI, De Luna- Romero M, Sanbonmatsu-Gámez S, Pérez-Ruiz M, Navarro-Marí JM, Carrillo-Vico A, Sánchez B, Ramos R, Cañizares J, Leal M, Pacheco YM. T-cell proliferative homeostatic alterations are negatively associated with the vaccine response against influenza in elderly people (en revisión) ........................................................ 19 4.2. Herrero-Fernández I, Pacheco YM, Genebat M, Rodriguez-Méndez MDM, Lozano MDC, Polaino MJ, Rosado-Sánchez I, Tarancón-Diez L, Muñoz-Fernández MÁ, Ruiz- Mateos E, Leal M. Association between a Suppressive Combined Antiretroviral Therapy Containing Maraviroc and the Hepatitis B Virus Vaccine Response. Antimicrob Agents Chemother. 2017;62:e02050-17 .................................................. 65 4.3. Herrero-Fernández I, Rosado-Sánchez I, Genebat M, Tarancón-Díez L, Rodríguez- Méndez MM, Pozo-Balado MM, Lozano C, Ruiz-Mateos E, Leal M, Pacheco YM. Improved CD4 T-cell profile in HIV-infected subjects on maraviroc-containing therapy is associated with better responsiveness to HBV vaccination. J Transl Med. 2018;16:238 ................................................................................................................. 79 - Anexo 1: Variables demográficas, clínicas e inmunológicas asociadas con la respuesta a la vacuna frente a VHB ......................................................................... 91 - Anexo 2: Asociación entre usPCR y título de anticuerpos frente a VHB ............... 93 5. Resumen global de los resultados ............................................................................... 94 6. Discusión global ........................................................................................................... 96 7. Conclusiones ..............................................................................................................104 8. Bibliografía .................................................................................................................106 9. Financiación ...............................................................................................................117 10. Otras publicaciones .................................................................................................119 5 1. RESUMEN 6 RESUMEN En la actualidad se está produciendo un cambio demográfico debido al envejecimiento de la población. Al aumentar la edad, el sistema inmunitario humano experimenta cambios característicos, conocidos como inmunosenescencia, que generan respuestas inmunológicas subóptimas. La iuoseeseia atual o iuoseeseia oológia se efiee al deteioo pogesio del sistea iuológio asociado a la edad. Debido a este proceso se produce una alteración homeostática del sistema inmunológico y se generan deficiencias en la función celular B y T. En las células T se originan cambios relevantes, como la reducción de células naïve, debido principalmente a la menor timopoyesis que tiene lugar durante el envejecimiento. Además, se produce por compensación homeostática una acumulación de células T efectoras y memoria con escasa diversidad de sus receptores, con una elevada proliferación, activación, senescencia y resistencia a la apoptosis. Debido a estas alteraciones homeostáticas también se produce una expansión de las células T reguladoras (Treg), células supresoras de la respuesta inmunitaria, que contribuye a la desregulación del sistema. En la población anciana se presenta asimismo una inflamación sistémica característica del envejecimiento, proceso conocido como inflammaging, ue taié difiulta la fuió iuitaia. Estas alteraciones conducen a que se generen respuestas inmunológicas subóptimas, provocando, entre otras complicaciones, baja efectividad de las vacunas, lo que contribuye a un aumento de la morbimortalidad de los ancianos. El virus de la gripe, o virus de la influenza, causa una elevada morbimortalidad en la población anciana. La vacuna frente a este virus es el método más efectivo frente a la infección y las complicaciones relacionadas con la enfermedad, sin embargo, los ancianos presentan una baja eficacia de la vacuna debido a las alteraciones de su sistema inmunológico. El objetivo de la presente Tesis Doctoral fue explorar la asociación entre distintos parámetros homeostáticos en la población anciana (función tímica, proliferación de las 7 células T, frecuencia y fenotipo de las Treg y biomarcadores de inflamación) con la respuesta a la vacuna frente a influenza. Este estudio se desaolló e el taajo T-cell proliferative homeostatic alterations are negatively associated with the vaccine response against influenza in elderly people e reisió. Observamos que las alteraciones homeostáticas proliferativas que se desarrollan en las subpoblaciones de maduración de las células T CD4 y CD8, incluyendo las Treg, se asocian a una menor respuesta a la vacuna frente a influenza. Estas alteraciones homeostáticas podrían estar íntimamente relacionadas con el aumento de inflamación sistémica y con la disminución de la función tímica asociadas al envejecimiento. Un espectro similar de alteraciones inmunológicas se produce en personas infectadas por el virus de la inmunodeficiencia humana (VIH), incluso las que están recibiendo un tratamiento antirretroviral combinado (TARc) supresor, proceso conocido como iuoseeseia peatua, u poeso peatuo y aetuado de la iuoseeseia oológia. Estos poesos opate astates asgos, peo no todos. Debido a la exposición al VIH y sus antígenos se produce una depleción masiva de linfocitos T CD4 y un gran desgaste inmunológico, con expansiones oligoclonales. Además, se produce una reducción de la función del timo y, por tanto, de células naïve, debido posiblemente al timotropismo del VIH. Por otro lado, se produce un aumento en los niveles de marcadores de inflamación sistémica. Además, en este escenario también se ha observado una expansión de células Treg y de células efectoras. Los sujetos infectados por VIH tienen un riesgo elevado de complicaciones hepáticas, puesto que hay una elevada tasa de coinfección por el Virus de la Hepatitis B (VHB) y por el Virus de la Hepatitis C (VHC). La coinfección por el VHB o el VHC aumenta notablemente la morbimortalidad hepática en estos pacientes VIH. La vacunación frente a VHB es la manera más importante de prevenir la coinfección, pero los individuos VIH muestran una baja respuesta a la vacuna. Además de la baja tasa de respuesta a la vacuna, en aquellos pacientes que responden a ella, la magnitud de los anticuerpos protectores es baja y, por consiguiente, se pierden rápidamente. 8 Estudios previos sugieren que Maraviroc (MVC), fármaco antirretroviral, posee propiedades inmunomoduladoras que pueden impactar de manera favorable en la respuesta inmunitaria y por tanto podrían favorecer la respuesta vacunal frente a VHB. Por ello, uno de los objetivos de la presente tesis doctoral fue analizar si un tratamiento antirretroviral combinado (TARc) que contenía MVC se asociaba a una mejor respuesta a la vacuna del VHB en pacientes infectados por VIH. Este objetivo se abordó en el trabajo Assoiatio etee a suppressie oied atiretroiral therapy containing maraviroc and the hepatitis B virus vaccine response Antimicrob Agents Chemother 2017; 62:e02050-17. Observamos que un TARc que incluía MVC se asociaba con una mayor magnitud de la respuesta a la vacuna frente a VHB en sujetos VIH menores de 50 años. Por tanto, un régimen conteniendo MVC durante el protocolo de inmunización podría ser una estrategia terapéutica adecuada para mejorar la respuesta vacunal en estos sujetos. Tras este trabajo se extendió su análisis, incluyendo variables inmunológicas para conocer los posibles factores, relacionados con la inmunosenescencia, que pudieran mediar en el impacto de MVC sobre la respuesta a la vacuna frente al VHB. Por tanto, exploramos el potencial efecto de un TARc que incluía MVC en diferentes parámetros relacionados con la inflamación, la función de las células T, incluidas las Treg, y de células dendríticas, que pudieran estar relacionados con la efectividad de la vacuna en la población VIH. Este análisis se desarrolló en el trabajo Iproed CD4 T-cell profile in HIV-infected subjects on maraviroc-containing therapy is associated with better responsiveness to HBV vaccination J Transl Med 2018; 16:238. Observamos que los pacientes tratados con un TARc incluyendo MVC mostraron un perfil inmunológico menos activado, con mayor contribución de las células T recientemente emigradas del timo y una menor frecuencia de células Treg, perfil que se asocia con una mejor respuesta a la vacuna frente al VHB. Estos datos nos llevan a especular que MVC podría tener un efecto regenerador sobre el sistema inmunitario, promoviendo la función tímica y disminuyendo la proliferación homeostática compensatoria, lo que le permitiría repercutir también en un menor estado proinflamatorio. 9 2. INTRODUCCIÓN 16 3. OBJETIVOS 17 OBJETIVOS  Objetivo 1: Explorar potenciales asociaciones entre distintos parámetros relacionados con la homeostasis de células T y la respuesta a la vacuna frente a influenza en sujetos ancianos. Con este fin, se consideraron principalmente los siguientes parámetros: función tímica, proliferación de las células T, frecuencia y fenotipo de las Treg y marcadores de inflamación. Este objetivo se abordó en el taajo: T-cell proliferative homeostatic alterations are negatively associated with the vaccine response against influenza in elderly people (en revisión).  Objetivo 2: Determinar si un TARc incluyendo MVC mejora la respuesta a la vacuna frente a VHB. Este objetivo se abordó en el trabajo: Association between a suppressive sombined antiretroviral therapy containing maraviroc and the hepatitis B virus vaccine response Antimicrob Agents Chemother 2017; 62:e02050-17.  Objetivo 3: Explorar el potencial efecto de un TARc incluyendo MVC sobre diferentes parámetros relacionados con la homeostasis de células T, potencialmente implicados en la respuesta vacunal frente a VHB Con este fin, se consideraron principalmente los siguientes parámetros: función tímica, proliferación de las células T y frecuencia y fenotipo de las Treg, marcadores de inflamación y fenotipo de células dendríticas. Este objetivo se abordó en el trabajo: Iproed CD4 T-cell profile in HIV-infected subjects on maraviroccontaining therapy is associated with better responsiveness to HBV vaccination J Transl Med 2018; 16:238. 18 4. MATERIAL, MÉTODOS Y RESULTADOS 19 4.1. T-cell proliferative homeostatic alterations are negatively associated with the vaccine response against influenza in elderly people Herrero-Fernández I, Rosado-Sánchez I, Álvarez-Ríos AI, Galvá MI, De Luna-Romero M, Sanbonmatsu-Gámez S, Pérez-Ruiz M, Navarro-Marí JM, Carrillo-Vico A, Sánchez B, Ramos R, Cañizares J, Leal M, Pacheco YM. (En revisión) 1 Title: Homeostatic T-cell proliferation was negatively associated with the vaccine 1 response against influenza in a cohort of elderly people. 2 Authors names: Herrero-Fernández I1, Rosado-Sánchez I1, Álvarez-Ríos AI1,2, Galvá 3 MI3, De Luna-Romero M1, Sanbonmatsu-Gámez S4, Pérez-Ruiz M4, Navarro-Marí JM4, 4 Carrillo-Vico A1,5, Sánchez B6, Ramos R3, Cañizares J3, Leal M1,7* and Pacheco YM1*≠. 5 Author email addresses: Herrero-Fernández I ([email protected]), Rosado-6 Sánchez I ([email protected]), Álvarez-Ríos AI ([email protected]), 7 Galvá MI (mariai.ga[email protected]), De Luna-Romero M 8 (martadelun[email protected]), Sanbonmatsu-Gámez S 9 ([email protected]), Pérez-Ruiz M 10 ([email protected]), Navarro-Marí JM 11 ([email protected]), Carrillo-Vico A ([email protected]), Sánchez B 12 ([email protected]), Ramos R ([email protected]), 13 Cañizares J ([email protected]), Leal M ([email protected]) and 14 Pacheco YM (ypacheco-i[email protected]). 15 Affiliations: 16 1. Institute of Biomedicine of Seville (IBiS), Virgen del Rocío University Hospital 17 (HUVR)/CSIC/University of Seville, Seville, Spain 18 2. Department of Clinical Biochemistry, Virgen del Rocío University Hospital, Seville, 19 Spain. 20 3. Heliopolis Nursing Home, Seville, Spain 21 4. Servicio de Microbiología, Hospital Universitario Virgen de las Nieves, Instituto de 22 Investigación Biosanitaria Ibs Granada, Granada, Spain. 23 2 5. Department of Medical Biochemistry, Molecular Biology and Immunology, 24 University of Seville, Seville, Spain. 25 6. Immunology Service, Instituto de Biomedicina de Sevilla, Hospital Universitario 26 Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain 27 7. Hospital Viamed, Santa Ángela de la Cruz, Seville, Spain 28 *Both authors contributed equally 29 30 ≠Corresponding author’s contact details: 31 Yolanda M. Pacheco, Ph.D. 32 Laboratory of Immunology, Institute of Biomedicine of Seville (IBiS)/UGC Clinical 33 Laboratories, Virgen del Rocío University Hospital, Avda. Manuel Siurot s/n. PC 34 41013, Seville, Spain. Phone number: +34955923120. Email: ypacheco[email protected]. 35 36 Running Title: Proliferating T-cells and the influenza vaccine response 37 Keywords: Treg, Ki67, inflammation, thymic function, TREC 38 Abstract word count: 202 39 Text word count: 3631 40 References: 47 41 Number of inserts: 4 (1 Table + 3 Figures) + 5 Additional files (4 Supplementary Tables 42 + 4 Supplementary Figures) 43 3 ABSTRACT 44 Background: Seasonal influenza virus infection is a significant cause of morbimortality 45 in the elderly. However, there is poor vaccine efficacy in this population due to 46 immunosenescence. We aimed to explore several homeostatic parameters in the elderly 47 that could impact influenza vaccine responsiveness. 48 Methods: Subjects (>60 years old) who were vaccinated against influenza virus were 49 included, and the vaccine response was measured by a haemagglutination inhibition 50 (HAI) test. At baseline, peripheral CD4 and CD8 T-cells were phenotypically 51 characterized. Thymic function and the levels of different inflammation-related 52 biomarkers, including Lipopolysaccharide Binding Protein (LBP) and anti-53 cytomegalovirus (CMV) IgG antibodies, were also measured. 54 Results: Influenza vaccine non-responders showed a higher frequency of regulatory T-55 cells (Tregs) before vaccination than responders (1.49 [1.08-1.85] vs. 1.12 [0.94-1.63], 56 respectively, p=0.061), as well as higher expression of the proliferation marker Ki67 in 57 Tregs and different CD4 and CD8 T-cell maturational subsets. The levels of 58 inflammation-related biomarkers correlated with the frequencies of different 59 proliferating T-cell subsets and with thymic function (e.g., thymic function with D-60 dimers, r=-0.442, p=0.001). 61 Conclusions: Age-related homeostatic dysregulation involving the proliferation of CD4 62 and CD8 T-cell subsets, including Tregs, was related to a limited responsiveness to 63 influenza vaccination and a higher inflammatory status in a cohort of elderly people. 64 Keywords: Treg, Ki67, inflammation, thymic function, TREC 65 4 BACKGROUND 66 The seasonal influenza virus (flu) is a significant cause of morbidity and mortality in 67 older adults [1]. The World Health Organization (WHO) estimates that 3-5 million 68 cases of severe influenza illness and up to 650,000 deaths related to respiratory diseases 69 are linked to seasonal flu each year [2], with the highest mortality rates occurring in the 70 elderly [3]. Despite vaccination remaining the most effective approach for the 71 prevention of influenza infection and influenza-related complications, there is poor 72 vaccine efficacy in the elderly [4], which is due to the age-associated dysregulation of 73 immune function known as immunosenescence [5,6]. 74 Immunosenescence affects both the innate and adaptive branches of the immune system. 75 Thus, age-related alterations, such as those affecting Toll-like receptors [7], reduced 76 telomerase activity [8] and deficiencies in B and T-cell functions [9,10], have been 77 associated with influenza vaccine responsiveness. Moreover, elderly people exhibit a 78 chronic inflammatory status, called inflammaging, with increased levels of circulating 79 inflammatory mediators such as pro-inflammatory cytokines and acute phase proteins, 80 e.g., interleukin-6 (IL-6) and C-reactive protein (CRP), respectively, that disturb 81 vaccine responses [11], specifically, the influenza vaccine response [12,13]. Several 82 factors, mainly persistent stressors such as translocated microbial products (LPS) and 83 cytomegalovirus coinfection (CMV) but also the age-related increasing activation of the 84 coagulation system (D-dimers), have been proposed to contribute to this age-related 85 inflammation [14]. Interestingly, regarding the adaptive immune system, naïve T-cells 86 retain their proliferative capacity in both aged mice and humans [15] and naïve T-cell 87 proliferation can even be enhanced in the elderly because of the age-dependent loss of 88 thymic output [16-18]. This immunosenescence-related homeostatic dysregulation 89 (mainly the thymic output – compensatory peripheral T-cell proliferation axis) could 90 5 also be related to inflammaging and even affect immune competence in the elderly. 91 However, the potential role of this homeostatic dysregulation in vaccine responsiveness 92 is mostly unexplored. 93 The proliferation and cytokine secretion of CD4 and CD8 T-cells is regulated by 94 regulatory T-cells (Tregs), which are also involved in the suppression of antigen 95 presenting cells (APC) and B cells [19]. In fact, Tregs suppress the B cell 96 immunoglobulin class switching within germinal centres of human lymphoid tissue 97 [20]. Because of that observation, Tregs have been explored in several immunization 98 models in animals [21,22] and in humans [23-24]. Particularly, in the context of 99 influenza vaccination, despite Tregs being known to expand after vaccination, which 100 possibly attenuates the production of anti-influenza antibodies [26,27], the role of 101 baseline Tregs remains mostly unexplored. However, this is an interesting question 102 since Treg frequency increases with age [28], probably as another consequence of age-103 dependent homeostatic dysregulation [29,30]. 104 In the present work, we aimed to further explore the potential association between 105 several immune homeostatic parameters in the elderly, such as thymic function, T-cell 106 proliferation, Tregs and several inflammation- and coagulation-related markers, and 107 influenza vaccine responsiveness. 108 RESULTS 109 Rates of vaccine response 110 Sixty subjects were included in this study. The demographic and clinical characteristics 111 of these subjects are summarized in Table 1. Briefly, 24/60 (40%) of the subjects were 112 men, and the median age was 79 [70-87] years. Before vaccination, the subjects showed 113 a median CD4/CD8 T-cell ratio of 1.8 [1.2-2.3] and a broad range of thymic function 114 6 measured as an sj/-TREC ratio of 32 [0-50]. Seroprotection was present in 48/60 115 (80%) of the subjects before vaccination and in 59/60 (98%) of the subjects after 116 vaccination. A vaccine response was observed in 27/60 (45%) of the subjects. The 117 baseline and post-vaccination HAI titres for the whole group, as well as the titres for the 118 responder and non-responder groups, are shown in Figure S1. The timing of the post-119 vaccination sampling did not affect the HAI titres or the seroconversion-fold data (data 120 not shown). 121 Levels of inflammation-related markers according to vaccine responsiveness 122 The non-responders did not differ from the responders in terms of age, sex, CD4/CD8 123 ratio, sj/-TREC ratio or anti-cytomegalovirus (CMV) titre (Table 1). Interestingly, 124 several nonsignificant differences in inflammation-related markers were observed. 125 Specifically, the non-responders showed higher levels of D-dimers (875 [445-1425] vs 126 620 [438-918], respectively; p=0.082), a higher % neutrophils (62.8 [54.3-66.9] vs 58.0 127 [52.4-62.5], respectively; p=0.089) and a higher neutrophil to lymphocyte ratio (NLR) 128 (2.6 [1.7-3.0] vs 1.9 [1.5-2.4], respectively; p=0.056) than the responders. However, the 129 non-responders showed lower % lymphocytes (24.9 [22.4-31.9] vs 29.0 [25.0-34.1], 130 respectively; p=0.063) and % eosinophils (3.15 [1.73-4.45] vs 3.70 [3.20-4.20], 131 respectively; p=0.094). 132 Frequency of Tregs and Tregs expressing Ki67 according to vaccine 133 responsiveness 134 We explored the frequencies of total-Tregs and Treg subsets and the expression of 135 different activation, proliferation and suppression markers in relation to influenza 136 vaccine responsiveness (Figure 1 and Table S1). We observed a higher frequency of 137 total-Tregs in the non-responders compared with the responders; however, the 138 13 as a compensatory mechanism) are relevant to the underlying mechanisms of 287 progression to death in elderly people. 288 CONCLUSIONS 289 In summary, age-dependent homeostatic dysregulation involving the proliferation of 290 CD4 and CD8 T-cell subsets, including Tregs, was related to a reduced responsiveness 291 to influenza vaccination as well as to a higher inflammatory status in an elderly 292 population. Despite the statistical weakness of our results, our data support and extend 293 previous observations from ageing studies of other human T-cell subsets and suggest 294 that further research on the mechanisms underlying such relationships in the elderly 295 could help to find better strategies to produce a proper vaccine response against 296 influenza in this compromised population. Deepening this knowledge will also be useful 297 to further understand how immunosenescence limits immune capacities. 298 LIST OF ABBREVIATIONS 299 APC: Antigen Presenting Cells 300 CM: Central Memory 301 CMV: Cytomegalovirus 302 ELISA: Enzyme-Linked Immunosorbent Assays 303 EM: Effector Memory 304 ESR: Erythrocyte Sedimentation Rate 305 eTreg: Effector Treg 306 HA: Haemagglutinin 307 HAI: Haemagglutination Inhibition 308 HBV: Hepatitis B Virus 309 HIV: Human Immunodeficiency Virus 310 14 hsCRP: High Sensitivity C Reactive Protein 311 IL-6: Interleukin-6 312 IQR: Interquartile Ranges 313 LBP: Lipopolysaccharide Binding Protein 314 MCV: Mean Corpuscular Volume 315 MPV: Mean Platelet Volume 316 NLR: Neutrophil to Lymphocyte Ratio 317 nTreg: Naïve Treg 318 PBMCs: Peripheral Blood Mononuclear Cells 319 PLR: Platelet to Lymphocyte Ratio 320 RBCs: Red Blood Cells 321 RDE: Receptor Destroying Enzyme 322 RTEs: Recent Thymic Emigrants 323 sCD163: Soluble CD163 324 sj/-TREC: Signal Joint/ T-Cell Receptor Excision Circles 325 TemRA: Terminally Differentiated Effector Memory 326 Treg: Regulatory T-cell 327 WHO: World Health Organization 328 METHODS 329 Study design 330 We included elderly subjects from the Heliopolis Nursing Home, Seville, who were 331 going to be vaccinated against influenza virus during November 2015 (the 2015-2016 332 campaign). Among these subjects, those older than 60 years, without cognitive 333 impairment and able to sign the informed consent were included in this study. Subjects 334 treated with antitumour therapy or any treatment that could influence their immune 335 15 status (mainly corticosteroids) during the preceding 6 months were excluded. The 336 vaccination protocol (Figure S2) consisted of one intradermal dose of the trivalent 337 influenza vaccine for the Northern Hemisphere (Intanza 15 µg, Sanofi Pasteur MSD, 338 Lyon, France) with split and inactivated viruses of the strains: A/California/7/2009 339 H1N1pdm09, A/Switzerland/9715293/2013 H3N2 and B/Phuket/30731/2013 Yamagata 340 lineage. Blood samples were collected pre-vaccination (from 29 to 0 days before the 341 administration of the vaccine) and post-vaccination (from 12 to 33 days after 342 vaccination) and processed at the Institute of Biomedicine of Seville, Virgen del Rocío 343 University Hospital. Deaths occurring within one year after vaccination were recorded, 344 except for one subject who was lost to follow-up due to a residency change. The study 345 was approved by the Ethics Committee of the Virgen del Rocío University Hospital. 346 Haemagglutination inhibition (HAI) test 347 Influenza vaccine responses were measured at the Microbiology Service of the Virgen 348 de las Nieves University Hospital, Granada through an HAI test analysis. Pre-349 vaccination and post-vaccination sera were tested for HAI titres. The standardized 350 antigen for the HAI test was prepared using the 2015-2016 trivalent influenza vaccine 351 for the Northern Hemisphere (Influvac, Mylan Pharmaceuticals, Barcelona, Spain). The 352 standardized antigen contained 4 haemagglutinin (HA) units per 25 µl of each of the 353 following inactivated strains: A/California/7/2009 H1N1pdm09, 354 A/Switzerland/9715293/2013 H3N2 and B/Phuket/30731/2013 Yamagata lineage. The 355 HAI tests were performed with chicken red blood cells (RBCs) according to the WHO 356 standard procedures [31]. Briefly, serum samples were pre-treated with Receptor 357 Destroying Enzyme (RDE II Seiken, Denka Seiken Co Ltd, Tokyo, Japan) in order to 358 inactivate non-specific haemagglutination inhibitors according to the manufacturer’s 359 instructions. The RDE-treated sera were diluted 1:10 and then 25 µl was diluted 2-fold 360 16 in PBS and incubated at room temperature for 15 minutes with 25 µl of standardized 361 antigen. Then, 50 µL of standardized RBCs were added to each well and incubated for 362 30 minutes at room temperature. The HAI titre was the last dilution at which 363 haemagglutination was inhibited. Seroprotection was defined as an HAI titre 40. A 364 positive response was defined as a 4-fold or greater increase in the HAI titre between 365 the pre- and post-vaccination serum samples [43]. 366 Flow cytometry 367 Peripheral blood mononuclear cells (PBMCs) collected pre-vaccination were isolated 368 from fresh blood and cryopreserved until analysis. The characterization of peripheral 369 CD4 and CD8 T-cells was performed according to the distribution of their maturational 370 subsets [naïve (CD27+CD45RA+), central memory (CD27+CD45RA-), effector memory 371 (CD27-CD45RA-), terminally differentiated effector memory (TemRA) (CD27- 372 CD45RA+) and recent thymic emigrants (RTEs; naïve CD31+)]. Our gating strategy is 373 shown in Figure S3. We measured the expression of an activation marker (HLA-DR), a 374 senescence marker (CD57), an apoptosis susceptibility marker (CD95), a proliferation 375 marker (Ki67) and a suppression marker (CTLA-4). Representative FACS plots for 376 Ki67 staining are shown in Figure S4. We also identified total-Tregs (CD25hiFoxP3+), 377 naïve-Tregs (nTregs, CD45RA+FoxP3lo), effector-Tregs (eTregs, CD45RA-FoxP3hi) 378 and non-Tregs (CD45RA-FoxP3lo) as previously described by Miyara et al. [44]. We 379 studied the expression of the abovementioned activation, proliferation and suppression 380 markers and a functional marker (CD39) on these Treg subsets. 381 For immunophenotyping, PBMCs were thawed and stained with the following surface 382 antibodies: anti-CD31 PE-CF594, anti-CD56 BV510, anti-CD25 BV605, anti-CD45RA 383 BV650, anti-CD4 BV786, anti-CD3 APC-H7 (BD Biosciences, USA), anti-CD39 384 FITC, anti-CD57 PE-Cy7, anti-HLA-DR BV570, anti-CD95 BV711 and anti-CD27 385 17 AF700 (BioLegend, USA). For intracellular staining, the cells were then fixed and 386 permeabilized according to the manufacturer's instructions (FoxP3/Transcription Factor 387 Staining Buffer, eBioscience, USA) and intracellularly stained [anti-Ki67 PerCP-Cy5.5, 388 anti-FoxP3 PE and anti-CTLA4 APC antibodies (BD Biosciences, USA)]. In each 389 experiment, isotype controls for the antibodies specific for CD39, CD31, CD25, CD95, 390 Ki67, FoxP3 and CTLA4 were included. The identification of viable cells was 391 performed using LIVE/DEAD fixable Aqua Blue Dead Cell Stain (Life Technologies, 392 USA). One million cells from each sample were stained, and a minimum of 100,000 393 total lymphocyte events were acquired. Flow cytometry was performed on an LSR 394 Fortessa (BD Biosciences, USA). Analyses were performed using FlowJo version 9.3 395 (TreeStar). 396 sj/-TREC ratio quantification 397 Thymic function was determined with pre-vaccination PBMC DNA by quantifying the 398 sj/-TREC ratio with a technique previously optimized by our group [43], with minor 399 modifications. A schematic representation of the sj/-TREC ratio quantification 400 protocol is shown in the original report [45]. Briefly, in the same PCR reaction tube, the 401 six dJ-TREC from cluster one were amplified, whereas the sj-TREC was amplified in 402 a different PCR reaction tube. Twenty amplification rounds were performed to 403 guarantee an accurate quantification at the real-time PCR step. All amplicons, dJ and 404 sj-TREC, were then amplified together in a second round of PCR using the 405 LightCycler 480 System (Roche, Mannheim, Germany). We defined an sj/-TREC 406 ratio value lower than 10 as thymic function failure, since we previously found that this 407 cutoff could forecast survival in a cohort of elderly people [39] as well as other clinical 408 endpoints such as cytomegalovirus disease after solid organ transplantation [46] or HIV 409 disease progression [47]. 410 18 Laboratory measurements and assaying soluble biomarkers 411 All determinations were performed with pre-vaccination samples. Absolute numbers of 412 CD4+ and CD8+ T-cells and percentages of lymphocytes, monocytes, neutrophils, 413 basophils, eosinophils and platelets were determined with an Epics XL-MCL flow 414 cytometer (Beckman-Coulter, Brea, California). The high sensitivity C-reactive protein 415 (hsCRP) and 2-microglobulin levels were determined with an immunoturbidimetric 416 sera assay using Cobas 701 (Roche Diagnostics, Mannheim, Germany). The D-dimer 417 levels were measured with an automated latex enhanced immunoassay using plasma 418 samples (HemosIL D-Dimer HS 500, Instrumentation Laboratory, Bedford, 419 Massachusetts). The mean corpuscular volume (MCV), mean platelet volume (MPV) 420 and erythrocyte sedimentation rate (ESR) were determined with a Sysmex XN-200 421 analyser (Sysmex, Kobe, Japan). The platelet to lymphocytes ratio (PLR) and neutrophil 422 to lymphocyte ratio (NLR) were calculated as inflammatory indices. 423 Serum and plasma samples were aliquoted and stored at -20°C until subsequent analysis 424 of the levels of Interleukin-6 (IL-6), soluble CD163 (sCD163), and Lipopolysaccharide 425 Binding Protein (LBP) as well as anti-CMV IgG antibody titres by colorimetric 426 enzyme-linked immunosorbent assays (ELISA) according to manufacturer’s 427 instructions. Specifically, the following kits were used: IL-6 (Quantikine HS ELISA, 428 R&D Systems, Minneapolis, Minnesota), sCD163 (MacroCD163TM, IQProducts, 429 Groningen, The Netherlands), LBP (Human ELISA kit, Hycult Biotech, Uden, The 430 Netherlands), and anti-CMV IgG (Cytomegalovirus IgG ELISA Kit, Abnova, Taiwan, 431 China). 432 Statistical analysis 433 Continuous variables were recorded as medians and interquartile ranges [IQR], and 434 categorical variables were recorded as the number of cases and percentages. 435 19 Comparisons among groups were made using the nonparametric Mann–Whitney U-test 436 for continuous variables and the χ2 or Fisher exact test for categorical variables. 437 Correlations were assessed using Spearman's rho correlation coefficient. A p value 438 <0.05 was considered statistically significant. Statistical analyses were performed using 439 SPSS software (version 22; IBM SPSS, Chicago, USA), and graphs were generated 440 using Prism (version 5, GraphPad Software, Inc.). 441 DECLARATIONS 442 - ETHICS APPROVAL AND CONSENT TO PARTICIPATE 443 This study was approved by the Ethics Committee of the Virgen del Rocío University 444 Hospital, and all subjects included in the study signed an informed consent form to 445 participate. 446 - CONSENT FOR PUBLICATION 447 Not applicable 448 - AVAILABILITY OF DATA AND MATERIAL 449 The corresponding author has taken custody of all data generated during this work. The 450 FlowJo files generated during flow cytometry experiments as well as the clinical 451 analytics and the database generated for statistical analyses are available upon request, 452 respecting individual data protection. 453 - COMPETING INTERESTS 454 The authors declare that they have no competing interests. 455 - FUNDING 456 20 This work was supported by grants from the Fondo de Investigación 457 Sanitaria (FIS; PI14/01693), which is co-funded by Fondos Europeos para el Desarrollo 458 Regional (FEDER) and the Junta de Andalucía, Consejería de Economía, Innovación, 459 Ciencia y Empleo (Proyecto de Investigación de Excelencia; CTS2593). The Spanish 460 AIDS Research Network of Excellence also supported this study (RD16/0025/0019). 461 YM.P was supported by the Consejería de Salud y Bienestar Social of Junta de 462 Andalucía through the ‘‘Nicolás Monardes’’ programme (C-0013-2017). The funders 463 had no role in study design, data collection and interpretation, or the decision to submit 464 the work for publication. 465 - AUTHORS’ CONTRIBUTIONS 466 I. H-F was globally responsible for sample handling, performed flow cytometry and 467 ELISA determinations, analysed data and wrote the draft. I. R-S performed flow 468 cytometry and ELISA determinations and helped with data analysis. A. A-R, A C and B 469 S performed haematological determinations. MI G, R R and J C assisted participants 470 during the vaccination protocol and collected samples. M De L-R performed thymic 471 function determinations. S S-G, M P-R and JM N-M performed vaccine response titre 472 determinations. M L and YM P designed and supervised the study. YM P conceived the 473 study and advised the data interpretation and writing. All authors critically reviewed the 474 final manuscript. 475 - ACKNOWLEDGEMENTS 476 The authors express their most sincere thanks to all the subjects included in this study. 477 We also thank Mª Antonia Abad and Mª del Mar Rodríguez for their technical 478 assistance and Juan Manuel Praena for assistance with the statistical analysis. 479 21 - AUTHORS' INFORMATION (OPTIONAL) 480 Not applicable 481 REFERENCES 482 1. Gasparini R, Amicizia D, Lai PL, Panatto D. Clinical and socioeconomic impact of 483 seasonal and pandemic influenza in adults and the elderly. Hum Vaccin 484 Immunother. 2012;8:21-28. 485 2. World Health Organization: Influenza seasonal. http://www.who.int/en/news-486 room/fact-sheets/detail/influenza-(seasonal). Accessed 27 August 2018. 487 3. Iuliano AD, Roguski KM, Chang HH, Muscatello DJ, Palekar R, Tempia S et al. 488 Lancet. Estimates of global seasonal influenza-associated respiratory mortality: a 489 modelling study. 2018;391:1285-1300. 490 4. Goodwin K, Viboud C, Simonsen L. Antibody response to influenza vaccination in 491 the elderly: a quantitative review. Vaccine. 2006;24:1159-1169. 492 5. Haq K, McElhaney JE. Immunosenescence: influenza vaccination and the elderly. 493 Curr Opin Immunol. 2014;29C:38-42. 494 6. Goronzy JJ, Weyand CM. Understanding immunosenescence to improve responses to 495 vaccines. Nat Immunol. 2013;14:428-436. 496 7. Panda A, Qian F, Mohanty S, van Duin D, Newman FK, Zhang L et al. Age-497 associated decrease in TLR function in primary human dendritic cells predicts influenza 498 vaccine response. J Immunol. 2010;184:2518-2527. 499 22 8. Najarro K, Nguyen H, Chen G, Xu M, Alcorta S, Yao X et al. Telomere length as an 500 indicator of the robustness of B- and T-cell response to influenza in older adults. J 501 Infect Dis. 2015;212:1261-1269. 502 9. Frasca D, Blomberg BB. B cell function and influenza vaccine responses in healthy 503 aging and disease. Curr Opin Immunol. 2014;29:112–118 504 10. Saurwein-Teissl M, Lung TL, Marx F, Gschösser C, Asch E, Blasko I et al. Lack of 505 antibody production following immunization in old age: association with 506 CD8(+)CD28(-) T-cell clonal expansions and an imbalance in the production of Th1 507 and Th2 cytokines. J Immunol. 2002;168:5893-5899. 508 11. Pinti M, Appay V, Campisi J, Frasca D, Fülöp T, Sauce D et al. Aging of the 509 immune system – focus on inflammation and vaccination. Eur J 510 Immunol. 2016;46:2286-2301 511 12. Trzonkowski P, Myśliwska J, Szmit E, Wieckiewicz J, Lukaszuk K, Brydak LB et 512 al. Association between cytomegalovirus infection, enhanced proinflammatory response 513 and low level of anti-hemagglutinins during the anti-influenza vaccination--an impact of 514 immunosenescence. Vaccine. 2003;21:3826-3836. 515 13. Parmigiani A, Alcaide ML, Freguja R Pallikkuth S, Frasca D, Fischl MA, Pahwa S. 516 Impaired Antibody Response to Influenza Vaccine in HIV-Infected and Uninfected 517 Aging Women Is Associated with Immune Activation and Inflammation. PloS one. 518 2013;8:e79816. 519 14. Franceschi C, Campisi J. Chronic inflammation (inflammaging) and its potential 520 contribution to age-associated diseases. J Gerontol A Biol Sci Med Sci. 2014;69:S4-9. 521 29 Protein; hsCRP, high sensitivity C-Reactive Protein; B2M, 2-microglobulin; sCD163, 635 soluble CD163; MCV, mean corpuscular volume; MPV, mean platelet volume; ESR, 636 erythrocyte sedimentation rate; PLR, platelet to lymphocyte ratio; and NLR, neutrophil 637 to lymphocyte ratio. (#) Thymic failure is defined as an sj/ TREC ratio<10. 638 FIGURE LEGENDS 639 Figure 1. Characterization of Treg subsets in relation to the response to the 640 influenza vaccine. a-d) Frequencies of Treg subsets. e-h) Frequencies of Treg subsets 641 expressing the proliferation marker Ki67+. A total of 60 subjects, with a median age of 642 79 [70-87] years old (median [IQR]). Comparisons between the groups of vaccine non-643 responders (n=33) and responders (n=27) were made using the nonparametric Mann–644 Whitney U test. Variables with a p value <0.05 were considered statistically significant 645 and are shown in bold. Note: nTreg, naïve-Treg; and eTreg, effector-Treg. 646 Figure 2. Maturational subsets of CD4+ and CD8+ T-cells expressing Ki67+. a-d) 647 Frequencies of CD4+ maturational subsets expressing the proliferation marker Ki67+. e-648 h) Frequencies of CD8+ maturational subsets expressing the proliferation marker Ki67+. 649 Comparisons between the groups of vaccine non-responders (n=33) and responders 650 (n=27) were made using the nonparametric Mann–Whitney U test. Variables with a p 651 value <0.05 were considered statistically significant and are shown in bold. Note: CM, 652 central memory; EM, effector memory; and TemRA, terminally differentiated effector 653 memory. 654 Figure 3. Correlations between the sj/ TREC ratio and inflammation-related 655 biomarkers. a) Correlation between sj/ TREC and D-dimers. b) Correlation between 656 sj/ TREC and the ESR. c) Correlation between sj/ TREC and the PLR. d) Correlation 657 30 between sj/ TREC and hsCRP. A total of 60 subjects. Correlations were assessed using 658 Spearman's rho correlation coefficient. Variables with a p value <0.1 are shown in 659 italics. Variables with a p value <0.05 were considered statistically significant and are 660 shown in bold. Note: ESR, erythrocyte sedimentation rate; PLR, platelets to lymphocyte 661 ratio; and hsCRP, high sensitivity C-Reactive Protein. 662 ADDITIONAL MATERIAL 663 Table S1. Characterization of Treg subsets in relation to the response to the 664 influenza vaccine. 665 Parameter Non-Responders N=33 Responders N=27 p total-Treg 1.49 [1.08-1.85] 1.12 [0.94-1.63] 0.061 HLADR+ 12.30 [7.51-17.60] 11.30 [9.48-14.50] 0.888 Ki67+ 16.91 [11.58-19.95] 14.75 [11.55-16.88] 0.204 CTLA4+ 60.56 [49.41-69.89] 56.14 [49.29-60.96] 0.262 CD39+ 76.81 [43.54-85.85] 65.90 [45.46-79.59] 0.547 nTreg 6.42 [4.31-8.57] 5.54 [3.55-8.30] 0.336 HLADR+ 0.80 [0.48-1.11] 0.87 [0.47-1.04] 0.795 Ki67+ 38.90 [19.20-42.85] 19.50 [16.10-35.60] 0.025 CTLA4+ 6.57 [5.85-8.00] 6.57 [6.06-7.92] 0.797 CD39+ 27.90 [12.60-42.35] 35.30 [12.90-42.20] 0.733 eTreg 2.86 [1.84-4.33] 2.87 [2.12-4.16] 0.873 HLADR+ 9.270 [4.470-14.60] 6.81 [4.59-11.30] 0.705 Ki67+ 10.90 [6.46-19.00] 10.00 [7.52-12.90] 0.683 CTLA4+ 48.20 [33.65-56.25] 37.00 [31.20-48.20] 0.152 CD39+ 48.00 [31.40-61.90] 43.00 [24.60-60.70] 0.499 nonTreg 20.29 [14.84-26.52] 21.18 [17.83-30.95] 0.353 HLADR+ 2.06 [1.19-3.56] 1.59 [1.35-3.24] 0.688 Ki67+ 38.50 [24.95-44.10] 27.70 [17.90-40.40] 0.053 CTLA4+ 10.60 [7.65-15.70] 9.20 [7.93-12.30] 0.365 CD39+ 33.20 [21.85-39.85] 24.00 [17.00-42.90] 0.305 31 Percentage of cells expressing each marker among the indicated subset. Continuous 666 variables are expressed as median values [IQR]. Comparisons between the groups were 667 made using the nonparametric Mann–Whitney U test. Variables with a p value <0.1 are 668 shown in italics. Variables with a p value <0.05 were considered statistically significant 669 and are shown in bold. Note: nTreg, naïve-Treg; and eTreg, effector-Treg. 670 671 32 Table S2. Comparison of CD4 and CD8 T-cell subsets in groups defined by the 672 vaccine response to the influenza vaccine. 673 Parameter Non-Responders N=33 Responders N=27 p CD4 CD4 RTE 52.20 [39.95-58.65] 53.00 [43.30-66.00] 0.199 CD4 naïve 28.50 [18.45-43.75] 22.70 [14.40-43.60] 0.385 CD4 CM 32.60 [22.25-41.35] 34.00 [29.50-45.20] 0.256 CD4 EM 32.00 [16.75-39.20] 27.70 [18.90-43.10] 0.761 CD4 TemRA 2.59 [1.45-5.83] 1.94 [1.00-4.85] 0.418 CD4 HLADR+ 1.24 [0.76-2.52] 1.19 [0.74-2.18] 0.923 CD4 Ki67+ 2.30 [1.85-2.80] 2.02 [1.56-2.68] 0.237 CD4 CD57+ 10.49 [5.88-21.22] 9.67 [4.27-18.04] 0.377 CD4 CD95+ 66.20 [49.15-80.50] 70.80 [53.40-86.20] 0.645 CD4 CTLA4+ 8.035 [6.38-10.68] 7.98 [6.90-9.58] 0.700 CD8 CD8 RTE 96.20 [93.85-97.85] 95.50 [90.00-98.70] 0.824 CD8 naïve 6.985 [4.80-11.40] 6.08 [3.50-8.82] 0.216 CD8 CM 15.70 [9.455-23.15] 19.20 [12.90-27.30] 0.373 CD8 EM 25.60 [18.65-36.05] 24.30 [17.40-35.30] 0.778 CD8 TemRA 33.90 [26.75-55.55] 35.30 [23.20-52.90] 0.677 CD8 HLADR+ 2.53 [1.47-4.07] 3.14 [1.76-4.21] 0.325 CD8 Ki67+ 9.48 [7.94-12.05] 9.66 [6.55-12.35] 0.325 CD8 CD57+ 55.00 [40.50-70.10] 52.70 [39.80-66.00] 0.744 CD8 CD95+ 97.80 [96.35-98.30] 97.90 [96.18-98.83] 0.609 Frequencies of the maturational subsets and the percentage of cells expressing each 674 marker among the CD4 and CD8 T-cell subsets. Continuous variables are expressed as 675 median values [IQR]. Comparisons between the groups were made using the 676 nonparametric Mann–Whitney U test. Variables with a p value <0.05 were considered 677 statistically significant and are shown in bold. Note: RTE, recent thymic emigrants; 678 33 CM, central memory; EM, effector memory; and TemRA, terminally differentiated 679 effector memory. 680 34 Table S3. Associations among different inflammation-related and haematological parameters and the expression of Ki67 in T-cell 681 subsets. 682 LBP (ng/mL) hsCRP (mg/L) B2M (µg/mL) DD (µg/L) % Lymph % Mono % Neutro % Baso % Eosino Platelets (x10e9/L) MCV (fL) MPV (fL) PLR NLR % total- Treg 0.329 0.013 0.235 0.071 -0.241 0.064 -0.221 0.089 % nTreg Ki67+ 0.254 0.059 -0.217 0.095 0.430 0.001 -0.223 0.087 -0.550 <0.001 % eTreg Ki67+ 0.301 0.020 0.254 0.059 0.269 0.038 -0.242 0.062 % nonTreg Ki67+ -0.227 0.081 0.401 0.001 -0.243 0.063 -0.411 0.001 0.220 0.091 % CD4 Ki67+ 0.332 0.010 0.446 0.001 0.264 0.041 0.278 0.031 % CD4 Naïve Ki67+ -0.266 0.040 0.494 <0.001 0.291 0.024 -0.223 0.087 -0.510 <0.001 0.247 0.057 % CD4 CM Ki67+ -0.233 0.073 0.225 0.084 0.424 0.001 -0.249 0.057 0.239 0.066 -0.415 0.001 0.225 0.083 % CD4 EM Ki67+ -0.310 0.016 0.307 0.017 0.425 0.001 -0.233 0.076 0.249 0.055 -0.470 <0.001 0.239 0.066 0.309 0.016 % CD4 TemRA Ki67+ 0.295 0.029 0.360 0.005 -0.224 0.090 0.231 0.079 -0.469 <0.001 % CD8 Ki67+ 0.277 0.041 -0.219 0.096 % CD8 Naïve Ki67+ 0.269 0.045 0.397 0.002 0.217 0.095 -0.297 0.021 -0.517 <0.001 0.242 0.062 35 % CD8 CM Ki67+ 0.406 0.001 0.235 0.071 -0.298 0.021 -0.531 <0.001 0.231 0.076 % CD8 EM Ki67+ 0.224 0.088 0.408 0.001 0.267 0.039 -0.327 0.011 -0.551 <0.001 0.217 0.096 % CD8 TemRA Ki67+ 0.434 0.001 0.247 0.057 -0.303 0.019 -0.550 <0.001 36 Correlations were assessed using Spearman's rho correlation coefficient. Variables with a p value <0.1 are shown in italics. Variables with a p value <0.05 were considered statistically significant and are shown in bold. N=60. Note: LBP, Lipopolysaccharide Binding Protein; hsCRP, high sensitivity C-reactive protein; B2M, 2-microglobulin; DD, D-Dimers; Lymph, lymphocytes; Mono, monocytes; Neutro, neutrophils; Baso, basophils; Eosino, eosinophils; MCV, mean corpuscular volume; MPV, mean platelet volume; PLR, platelet to lymphocyte ratio; NLR, neutrophils to lymphocyte ratio; nTreg, naïve-Treg; eTreg, effector-Treg; CM, central memory; EM, effector memory; and TemRA, terminally differentiated effector memory. Table S4. Inflammation-related biomarkers and Ki67 expression in the T-cells of the subjects who died during the follow-up-year. Death (YES) N=6 Death (NO) N=53 p sj/ TREC ratio 0 [0-0] 34 [10-51] 0.001 Thymic failure (#) 6 (100) 13 (25) <0.001 hsCRP (mg/L) 5.10 [3.70-7.58] 2.60 [1.25-4.30] 0.012 PLR 152 [152-229] 116 [87-166] 0.318 NLR 2.75 [2.30-3.57] 2.12 [1.63-2.85] 0.153 β2-microglobulin (µg/mL) 3.50 [2.45-5.40] 2.50 [2.03-3.30] 0.076 % nTreg Ki67+ 49.65 [34.78-51.15] 29.60 [16.70-39.65] 0.007 % eTreg Ki67+ 18.75 [11.49-23.23] 9.96 [6.50-13.95] 0.034 % nonTreg Ki67+ 45.05 [34.93-51.60] 36.10 [20.65-41.20] 0.025 % CD4 Ki67+ 2.65 [2.28-3.20] 2.08 [1.72-2.69] 0.096 % CD4 N Ki67+ 32.65 [20.39-42.28] 21.70 [12.00-30.35] 0.160 % CD4 CM Ki67+ 39.30 [29.70-48.78] 28.20 [19.15-36.90] 0.068 % CD4 EM Ki67+ 39.30 [30.80-44.05] 28.50 [19.75-35.60] 0.041 % CD4 TemRA Ki67+ 39.60 [30.93-42.73] 25.50 [18.75-33.30] 0.033 % CD8 Ki67+ 7.60 [4.20-12.35] 9.86 [7.79-12.18] 0.242 % CD8 N Ki67+ 47.55 [32.12-55.28] 15.20 [8.52-42.45] 0.039 % CD8 CM Ki67+ 38.15 [26.11-49.18] 17.70 [14.45-34.10] 0.057 % CD8 EM Ki67+ 42.15 [30.74-48.70] 20.40 [15.75-36.60] 0.020 % CD8 TemRA Ki67+ 41.05 [24.07-41.95] 18.70 [12.30-32.55] 0.032 37 Comparisons between the groups were made using the nonparametric Mann–Whitney U test. Variables with a p value <0.1 are shown in italics. Variables with a p value <0.05 were considered statistically significant and are shown in bold. Note: hsCRP, high sensitivity C-Reactive Protein; PLR, platelets to lymphocyte ratio; NLR, neutrophils to lymphocyte ratio; nTreg, naïve-Treg; eTreg, effector-Treg; N, naïve; CM, central memory; EM, effector memory; and TemRA, terminally differentiated effector memory. (#) Thymic failure is defined as an sj/ TREC ratio<10. ADDITIONAL FIGURE LEGENDS Figure S1. Baseline and post-vaccination HAI titres. Data from the Haemagglutination Inhibition (HAI) test, which was performed at baseline (circles) and post-vaccination (squares), are shown as data for the whole population (n=60) and the groups of influenza vaccine non-responders (n=33) and responders (n=27). Median [IQR] values are included in the data cells below each case. Figure S2. Protocol. Subjects were vaccinated with one intradermal dose of the trivalent influenza vaccine Intanza (15 µg). Blood samples were collected prevaccination (from 29 to 0 days before the administration of the vaccine) and postvaccination (from 12 to 33 days after vaccination). HAI titres were measured in the prevaccination and post-vaccination samples. T-cell immunophenotypes and soluble biomarkers were measured in the pre-vaccination samples. Deaths occurring within one year after vaccination were recorded. Figure S3. Gating strategy for the T-cell subsets. The gating strategy for the different CD4 T-cell subsets (naïve, central memory, effector memory and TemRA) depending on their expression of CD27 and CD45RA is represented. 38 Figure S4. Representative FACS plots of ki67 staining. Treg subsets (naïve and effector Treg) and the non-Treg subsets were gated on CD4 T-cells depending on their expression of CD45RA and FoxP3. Then, the percentage of Ki67+ T-cells from each subset was quantified by using isotype control as it is shown in representative histograms. Supplementary Figure 4 n-Treg e-Treg non-Treg 49.9 55.3 24.4 Isotype control Ki67 expression 65 4.2. Association between a suppressive combined antiretroviral therapy containing maraviroc and the hepatitis B virus vaccine response Herrero-Fernández I, Pacheco YM, Genebat M, Rodriguez- Méndez MDM, Lozano MDC, Polaino MJ, Rosado-Sánchez I, Tarancón-Diez L, Muñoz-Fernández MÁ, Ruiz-Mateos E, Leal M. Antimicrob Agents Chemother 2017; 62: e02050-17 Association between a Suppressive Combined Antiretroviral Therapy Containing Maraviroc and the Hepatitis B Virus Vaccine Response Inés Herrero-Fernández, a Yolanda M. Pacheco, a Miguel Genebat, a María del Mar Rodriguez-Méndez, a María del Carmen Lozano, b María José Polaino, a Isaac Rosado-Sánchez, a Laura Tarancón-Diez, a María Ángeles Muñoz-Fernández, c,d Ezequiel Ruiz-Mateos, a Manuel Leal a a Laboratory of Immunovirology, Institute of Biomedicine of Seville (IBiS), Virgen del Rocío University Hospital/ CSIC/University of Seville, Seville, Spain b Microbiology Service, Virgen del Rocío University Hospital, Seville, Spain c Molecular Immunobiology Laboratory, General Universitary Hospital Gregorio Marañon, Health Research Institute Gregorio Marañon, Spanish HIV HGM BioBank, Madrid, Spain d Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain ABSTRACT The response to the HBV vaccine in HIV-infected patients is deficient. Our aim was to analyze whether a suppressive combined antiretroviral treatment (cART) containing maraviroc (MVC-cART) was associated with a better response to HBV vaccine. Fifty-seven patients on suppressor cART were administered the HBV vaccine. The final response, the early response, and the maintenance of the response were assessed. An anti-HBs titer of ⬎10 mIU/ml was considered a positive response. A subgroup of subjects was simultaneously vaccinated against hepatitis A virus (HAV). Lineal regression analyses were performed to determine demographic, clinical, and immunological factors associated with the anti-HBs titer. Vaccine response was achieved in 90% of the subjects. After 1 year, 81% maintained protective titers. Only simultaneous HAV vaccination was independently associated with the magnitude of the response in anti-HBs titers, with a Pvalue of 0.045 and a regression coefficient (B) [95% confident interval (CI)] of 236 [5 to 468]. In subjects ⱕ50 years old (n⫽42), MVC-cART was independently associated with the magnitude of the response (P⫽0.009; B [95% CI], 297 [79 to 516]) together with previous vaccination and simultaneous HAV vaccination. High rates of HBV vaccine response can be achieved by revaccination, simultaneous HAV vaccination, and administration of cARTs including MVC. MVC may be considered for future vaccination protocols in patients on suppressive cART. KEYWORDS maraviroc, vaccine, HIV infection, combined antiretroviral treatment, hepatitis A virus, hepatitis B virus HIV accelerates viral hepatitis-related liver damage, early cirrhosis, and end-stage liver disease (1, 2). Direct antiviral agents for hepatitis C virus (HCV) infection are paving the way for HCV eradication in HIV-coinfected patients. For hepatitis B virus (HBV) and hepatitis A virus (HAV), the vaccine is the most effective method to prevent the morbimortality associated with these infections, specially in HIV-infected populations. However, the response to HBV vaccine following the classic vaccination schedule in HIV-infected patients is deficient (3). In addition to the insufficient number of patients responding to these vaccines, in those who respond, the magnitude of protective antibody titers is low and is subsequently lost quickly, which probably restricts the protection of these patients in the long term (4). For this reason, interventions to improve the response to HBV vaccine in HIV-infected patients are urgently needed. Received 5 October 2017 Accepted 23 October 2017 Accepted manuscript posted online 30 October 2017 Citation Herrero-Fernández I, Pacheco YM, Genebat M, del Mar Rodriguez-Méndez M, del Carmen Lozano M, Polaino MJ, Rosado- Sánchez I, Tarancón-Diez L, Muñoz-Fernández MA, Ruiz-Mateos E, Leal M. 2018. Association between a suppressive combined antiretroviral therapy containing maraviroc and the hepatitis B virus vaccine response. Antimicrob Agents Chemother 62:e02050-17. https://doi.org/10 .1128/AAC.02050-17. Copyright © 2017 American Society for Microbiology. All Rights Reserved. Address correspondence to Yolanda M. Pacheco, [email protected]. I.H.-F., Y.M.P., E.R.-M., and M.L. contributed equally to this work. ANTIVIRAL AGENTS crossm January 2018 Volume 62 Issue 1 e02050-17 aac.asm.org 1Antimicrobial Agents and Chemotherapy Apart from controlling for traditional clinical factors associated with lower rates of response such as CD4 T-cell counts and viral load levels (5), the number of doses and the immunization schedule seem to be important too (6, 7). However, no attention has been paid regarding the type of antiretroviral treatment in relation to the rate and magnitude of response. We hypothesize that a combined antiretroviral treatment (cART) containing the CCR5 antagonist maraviroc (MVC) might increase the rates of response to HBV vaccine in HIV-infected patients on suppressive therapy (cART). In support of this hypothesis, we have observed that a switch from a suppressive MVC-free cART to an MVC-containing cART decreased the levels of inflammatory biomarkers (8). In addition, a beneficial profile in HIV disease progression markers such as soluble CD14 (sCD14) and T-cell immunosenescence has been observed in a cART including MVC (9). Finally, we have shown that MVC is able to reduce the frequency of regulatory T-cell (Tregs) (10) and also that a higher frequency of Tregs was associated with a lower rate and magnitude of response to HBV vaccine in HIV-infected patients (11). Altogether, these immunomodulatory effects of MVC may allow synchronized and coordinated innate and adaptive immune responses, which may facilitate a better response to vaccines. In fact, MVC has been shown to enhance meningococcal neoimmunization and improve the response to recall antigens, such as tetanus toxoid (12). Thus, the aim of the present work was to analyze whether a suppressive MVC-containing cART was associated with a better response to HBV vaccine in HIV-infected patients. RESULTS Characteristics of the study subjects. Fifty-seven HIV-infected subjects were included in the study, whose vaccination protocol is summarized in Fig. 1; three subjects were lost between month 6 and the year of follow-up. Demographic, clinical, and immunological characteristics of the subjects are summarized in Table 1. Briefly, most were men (79%) with a median age of 44 years. During the vaccination, 46% of the subjects were receiving a nucleoside-reverse transcriptase inhibitor (NRTI)-containing cART (NRTI-cART) consisting of two NRTIs and one protease inhibitor (PI) or two NRTIs and one non-NRTI. On the other hand, nearly one-half of the subjects were receiving a maraviroc-containing cART (MVC-cART) consisting of MVC and a boosted PI or MVC and two NRTIs. All subjects on an MVC-cART had been previously tested for CCR5-tropic virus. Approximately one-third of the cohort had been previously vaccinated against HBV, and one-third received simultaneous HBV and HAV vaccinations. Rates of vaccine response and antibody titers. The vaccine response (antiHBs ⬎10 mIU/ml at month 6) was observed in 51/57 (90%) subjects. The mean of anti-HB titers at month 6 in this cohort was 644 (101 to 1,000). Early responses were observed in 20/57 (35%) subjects after the first dose (month 1) and in 42/57 (74%) of the subjects after the second dose (month 3) (Fig. 2). Vaccine response was maintained in 44/54 (81%) subjects after 1 year of follow-up. Maraviroc-containing cART, previous vaccination, and simultaneous HAV vaccination were independently associated with the magnitude of the vaccine response in subjects younger than 50 years. The associations of different demo- FIG 1 Vaccination protocol. Subjects received 3 intramuscular (deltoid region) double doses (40 ␮ g) of the recombinant Engerix-B vaccine (GlaxoSmithKline, Brentford, United Kingdom) at 0, 1, and 3 months. The vaccine response was measured 6 months after the first dose. We measured the early response at months 1 and 3 after the first dose and the maintenance of the response 1 year after the first vaccination dose. Herrero-Fernández et al. Antimicrobial Agents and Chemotherapy January 2018 Volume 62 Issue 1 e02050-17 aac.asm.org 2 graphic, clinical, and immunological characteristics with the magnitude of the final response (anti-HBs titer) are shown in Table 2. In the unadjusted model, female sex and simultaneous HAV vaccination were associated with the antibody titer; in addition, MVC-cART was positively associated with the magnitude of the response with borderline significance. In fact, the antibody titer was significantly higher in the group of subjects receiving MVC-cART (1,000 [320 to 1,000] versus 704 [36 to 1,000] mIU/ml; P⫽ 0.048); these groups did not differ in the parameters of the study with the exception of age (42 [31 to 48] versus 47 [41 to 54] years; P⫽0.012) and NRTI-containing cART (5 [18] versus 21 [72] subjects; P⫽⬍0.001) (see Table S1 in the supplemental material). Only simultaneous HAV vaccination was independently associated with the magnitude of the response in anti-HBs titers (B), with a Pvalue of 0.045 and a regression coefficient (B) [95% confidence interval (CI)] of 236 [5 to 468], after adjusting for sex, CD4 T-cell counts, previous vaccination, and MVC-cART. Interestingly, we observed that the effect of maraviroc was enhanced in subjects younger than 50 years (n⫽42). After adjusting for sex, previous AIDS, previous vaccination, and simultaneous HAV vaccination, MVC-cART was independently TABLE 1 Demographic, clinical, and immunological characteristics of the studied subjects Characteristic, unit (nⴝ57) n(%) or median value (IQR) Male sex, n45 (79) Age, yr 44 (36–50) Nadir CD4 ⫹ T-cell count, cells/mm 3 257 (152–396) CD4 ⫹ T-cell count, cells/mm 3 696 (552–887) CD8 ⫹ T-cell count, cells/mm 3 647 (494–972) CD4 ⫹ /CD8 ⫹ ratio 1.02 (0.79–1.48) Time since diagnosis, mo 95 (42–217) Sexual transmission, n51 (90) Previous AIDS, n4 (7) Previous HCV coinfection, n7 (12) NRTI-containing cART, n26 (46) MVC-containing cART, n28 (49) Previous HBV vaccination, n a 19 (33) Simultaneous HAV vaccination, n19 (33) CCR5 WT/Δ32, n10 (17) hsCRP, mg/liter 1 (0.6–2) a Previous HBV vaccination data were available for only 56 subjects. FIG 2 Rates of vaccine response. Percentage of subjects responding at month 1 (early first-dose response), month 3 (early second-dose response), and month 6 (final-vaccination response) and maintaining response at 1 year after the first dose. The numbers inside the bars quantify the subjects with positive vaccine response among the subjects with the vaccine response data recorded. The vaccine response was considered positive when the anti-HBs titer was ⱖ10 mIU/ml. Maraviroc and HBV Vaccine Antimicrobial Agents and Chemotherapy January 2018 Volume 62 Issue 1 e02050-17 aac.asm.org 3 associated with the magnitude of the response (P⫽0.009; B [95% CI], 297 [79 to 516]) (Table 3). The early response was favored mainly by previous vaccination, and an additional effect of maraviroc was also observed in subjects younger than 50 years. We also explored the potential associations of study variables with the early response after the first dose (month 1) (see Table S2 in the supplemental material) and after the second dose (month 3) (see Table S3 in the supplemental material). At both time points, to be previously vaccinated against HBV was independently associated with the antibody titer (P⫽⬍0.001; B [95% CI], 363 [216 to 510]) and (P⫽⬍0.001; B [95% CI], 483 [295 to 670]), respectively. At month 3, the sex was also independently associated with the antibody titer (P⫽0.039; B [95% CI], ⫺228 [⫺444 to ⫺11]). When we restricted these analyses to subjects with age ⱕ50 years (n⫽42), in the early response to the second dose (month 3), we observed an independent association with the MVC-cART (P⫽0.041; B [95% CI], 206 [8 to 403]), together with that of the TABLE 2 Multivariate analysis of factors associated with the magnitude of HBV vaccine response a Characteristic, unit (nⴝ57) Unadjusted Pvalue; B (95% CI) Adjusted Pvalue; B (95% CI) Male sex, n(%) 0.013; ⴚ344 (ⴚ615–ⴚ74) 0.085; ⫺237 (⫺509–33) Age, yr 0.347; ⫺5(⫺17–6) Nadir CD4 ⫹ T-cell count, cells/mm 3 0.685; 0.15 (⫺0.60–0.90) CD4 ⫹ T-cell count, cells/mm 3 0.122; 0.37 (ⴚ0.10–0.85) 0.343; 0.21 (⫺0.24–0.67) CD8 ⫹ T-cell count, cells/mm 3 0.569; ⫺0.09 (⫺0.42–0.23) CD4 ⫹ /CD8 ⫹ ratio 0.265; 107 (⫺83–299) Time since diagnosis, mo 0.438; 0.46 (⫺0.73–1.66) Sexual transmission, n0.906; 22 (⫺357–402) Previous AIDS, n0.745; ⫺74 (⫺529–381) Previous HCV coinfection, n0.831; 38 (⫺317–393) NRTI containing cART, n0.193; 152 (⫺79–385) MVC containing cART, n0.060; 217 (ⴚ9–444) 0.107; 172 (⫺38–383) Previous HBV vaccination, n b (%) 0.119; 191 (ⴚ50–434) 0.070; 205 (⫺17–428) Simultaneous HAV vaccination, n(%) 0.022; 282 (43–521) 0.045; 236 (5–468) CCR5 WT/Δ32, n(%) 0.630; 73 (⫺232–379) hsCRP, mg/liter 0.354; 3 (⫺4–11) a Different factors were associated with the anti-HBs titer. All demographic, clinical, and immunological variables with a Pvalue of ⬍0.15 in the unadjusted model were included in the adjusted model and are shown in bold. Male sex, CD4 ⫹ T-cell count, MVC-containing cART, previous HBV vaccination, and simultaneous HAV vaccination were included in the multivariate model. Variables with a Pvalue of ⬍0.05 in the adjusted model were considered statistically significant and are shown in bold. b Previous HBV vaccination data were available for 56 of 57 subjects. TABLE 3 Multivariate analysis of factors associated with the magnitude of vaccine response in subjects younger than 50 years a Characteristic (nⴝ42) Unadjusted Pvalue; B (95% CI) Adjusted Pvalue; B (95% CI) Male sex, n(%) 0.055; ⴚ294 (ⴚ595–7) 0.132; ⫺196 (⫺454–61) Age, yr 0.583; ⫺4(⫺22–12) Nadir CD4 ⫹ T-cell count, cells/mm 3 0.349; 0.39 (⫺0.45–1.24) CD4 ⫹ T-cell count, cells/mm 3 0.195; 0.36 (⫺0.19–0.93) CD8 ⫹ T-cell count, cells/mm 3 0.448; ⫺0.14 (⫺0.54–0.24) CD4 ⫹ /CD8 ⫹ ratio 0.206; 138 (⫺79–356) Time since diagnosis, mo 0.251; 0.79 (⫺0.58–2.16) Sexual transmission, n(%) 0.579; 114 (⫺298–527) Previous AIDS, n(%) 0.074; ⴚ548 (ⴚ1152–56) 0.103; ⫺421 (⫺933–90) Previous HCV coinfection, n(%) 0.453; 154 (⫺257–566) NRTI containing cART, n(%) 0.351; 127 (⫺145–399) MVC containing cART, n(%) 0.008; 344 (96–593) 0.009; 297 (79–516) Previous HBV vaccination, n(%) b 0.082; 247 (ⴚ32–527) 0.047; 239 (3–474) Simultaneous HAV vaccination, n(%) 0.029; 287 (30–544) 0.037; 238 (15–462) CCR5 WT/Δ32, n(%) 0.398; 137 (⫺187–462) hsCRP, mg/liter 0.643; 2 (⫺6–10) a Different factors were associated with the anti-HBs titer in subjects less than 50 years. All demographic, clinical, and immunological variables with a Pvalue of ⬍0.15 in the unadjusted model were included in the adjusted model and are shown in bold. Male sex, previous AIDS, MVC-containing cART, previous HBV vaccination, and simultaneous HAV vaccination were included in the multivariate model. Variables with a Pvalue of ⬍0.05 in the adjusted model were considered statistically significant and are shown in bold. b Previous HBV vaccination data were available for 41 of 42 subjects. Herrero-Fernández et al. Antimicrobial Agents and Chemotherapy January 2018 Volume 62 Issue 1 e02050-17 aac.asm.org 4 previous vaccination (P⫽⬍0.001; B [95% CI], 539 [328 to 751]) (see Table S4 in the supplemental material). Female sex, previous vaccination, and simultaneous vaccination were associated with the maintenance of the antibody titer 1 year after the vaccination. We additionally analyzed the factors associated with the antibody titer maintenance after 1 year after the first dose (Table 4). Adjusted analyses revealed that female sex (P⫽ 0.013; B [95% CI], ⫺304 [⫺543 to ⫺66]), the previous vaccination (P⫽0.027; B [95% CI], 221 [26 to 415]), and the simultaneous HAV vaccination (P⫽0.008; B [95% CI], 292 [80 to 503]) were independently associated with the antibody titer. At this time point, the restriction to subjects with age ⱕ50 (n⫽42) did not show association of MVC-cART with the antibody titer maintenance (P⫽0.236; B [95% CI], 126 [⫺86 to 340]), whereas only the sex P⫽0.036; B (95% CI), ⫺267 (⫺518 to ⫺17) and the simultaneous HAV vaccination were independently associated (P⫽0.012; B [95% CI], 287 [67 to 506]). DISCUSSION In this work, we show that the type of cART, specifically those combinations including the CCR5 antagonist MVC, was associated with the magnitude of the high rate of response (90%) to the HBV vaccine in subjects younger than 50 years old. Additionally, we observed that simultaneous HAV vaccination, previous HBV vaccination, and female sex were also associated with the magnitude of response in the overall population of HIV-infected patients on suppressive cART. The association of the type of cART with the magnitude of the response only in subjects under 50 years old is especially important since the HIV-infected population is becoming older, with increased morbidity and mortality rates in people over 50 years old (13). The results presented here point to an early HBV vaccination in people under 50 years old, for which an additional strategy using a MVC-cART can be adopted to increase the magnitude of the response. The increased damage of immune function with age, also known as immunosenescence, decreases response to vaccines among other functions (13) and is accentuated or presents unique features in HIV infection scenario (14, 15). This age-related immune deterioration, in people older than 50, may compromise the beneficial immune profile exerted by MVC. The reasons why an MVC-cART was able to increase the early and final magnitudes of the HBV vaccine response are unknown but may reside in the very diverse immunomodulatory properties of this CCR5 antagonist. For instance, MVC has been shown to ameliorate graft-versus-host disease in the allogeneic hematopoietic stem cell transplantation TABLE 4 Analysis of factors associated with titer maintenance at 1 year a Characteristic (nⴝ54) Unadjusted Pvalue; B (95% CI) Adjusted Pvalue; B (95% CI) Male sex, n(%) 0.003; ⴚ396 (ⴚ647–ⴚ144) 0.013; ⴚ304 (ⴚ543–ⴚ66) Age, yr 0.431; ⫺4(⫺16–7) Nadir CD4 ⫹ T-cell count, cells/mm 3 0.354; ⫺0.32 (⫺1.02–0.37) CD4 ⫹ T-cell count, cells/mm 3 0.307; 0.23 (⫺0.22–0.68) CD8 ⫹ T-cell count, cells/mm 3 0.714; ⫺0.05 (⫺0.36–0.25) Ratio CD4 ⫹ /CD8 ⫹ 0.690; 36 (⫺145–218) Time since diagnosis, mo 0.228; 0.68 (⫺0.44–1.81) Sexual transmission, n(%) 0.919; 17 (⫺333–369) Previous AIDS, n(%) 0.897; ⫺27 (⫺449–394) Previous HCV coinfection, n(%) 0.842; ⫺33 (⫺362–296) NRTI containing cART, n(%) 0.327; 109 (⫺112–331) MVC containing cART, n(%) 0.325; 109 (⫺111–330) Previous HBV vaccination, n(%) b 0.073; 204 (ⴚ20–430) 0.027; 221 (26–415) Simultaneous HAV vaccination, n(%) 0.003; 350 (128–572) 0.008; 292 (80–503) CCR5 WT/Δ32, n(%) 0.838; ⫺29 (⫺313–255) hsCRP, mg/liter 0.256; 4 (⫺3–11) a Factors associated with the anti-HBs maintenance 1 year after the first dose. All demographic, clinical, and immunological variables with a Pvalue of ⬍0.15 in the unadjusted model were included in the adjusted model and are shown in bold. Male sex, previous HBV vaccination, and simultaneous HAV vaccination were included in the multivariate model. Variables with a Pvalue of ⬍0.05 in the adjusted model were considered statistically significant and are shown in bold. b Previous HBV vaccination data were available for 53 of 54 subjects. Maraviroc and HBV Vaccine Antimicrobial Agents and Chemotherapy January 2018 Volume 62 Issue 1 e02050-17 aac.asm.org 5 through inhibition of lymphocyte trafficking (16, 17). In addition, MVC suppressed tumor growth and induced cell apoptosis in the setting of acute lymphoblastic leukemia cells (18). In the scenario of HIV infection, in two clinical trials, MVC has been associated with improved duodenal immunity and reduction of bone loss in antiretroviral-naive HIV-infected patients (19, 20). Specifically, MVC may exert a better anti-inflammatory profile that may enhance the immune response after vaccination, as we have previously shown a decrease in levels of ␤ 2-microglobulin, soluble CD40L, and sCD14 after switching from an MVC-free cART to an MVC-cART (8). These data, together with the effect on reducing the Treg frequency (10, 21), which have been negatively associated with the rate of HBV response in patients on cART (11), support our results. Additionally, these results are in accordance with the beneficial effects of MVC in other settings of vaccination, such as the improved response to meningococcal neoimmunizations and the accelerated lymphoproliferation to tetanus toxoid boost (12). The absence of a CCR5 WT/Δ32 variant (see Materials and Methods) association with the magnitude of the HBV vaccine response suggests that this effect is drug specific or that the population size is too small to derive a definitive conclusion. Further research is encouraged to explore potential immunological mechanisms subjacent to the effect of MVC. In this study, we observed that double doses (40 ␮ g) on a rapid schedule (0, 1, and 3 months) of HBV vaccination are effective in reaching a high rate of response in HIV-infected patients on cART. This is in accordance to previous studies with all patients on cART that have shown improved response using a double dose of intramuscular vaccine compared to a single dose (20 ␮ g) (7, 22). Despite the fact that most of the patients were male, the characteristics of the included subjects, who had relatively high CD4 T-cell counts (⬎300 CD4 T-cells/mm 3 ) and undetectable viral loads because of cART, are in accordance with previous works (23–25) as determinants to reach a high rate of response in HIV-infected people. Regarding the factors associated with the magnitude of the response, simultaneous vaccination against HAV was associated not only with the final response but also with the maintenance of the magnitude after 1 year. There are limited data about this synergistic effect that suggest an increased rate of response in HIV-infected patients vaccinated with the combined HAV/HBV vaccine (26, 27). The reasons for the improved response, in our case after simultaneous vaccinations, are unknown at the moment. We can speculate about the increased amount of adjuvant, aluminum hydroxide, which was present in both vaccines and may favor innate and adaptive immune responses (28), and/or, alternatively, the increased immune response due to cross-reactivity of both types of viral antigens. In addition to this added value, HAV vaccination itself is very important now due to the recent outbreaks of HAV infection in men who have sex with men in HIV-infected populations (29). Globally, two other important factors associated with the magnitude of the response were revaccination and female sex. The revaccination has been effective in several cohorts of nonresponders (26, 27, 30, 31). There is no global recommendation about which schedule to use. Although we do not know if the 33% of patients with antiHBs titers of ⬍10 mUI/ml were nonresponders to the first vaccine or if these titers were lost during the follow-up, revaccination yielded a high response in these subjects. These results point out the presence of an anamnestic response favoring a higher magnitude of response in individuals revaccinated, which raises the question of whether these subjects are actually protected even with antiHBs levels of ⬍10 mIU/ml (32). In any case, these data support a periodical supervision of antiHBs titers to determine when new boosters are needed. The influence of sex in the magnitude of the response is in accordance with previous works involving HIV-infected patients (5, 7, 30); however, the mechanisms behind a high response in females are poorly understood, but the association of sex steroids with an improved innate and adaptive immunity in women may explain it (33). This study has some limitations. We were not able to analyze factors associated with the rate of response due to the fact that 90% of the subjects responded to the vaccine. Herrero-Fernández et al. Antimicrobial Agents and Chemotherapy January 2018 Volume 62 Issue 1 e02050-17 aac.asm.org 6 In addition, a deleterious effect has been attributed to NRTIs favoring a short telomere length in T cells (34), which has been associated with a paucity in vaccine response (35). Ideally, an MVC-cART with no NRTIs may be the best regimen for reaching the highest magnitude of response; however, that combination was not represented well enough to make any statistically significant contrast. In addition, we had to restrict the cohort to patients younger than 50 years old, and a larger cohort is needed to confirm our observations. In summary, an early and high magnitude of response after HBV vaccination can be achieved with revaccination, simultaneous HAV vaccine, and the administration of a suppressive cART containing MVC. This CCR5 antagonist may act as an immune booster beyond its antiviral effect, enabling a favorable immune milieu to increase the magnitude of the HBV vaccine response. The inclusion of this drug in further HBV vaccination protocols in HIV-infected patients on suppressive cART should be considered. MATERIALS AND METHODS Study participants. In this observational study, 57 HIV-infected subjects were included from the Assistance Vaccination Program from the Virgen del Rocío University Hospital, Seville, Spain, between April 2013 and July 2015. Inclusion criteria were (i) being on suppressive cART (undetectable viral load) during at least 6 months, (ii) having CD4 T-cell populations of ⬎300 cell counts/ ␮ l, (iii) having negative serology for HBsAg and anti-HBc, and (iv) having anti-HBs titers of ⬍10 mIU/ml. Written informed consent was obtained from all study participants. The study was approved by the ethics review board from the Virgen del Rocío University Hospital. Vaccination protocol. All subjects received 3 intramuscular (deltoid region) double doses (40 ␮ g) of the recombinant Engerix-B vaccine (GlaxoSmithKline, Brentford, United Kingdom) at 0, 1, and 3 months (Fig. 1). The vaccine response was measured 6 months after the first dose. Additionally, we measured the early response at months 1 and 3 after the first dose and the maintenance of the response 1 year after the first dose of vaccination. The vaccine response was considered positive when the anti-HBs titer was ⱖ10 mIU/ml. A group of subjects was simultaneously vaccinated at 0 and 6 months against HAV (simultaneous HAV vaccination) with two intramuscular doses of the vaccine Havrix-1440 (GlaxoSmithKline, Brentford, United Kingdom). This subgroup of subjects had negative serology for HAV. Clinical data and laboratory measurements. Demographic, clinical, and immunological data, including the presence of a 32-bp deletion in the CCR5 gene (CCR5Δ32) and high-sensitivity C-reactive protein (hsCRP) levels, were recorded in a database. Absolute CD4 and CD8 T cell counts were determined with an Epics XL-MCL flow cytometer (Beckman-Coulter, Brea, CA), according to the manufacturer’s instructions. Plasma HIV-1 RNA levels were measured using quantitative PCR (Cobas Ampliprep/Cobas TaqMan HIV-1 test; Roche Molecular Systems, Basel, Switzerland), according to the manufacturer’s protocol. The detection limit was 20 HIV-RNA copies/ml. Plasma samples were tested for HBV-related markers (HBsAg, anti-HBs, and anti-HBc) using an HBV enzyme-linked immunosorbent assay (ELISA; Siemens Healthcare Diagnosis, Malvern, PA). Qualitative PCR amplification was used for plasma HCV amplification (Cobas Amplicor; Roche Diagnosis); the test had a detection limit of 15 IU/ml. The hsCRP levels were determined with an immunoturbidimetric serum assay, using Cobas 701 (Roche Diagnostics, Mannheim, Germany). CCR5Δ32 deletion was assayed in DNA samples obtained from peripheral blood mononuclear cells (PBMCs) using the QIAamp blood kit (Qiagen) by PCR as previously described (36). Two fragments were generated, the wild-type (WT) fragment (185 bp) and the deleted fragment (153 bp), detected in 2% agarose gels. Heterozygosity (CCR5 WT/Δ32) was considered when the two fragments were present. Statistical analyses. Continuous variables were expressed as medians and interquartile ranges [IQR] and categorical variables as numbers and percentages. Comparisons between groups were made using the nonparametric Mann-Whitney Utest. Lineal regression analyses were performed to determine factors associated with the magnitude of response (absolute anti-HBs titer). Among covariates, we analyzed the type of cART as NRTI- or MVC-containing cART during the vaccination protocol. As a post hoc analysis, we restricted the study population to subjects younger than 50 years old. All demographic, clinical, and immunological variables with a Pvalue of ⬍0.15 in the unadjusted model were included in the adjusted model. Variables with a Pvalue of ⬍0.05 in the adjusted model were considered statistically significant. Statistical analyses were performed using SPSS software (version 22; IBM SPSS, Chicago, IL), and graphs were generated using Prism (version 5, GraphPad Software, Inc.). SUPPLEMENTAL MATERIAL Supplemental material for this article may be found at https://doi.org/10.1128/AAC .02050-17. SUPPLEMENTAL FILE 1, PDF file, 0.1 MB. Maraviroc and HBV Vaccine Antimicrobial Agents and Chemotherapy January 2018 Volume 62 Issue 1 e02050-17 aac.asm.org 7 ACKNOWLEDGMENTS This study was funded by an investigator-initiated research grant from ViiV Healthcare S.L. (grant number 205644) and by grants from the Fondo de Investigación Sanitaria (FIS; PI14/01693; PI16/01863), cofunded by Fondos Europeos para el Desarrollo Regional (FEDER) and the Junta de Andalucía, Consejería de Economía, Innovación, Ciencia y Empleo (Proyecto de Investigación de Excelencia; CTS2593). The Spanish AIDS Research Network of Excellence also supported this study (RD16/0025/0019). E.R.-M. and Y.M.P. were supported by the Fondo de Investigación Sanitaria through the ‘Miguel Servet’ programs (CPII014/00025 and CPII13/00037, respectively). Y.M.P. was supported by the Consejería de Salud y Bienestar Social of Junta de Andalucía through the ‘Nicolás Monardes’ program (C-0010/13). L.T.-D. was supported by Instituto de Salud Carlos III (PFIS program; FI00/00431). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. We express our most sincere thanks to all the subjects included in the study and to HIV Biobank of the Spanish AIDS Research Network. We thank also Magdalena Rodriguez and Marien Gutierrez Sancho for their assistance at the Day Care Hospital (Infectious Diseases Department), to Antonia Abad and Marta de Luna for their technical assistance, and to Juan Manuel Praena for statistical assistance. 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Serrano-Villar S, Sainz T, Ma ZM, Utay NS, Wook-Chun T, Mann S, Kashuba AD, Siewe B, Albanese A, Troia-Cancio P, Sinclair E, Somasunderam A, Yotter T, Deeks SG, Landay A, Pollard RB, Miller CJ, Moreno S, Asmuth DM. 2016. Effects of combined CCR5/integrase inhibitors-based regimen on mucosal immunity in HIV-infected patients naïve to antiret- Herrero-Fernández et al. Antimicrobial Agents and Chemotherapy January 2018 Volume 62 Issue 1 e02050-17 aac.asm.org 8 Herrero‑Fernándezetal. J Transl Med (2018) 16:238 https://doi.org/10.1186/s12967‑018‑1617‑1 RESEARCH Improved CD4 T cell profile inHIV‑infected subjects onmaraviroc‑containing therapy isassociated withbetter responsiveness toHBV vaccination Inés Herrero-Fernández1, Isaac Rosado-Sánchez1, Miguel Genebat1, Laura Tarancón-Díez1, María Mar Rodríguez-Méndez1, María Mar Pozo-Balado1, Carmen Lozano2, Ezequiel Ruiz-Mateos1, Manuel Leal1,3† and Yolanda M. Pacheco1*† Abstract Background: Maraviroc-containing combined antiretroviral therapy (MVC-cART) improved the response to the hepatitis B virus (HBV) vaccine in HIV-infected subjects younger than 50 years old. We aimed here to explore the effect of this antiretroviral therapy on different immunological parameters that could account for this effect. Methods: We analysed baseline samples of vaccinated subjects under 50 years old (n = 41). We characterized the maturational subsets and the expression of activation, senescence and prone-to-apoptosis markers on CD4 T-cells; we also quantified T-regulatory cells (Treg) and dendritic cell (DC) subsets. We used binary logistic regression to evaluate the immunological impact of MVC-cART, correlation with MVC exposure and linear regression for association with the magnitude of the HBV vaccine response. Results: HIV-infected subjects on MVC-cART prior to vaccination showed increased recent thymic emigrants levels and reduced myeloid-DC levels. A longer exposure to MVC-cART was associated with lower frequencies of Tregs and activated and proliferating CD4 T-cells. Furthermore, the frequencies of activated and proliferating CD4 T-cells were inversely associated with the magnitude of the HBV vaccine response. Conclusion: The beneficial effect of MVC-cART in the HBV vaccine response in subjects below 50 years old could be partially mediated by its reducing effect on the frequencies of activated and proliferating CD4 T-cells prior to vaccination. Keywords: Maraviroc (MVC), CD4 T-cell, Ki67, Activation, Treg, hsCRP, Inflammation, HBV vaccine, Dendritic cells (DC), Recent thymic emigrants (RTE) © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Open Access Journal of Translational Medicine *Correspondence: [email protected] †Manuel Leal and Yolanda M. Pacheco contributed equally to this work 1 Institute of Biomedicine of Seville (IBiS), Virgen del Rocío University Hospital (HUVR)/CSIC/University of Seville, Avda. Manuel Siurot s/n, PC 41013 Seville, Spain Full list of author information is available at the end of the article Page 2 of 8 Herrero‑Fernándezetal. J Transl Med (2018) 16:238 Background Human immunodeficiency virus (HIV)-infected subjects are at high risk for hepatitis B virus (HBV) infection and progression of severe, life-threatening hepatic complications, such as cirrhosis and hepatocellular carcinoma [1, 2]. To prevent the associated morbimortality, worldwide current guidelines recommend vaccination against HBV in all HIV-infected subjects susceptible to be coinfected by HBV, but the response rates are lower than in HIV- uninfected subjects [reviewed in 3]. The best-known predictors of vaccine efficacy are an undetectable viral load and CD4 T-cell counts above 350 cells/mm3 [3]. Thus, it is well assumed that successful combined antiretroviral therapy (cART) favours the vaccine response; however, the influence of the type of antiretroviral treatment has been scarcely explored until now. In this line, it was first described that maraviroc (MVC), a CCR5 antagonist, enhanced meningococcal neo-immunization and accelerated the response to tetanus boost [4]. More recently, we also reported that MVC-containing cART (MVC-cART) was associated with a better response against the HBV vaccine, at least in subjects younger than 50years old [5]. Nevertheless, the potential underlying mechanisms were unaddressed. Different antiretroviral combinations including MVC have comparatively proved their beneficial effects on the levels of inflammatory biomarkers [6, 7] and the T-cell immunophenotype [8]. In two clinical trials, an improvement of duodenal immunity and a reduction in bone loss has been associated with such combinations [9, 10]. Furthermore, MVC monotherapy also reduced the frequency of regulatory T-cells (Treg) in antiretroviral-naïve subjects [11], even improving the distribution of Treg subsets [12]. This could be relevant since we observed that Treg cells negatively impacted the HBV vaccine responsiveness in a previous cohort [13]. It is possible that MVC could enhance different functions required to mount an effective response following HBV vaccination, including antigen-presentation, T-cell help, regulatory T-cell suppression and B cell functions [14, 15]. In the present study, we aimed to explore the potential effect of MVC-cART in different parameters related to inflammation, T-cell function and dendritic cell subsets that could account for its effect on the HBV vaccine response; to this aim, we studied the same cohort of vaccinated subjects that had revealed a positive effect of such MVC-cART. Methods Study design, patients andsamples The vaccination protocol has been reported elsewhere [5]. Briefly, HIV-infected subjects from the Virgen del Rocío University Hospital were consecutively vaccinated against HBV. These subjects (a) were on suppressive cART (at least in the last 6 months), (b) had CD4 T-cell counts of > 300 cells/µl, (c) had negative serology for HBsAg and anti-HBc and (d) had anti-HBs titers of ≤ 10 mIU/ml. The vaccination protocol consisted of 3 intramuscular double doses (40 µg) of the recombinant Engerix-B vaccine (GlaxoSmithKline, Brentford, United Kingdom) at 0, 1, and 3 months. The vaccine response was measured 6months after the first dose. A group of subjects was simultaneously vaccinated at 0 and 6 months against hepatitis A virus (HAV) (simultaneous HAV vaccination) with two intramuscular doses of the vaccine Havrix-1440 (GlaxoSmithKline, Brentford, United Kingdom). This subgroup of subjects had a previous negative serology for HAV. Fresh blood samples were collected at baseline, just before the administration of the first vaccine dose. All patients gave informed consent to enter the study, which was approved by the Ethic Committee of our Hospital. We restricted the present analyses to subjects younger than 50years old (n = 41) from the total vaccinated population because the beneficial effect of MVC-cART on the vaccine response was observed in this population [5]. Laboratory measurements Absolute numbers of CD4 and CD8 T cells were determined with an Epics XL-MCL flow cytometer (Beckman- Coulter). Plasma HIV-1 RNA levels were measured using quantitative PCR (Cobas Ampliprep/Cobas TaqMan HIV-1 test; Roche Molecular Systems, Basel, Switzerland) with a detection limit of 20 HIV-RNA copies/ml. Plasma samples were tested for HBV-related markers (HBsAg, anti-HBs, and anti-HBc) using an HBV enzyme-linked immunosorbent assay (ELISA; Siemens Healthcare Diagnosis, Malvern, PA). Qualitative PCR amplification was used for plasma hepatitis C virus (HCV) amplification (Cobas Amplicor; Roche Diagnosis, Mannheim, Germany) with a detection limit of 15IU/ml. The highly sensitive C-reactive protein (hsCRP) levels were determined with an immunoturbidimetric serum assay using a Cobas 701 (Roche Diagnostics, Mannheim, Germany). Flow cytometry Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood before the first dose of vaccine and cryopreserved. For the immunophenotyping of cellular subsets, PBMCs were thawed and immediately stained with the following surface antibodies: anti-CD31 PE-CF594, anti-CD56 BV510, anti-CD25 BV605, anti-CD45RA BV650, anti-CD4 BV786, anti- CD3 APC-H7, Lin2 FITC (anti-CD3, anti-CD19, anti- CD20, anti-CD14 and anti-CD56), anti-CD11c BV650, and anti-HLA-DR BV711 (BD Biosciences, USA); Page 3 of 8 Herrero‑Fernándezetal. J Transl Med (2018) 16:238 anti-CD39 FITC, anti-CD57 PE-Cy7, anti-HLA-DR BV570, anti-CD95 BV711, and anti-CD27 AF700 (Bio- Legend, USA); and anti-CD123 AF700 (R&D, San Diego CA, USA). When necessary for intracellular staining, cells were fixed and permeabilized according to the manufacturer’s instructions (FoxP3/Transcription Factor Staining Buffer, Ebioscience, USA) and stained with the following intracellular antibodies: anti-Ki67 PerCP- Cy5.5, anti-FoxP3 PE and anti-CTLA-4 APC (BD Biosciences, USA). Isotype controls for CD39, CD31, CD25, CD95, Ki67, FoxP3 and CTLA4 were included in each experiment. We characterized peripheral CD4 T-cells according to the distribution of their maturational subsets [naïve (CD27+CD45RA+), central memory (CD27+CD45RA−), effector memory (CD27−CD45RA−) and TemRA (CD27−CD45RA+)], also including recent thymic emigrants (RTEs; naïve- CD31+) and the expression of activation (HLADR), cell-cycle entry (Ki67), senescence (CD57) and prone-to-apoptosis (CD95) markers. We also identified Tregs with classical markers (CD25hiFoxP3+) and their expression of the mentioned activation markers but also of functional markers (CD39, CTLA-4). We immunophenotyped myeloid dendritic cells (mDCs) as Lin2−HLA-DR+CD123−CD11c+ and plasmacytoid dendritic cells (pDCs) as Lin2−HLA-DR+CD11c−CD123+.Viable cells were identified using LIVE/DEAD fixable Aqua Blue Dead Cell Stain (Life Technologies, USA). One million cells of each sample were stained, and a minimum of 100,000 events of total lymphocytes and 150,000 dendritic cells were acquired. Flow cytometry was performed on an LSR Fortessa (BD Biosciences, USA). Analysis was performed using FlowJo version 9.3 (TreeStar). Statistical analysis Continuous variables were expressed as medians and interquartile ranges [IQRs] and categorical variables as the number of cases and percentages. Binary logistic regression was used to analyse the potential effect of MVC-cART on the clinical and immunological parameters. Variables with a p value < 0.1 in the univariate analysis were considered in multivariable models. Linear regression analyses were performed to determine factors associated with the magnitude of response (absolute anti- HBs titre). Correlations were assessed using the Spearman’s rho correlation coefficient. A p value of < 0.05 was considered statistically significant. Statistical analysis was performed using SPSS software (version 22; IBM SPSS, Chicago, USA), and graphs were generated using Prism (version 5, GraphPad Software, Inc.). Results Demographic, clinical andimmunological variables associated withMVC‑containing cART Around half of the population (51%) received MVC- cART, consisting of MVC and a boosted protease inhibitor (PI) or MVC and two nucleoside-reverse transcriptase inhibitors (NRTIs). We compared the demographic, clinical and immunological variables between patients receiving MVC-containing cART or MVC-lacking cART at the moment of vaccination (Table1). The age, CD4+/CD8+ ratio, time on HIVtreatment, %CD4+RTE and %mDCs had p values < 0.1 in the univariate analyses and were therefore included in the multivariate analysis. Notably, 19% of the subjects treated with MVC-cART were also receiving NRTIs, whereas 70% of the subjects on MVC- lacking cART were receiving NRTIs. Thus, the absence of NRTIs was highly collinear with the presence of MVC and was not included for adjustment in the multivariate analysis. As is shown, the CD4+/CD8+ ratio (p = 0.086; OR [95% CI], 0.19 [0.03–1.26]) showed a trend toward independent association; however, %CD4+RTE (p = 0.024; OR [95% CI], 1.20 [1.02–1.41]) and %mDCs (p = 0.048; OR [95% CI], 0.16 [0.02–0.98]) were independently associated with MVC-cART. Relationship betweenthetime ofexposure toMVC‑containing cART andimmunological variables Since we observed a high degree of variability in the time of exposure to MVC-cART prior to vaccination (median [IQR], 16 [5–38] months), we explored whether this fact could have affected the immunological variables of the study. This analysis was logically restricted to the MVC- cART group (N = 21) (Additional file 1: Table S1). We found significant negative correlations between the time of exposure to MVC-cART and the %CD4+Ki67+, the %CD4+HLA-DR+ and the %CD4+CD25hiFoxP3+ (Fig.1). On the other hand, since we expected to find a direct association between MVC-cART and the inflammationrelated marker hsCRP, we also explored potential correlations between the five immunological variables affected by MVC-cART in both a direct or a time-dependent way and the levels of hsCRP. hsCRP was only correlated with %CD4+RTE (r = − 0.326; p = 0.049) and with %CD4+HLA-DR+ with borderline significance (r = 0.316; p = 0.057) (Additional file2: Figure S1). Impact ofimmunological variables targeted byMVC‑cART onthemagnitude oftheHBV vaccine response To explore to what extent each of the immunological factors that were affected by MVC-cART could affect the magnitude of the response, we tested potential associations between these five variables and the magnitude of the HBV response. When analysed in the entire cohort Page 4 of 8 Herrero‑Fernándezetal. J Transl Med (2018) 16:238 Table 1 Demographic, clinical andimmunological variables associated withMVC-containing cART Continuous variables are expressed as median values [IQR], and categorical variables are expressed as the number of cases (%). All demographic, clinical and immunological variables with p values of < 0.1 in the unadjusted model, except NRTI‑containing cART*, were included in the adjusted model and are shown in bolditalics. Hence, age, CD4+/CD8+ ratio, time on HIV treatment, %CD4+RTE and %mDCs were included in the multivariate model (n = 38). Variables with p values of < 0.1 are shown in italics. Variables with p values of < 0.05 in the adjusted model were considered statistically significant and are shown in bolditalic. *The absence of NRTIs was collinear with the presence of MVC Demographic, clinical andimmunological variables (n = 41) MVC‑containing cART (N = 21) MVC‑lacking cART (N = 20) Unadjusted p value; OR[95% CI] Adjusted p value; OR[95% CI] Male sex, n (%) 15 (71) 16 (80) 0.525; 0.625 [0.147–2.659] Age (years) 36 [31–44] 44 [39–48] 0.024; 0.892 [0.800–0.985] 0.878; 1.01 [0.87–1.18] Nadir CD4+ T-cell count (cells/mm3) 294 [184–412] 264 [206–379] 0.539; 1.001 [0.997–1.005] CD4+ T-cell count (cells/mm3) 703 [565–869] 725 [529–911] 0.775; 1.000 [0.997–1.002] CD8+ T-cell count (cells/mm3) 781 [538–961] 596 [491–829] 0.109; 1.002 [1.000–1.004] CD4+/CD8+ ratio 0.9 [0.6–1.2] 1.1 [0.9–1.6] 0.084; 0.309 [0.082–1.170] 0.086; 0.20 [0.03–1.26] Time since diagnosis (months) 65 [32–212] 132 [67–235] 0.158; 0.995 [0.989–1.002] Time on HIV-treatment (months) 46 [33–147] 120 [64–201] 0.057; 0.992 [0.984–1.000] 0.105; 0.99 [0.97–1.00] Sexual transmission, n (%) 18 (86) 18 (90) 0.677; 1.500 [0.223–10.077] Previous AIDS, n (%) 1 (5) 1 (5) 0.972; 0.950 [0.055–16.293] Previous HCV coinfection, n (%) 4 (19) 1 (5) 0.199; 4.471 [0.454–44.011] NRTI containing cART, n (%)* 4 (19) 14 (70) 0.002; 0.101 [0.024–0.430] hsCRP (mg/l) 0.8 [0.5–1.1] 0.9 [0.6–1.5] 0.324; 0.612 [0.231–1.623] % CD4+ naive 44.1 [34.5–55.6] 44.9 [33.7–50.1] 0.829; 0.995 [0.954–1.039] % CD4+ RTE 78.0 [69.9–82.5] 69.3 [64.3–76.4] 0.016; 1.116 [1.021–1.220] 0.024; 1.20 [1.03–1.41] % CD4+ central memory 29.4 [23.3–33.2] 28.3 [22.9–42.5] 0.752; 0.989 [0.926–1.057] % CD4+ effector memory 20.0 [18.0–27.5] 22.0 [12.9–28.5] 0.968; 0.999 [0.936–1.065] % CD4+ TemRA 2.4 [1.1–4.3] 1.6 [0.8–4.4] 0.873; 1.017 [0.831–1.243] % CD4+ HLA-DR+1.8 [1.0–2.0] 1.6 [1.1–2.3] 0.880; 1.066 [0.464–2.451] % CD4+ Ki67+2.4 [2.0–3.2] 2.2 [2.1–2.6] 0.219; 1.629 [0.748–3.547] % CD4+ CD57+4.8 [3.5–8.0] 4.47 [2.1–10.8] 0.879; 0.990 [0.873–1.124] % CD4+ CD95+55.0 [40.7–66.0] 54.4 [44.1–63.9] 0.590; 1.011 [0.971–1.052] % CD4+ CD25hiFoxP3+1.2 [0.9–2.0] 1.5 [1.1–1.7] 0.992; 0.995 [0.358–2.765] % CD4+ CD25hiFoxP3+HLA-DR+13.9 [9.9–21.6] 16.1 [12.2–22.1] 0.543; 0.973 [0.889–1.064] % CD4+ CD25hiFoxP3+ki67+18.5 [13.5–25.2] 20.1 [17.6–25.0] 0.216; 0.930 [0.830–1.043] % CD4+ CD25hiFoxP3+CD39+82.0 [41.7–88.2] 83.7 [81.2–85.1] 0.716; 1.005 [0.980–1.029] % CD4+ CD25hiFoxP3+CTLA4+59.0 [46.2–65.2] 57.7 [47.8–69.2] 0.545; 0.986 [0.941–1.032] % mDCs 0.5 [0.3–0.8] 0.8 [0.6–1.4] 0.042; 0.226 [0.054–0.949] 0.048; 0.16 [0.03–0.98] % pDCs 0.2 [0.1–0.2] 0.2 [0.1–0.3] 0.529; 0.109 [0.000–108.297] 0 20 40 60 80 0 1 2 3 4 5 Time under MVC-cART (months) % CD4+ HLA-DR+ 0 20 40 60 80 0 1 2 3 4 Time under MVC-cART (months) % CD4 + CD25 hi FOXP3 + 0 20 40 60 80 0 2 4 6 Time under MVC-cART (months) % CD4 + ki67 + r= -0.489 p = 0.034 r= -0.477 p = 0.029 r= -0.523 p = 0.015 Fig. 1 Relationship between the time of exposure to MVC-cART and immunological variables. Only significant correlations between the time of exposure to MVC-cART and immunological variables are represented Page 5 of 8 Herrero‑Fernándezetal. J Transl Med (2018) 16:238 (n = 41) (Additional file3: TableS2), only the %CD4Ki67+ showed a negative association with the magnitude of the response, with borderline significance (p = 0.053; B [95% CI], − 199.5 [− 401.4 to 2.5]). However, when restricting to the population treated with MVC-cART (n = 21) (Table2), both the %CD4+Ki67+ (p = 0.027; B [95% CI], − 241.7 [– 452.8 to 30.5]) and the %CD4+HLA-DR+ (p = 0.038; B [95% CI], – 211.2 [– 409.6 to 12.8]) showed significant associations with the anti-HBs titres. Discussion We recently observed a beneficial effect of MVC-cART in the HBV vaccine response in a cohort of HIV-infected subjects younger than 50 years old [5]. We report now that HIV-infected subjects on MVC-cART have increased RTE but reduced mDC frequencies prior to vaccination. In addition, a longer time of exposure to MVC-cART was associated with lower frequencies of Tregs and activated and proliferating CD4 T-cells, with proliferating CD4 T-cells being inversely associated with the magnitude of the HBV vaccine response. In the response to the HBV vaccine, a peptide antigen administered intramuscularly, helper CD4 T-cell function plays a major role [14, 15], and it is well assumed that T-cell exhaustion and senescence related to HIV infection may result in response failure [16]. Antiretroviral treatment improves antigen-specific T-cell responses and recovers the T-cell repertoire [17]. In fact, the duration of cART was associated with the HBV vaccine response [18]. However, the specific effects of different antiretroviral families have been less studied. It is reasonable to expect a negative impact of NRTIs because they favour cellular senescence through inducing accelerated shortening of telomeres in peripheral T-cells [19]. In fact, telomere length has been associated with the response to influenza vaccine in elderly non-HIV-infected subjects [20]. Moreover, we have recently found a better profile in T-cells in subjects on NRTI-lacking regimens regarding cell survival and replicative senescence [21]. On the other hand, there is some controversy about the potential immunological effects of MVC-cART. While some authors have described no effects on inflammatory biomarkers [10, 22], others have comparatively demonstrated their beneficial effects on these markers [6] and on the T-cell immunophenotype [8]. We have now explored the immunological profile associated with MVC-cART in the context of the HBV vaccine response, finding a less activated and proliferative phenotype, a higher contribution of RTEs and a lower frequency of mDC and Treg cells. Notably, HBV vaccine responsiveness has been associated with most of these factors in other cohorts, including decreased activation of T-cells [23], a higher frequency of CD34+ precursors [24, 25] and a lower frequency of Tregs [13]. Dendritic cells are being targeted for improvement of HBV vaccine responsiveness [26]. As far as we know, no previous data link proliferative CD4 T-cells with vaccine response in this context. However, it is known that HIV-infected subjects have increased memory CD4 T-cell cycling, which has been proposed to be consequence of the inflammatory environment of HIV infection [27], and a compromised thymic output [28]. Since we found an increase of the RTE frequency along with a reduction of the frequency of proliferating CD4 T-cells associated with MVC-cART, we speculate a potential regenerative capacity for this regimen that requires further research. Indeed, proliferating CD4 T-cells in the absence of thymic output may show limited immunocompetence due to the constriction of TCR diversity [29]. Thus, this regimen could contribute to a potential enrichment of TCR diversity, which would favour the response against vaccine antigens. Along these lines, MVC positively impacted the response to different vaccine antigens in HIV-infected subjects [4, 5]. The effect on Treg cells also deserves discussion. We previously showed a net effect of MVC monotherapy in reducing Tregs in antiretroviral-naïve subjects [11]. In the present cohort of cART-experienced subjects, this reducing effect was dependent on the time of exposure. Notably, both cohorts differed very much, not only because of the presence/absence of treatment but also in age or time from diagnosis, among other factors. Thus, our current results strengthen the hypothesis that MVC exerts immunomodulatory effects through reducing Treg cells. Tregcells are being studied in several immunization models [30–32] because they suppress the proliferation and cytokine secretion of CD4 and CD8 T cells as well Table 2 Relationship betweenvariables affected byMCV- containing cART and the magnitude of the HBV vaccine response Continuous variables are expressed as median values [IQR]. Linear regression analyses were performed to determine variables associated with the magnitude of response (absolute anti‑HBs titre). Variables with p values of < 0.1 are shown in italics. Variables with p values of < 0.05 were considered statistically significant and are shown in bolditalic Immunological variables MVC‑cART (n = 21) Unadjusted p value; B (95% CI) % CD4+ RTE 78.0 [67.0–82.5] 0.599; 6.2 [− 18.0–30.3] % CD4+ HLA-DR+1.8 [1.0–2.0] 0.038; − 211.2 [− 409.6–12.8] % CD4+ ki67+2.4 [2.0–3.1] 0.027; − 241.7 [− 452.8–30.5] % CD4+ CD25hiFoxP3+1.2 [0.9–2.0] 0.889; − 17.0 [− 267.7– 233.78] % mDCs 0.5 [0.3–0.8] 0.272; 196.1 [− 166.3– 558.5] Page 6 of 8 Herrero‑Fernándezetal. J Transl Med (2018) 16:238 as monocytes, dendritic cells and B cells [33, 34]. In fact, Treg cells were found within germinal centres of human lymphoid tissues, suppressing the B cell immunoglobulin class switching needed to mount a proper antibody response [35]. In our cohort, the frequencies of activated and proliferating Treg cells, which could be highly suppressive, were inversely associated with the magnitude of the vaccine response (data not shown). Importantly, plasma levels of soluble inflammatory markers before vaccination negatively predicted responses to HAV, HBV, and tetanus vaccines in HCV and HIV infection [36]. Moreover, hsCRP levels were a significant predictor of herpes zoster vaccine response in elderly nursing home residents [37]. hsCRP levels were also inversely associated with the magnitude of the vaccine response in our cohort (data not shown), but we failed to observe a direct association between hsCRP levels and MVC-containing cART. However, we cannot exclude a potential effect of MCV-cART on other inflammatory cytokines, as previously reported [6]. Moreover, hsCRP was inversely associated with the frequency of RTEs and positively associated with activated CD4 T-cells, both of which were impacted by MVC-cART, suggesting a potential indirect effect of MVC-cART on the inflammatory state. As a limitation, the size of our cohort was restricted to the vaccinated population younger than 50years old, where the effect of MVC-cART on the magnitude of vaccine responsiveness was clear [5]. It is well-known that age limits HBV vaccine responsiveness [38]. Thus, it is reasonable to speculate that the added age-associated immunodeficiency could limit or mask the potential benefits of such antiretroviral therapy on the immunological profile. In this sense, aged people have lower thymic output concomitant with higher peripheral T-cell proliferation [27]. Interestingly, the group on MVC-cART had lower CD4/CD8 ratios, which have been reported to negatively impact the vaccine response [39]. This could be due to the shorter period of treatment in this group, which is critical for CD4/CD8 T-cell ratio normalization [40]. In any case, despite the lower CD4/CD8 ratio, the group on MVC-cART showed better vaccine responsiveness and improved CD4 T-cell profiles. Finally, we cannot discriminate among the particular effects due to the presence of MVC or to the absence of NRTIs in the cART, and thus, we can only draw conclusions about the beneficial effects of such combined therapy. Similar combined therapies are being explored in the clinical setting in an attempt to reduce toxicities and to improve immune reconstitution [41]. Conclusion The beneficial effect of MVC-cART in the HBV vaccine response in subjects below 50years old could be mediated at least partially by its reducing effect on the frequencies of activated and proliferating CD4 T-cells prior to vaccination. This fact could be related with a potential regenerative capacity of such therapy and deserves further research due to its relevance in the search for novel therapeutic targets that could improve immune function and vaccine responsiveness in HIV-infected subjects. Additional files Additional file1: TableS1. Relationship between the time of exposure to MVC-containing cART and immunological variables. Additional file2: Figure S1. Associations between hsCRP and T-cell immunological variables affected by MVC-cART. Only significant correlations between hsCRP and T-cell immunological variables are represented. Additional file3: TableS2. Relationship between variables modified by MCV-containing cART and the magnitude of the HBV vaccine response in the whole cohort. Abbreviations cART : combined antiretroviral therapy; DC: dendritic cell; ELISA: enzymelinked immunosorbent assay; HAV: hepatitis A virus; HBV: hepatitis B virus; HCV: hepatitis C virus; HIV: human immunodeficiency virus; hsCRP: highly sensitive C-reactive protein; mDC: myeloid-DC; MVC: maraviroc; MVC-cART : maraviroc-containing combined antiretroviral therapy; NRTI: nucleoside reverse-transcriptase inhibitors; PBMCs: peripheral blood mononuclear cells; pDC: plamacytoid dendritic cells; PI: protease inhibitor; RTE: recent thymic emigrants; Treg: regulatory T-cells. Authors’ contributions IH-F performed experiments, data analysis and interpretation and wrote the manuscript; IR-S, LT-D and ER-M performed experiments; MG and ML were the clinicians involved in the vaccination protocol and clinical assistance of the patients; MMR-M and MMP-B assisted in the vaccination protocol and sampling; CL performed anti-HBs titres; ML and YMP coordinated the study; YMP designed the study and participated in data analysis and interpretation and writing of the manuscript. All authors read and approved the final manuscript. Author details 1 Institute of Biomedicine of Seville (IBiS), Virgen del Rocío University Hospital (HUVR)/CSIC/University of Seville, Avda. Manuel Siurot s/n, PC 41013 Seville, Spain. 2 Microbiology Service, Virgen del Rocío University Hospital (HUVR), Seville, Spain. 3 Internal Medicine Service, Hospital Viamed Santa Ángela de la Cruz, Seville, Spain. Acknowledgements The authors express their most sincere thanks to all the subjects included in the study and to the HIV Biobank of the Spanish AIDS Research Network. We also thank Magdalena Rodriguez and Marien Gutierrez Sancho for their assistance at the Day Care Hospital (Infectious Diseases Department), to Mª Antonia Abad and Marta de Luna for their technical assistance and to Juan Manuel Praena for statistical assistance. Competing interests The authors declare that they have no competing interests. Availability of data and materials Not applicable. Page 7 of 8 Herrero‑Fernándezetal. J Transl Med (2018) 16:238 Consent for publication Not applicable. Ethics approval and consent to participate All participants signed informed consent forms at study entry. The study protocol was approved by the Comité de Ética de la Investigación (CEI) de los hospitales universitarios Vírgen Macarena-Virgen del Rocío. Funding This study was funded by grants from the Fondo de Investigación Sanitaria [FIS; PI14/01693; PI16/00684], co-funded by Fondos Europeos para el Desarrollo Regional (FEDER), and the Junta de Andalucía, Consejería de Economía, Innovación, Ciencia y Empleo [Proyecto de Investigación de Excelencia; CTS2593]. The Spanish AIDS Research Network of Excellence also supported this study (RD16/0025/0019). I.H-F and I.R-S were supported by an investigator sponsored research grant from ViiV Healthcare S.L. (Grant Number 205644). YM.P and E.R-M. were supported by the Consejería de Salud y Bienestar Social of Junta de Andalucía through the ‘‘Nicolás Monardes’’ programme [C-0013- 2017 and C-0032-2017, respectively]. L.T-D. was supported by Instituto de Salud Carlos III [PFIS programme; FI00/00431]. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. 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Caby F, Guihot A, Lambert-Niclot S, Guiguet M, Boutolleau D, Agher R, et al. Determinants of a low CD4/CD ratio in HIV-1 infected individuals despite long term viral suppression. Clin Infect Dis. 2016;62:1297–303. 41. Nozza S, Svicher V, Saracino A, d’Ettorre G, De Luca A, Maggiolo F, et al. State of the art of dual therapy in 2015. AIDS Rev. 2015;17:127–34. Supplementary Table 1. Relationship between the time of exposure to MVC-containing cART and immunological variables. Immunological variables (n=21) r p hsCRP (mg/L) 0.003 0.989 % CD4+ Naive 0.185 0.434 % CD4+ RTE -0.161 0.497 % CD4+ Central Memory -0.151 0.526 % CD4+ Effector Memory -0.085 0.723 % CD4+ TemRA -0.087 0.714 % CD4+ HLA-DR+ -0.489 0.034 % CD4+ Ki67+ -0.477 0.029 % CD4+ CD57+ 0.110 0.663 % CD4+ CD95+ -0.170 0.460 % CD4+ CD25hiFoxP3+ -0.523 0.015 % CD4+ CD25hiFoxP3+HLA-DR+ -0.126 0.586 % CD4+ CD25hiFoxP3+ki67+ -0.318 0.172 % CD4+ CD25hiFoxP3+CD39+ 0.116 0.617 % CD4+ CD25hiFoxP3+CTLA4+ 0.226 0.324 % mDCs -0.133 0.567 % pDCs 0.105 0.650 Correlations were assessed using Spearman's rho correlation coefficient. Variables with p values of <0.05 were considered statistically significant and are shown in bold. Supplementary Figure 1. 0 1 2 3 4 0 20 40 60 80 100 hsCRP (mg/L) %CD4+RTEs 01234 0 1 2 3 4 5 hsCRP (mg/L) %CD4+ HLA-DR+ r= -0.326 p= 0.049 r= 0.316 p= 0.057 96 6. DISCUSIÓN GLOBAL 97 DISCUSIÓN GLOBAL La presente Tesis Doctoral profundiza en el conocimiento del proceso de inmunosenescencia en el contexto de la respuesta vacunal. Por un lado, estudiamos la respuesta vacunal frente a la gripe en sujetos ancianos y, por otro, la respuesta a la vacuna frente a VHB en sujetos VIH, siendo ambos escenarios de inmunosenescencia cronológica y prematura, respectivamente. E el pie taajo, T-cell proliferative homeostatic alterations are negatively associated with the vaccine response against influenza in elderly people, encontramos que la respuesta a la vacuna frente a influenza en ancianos se asoció con las alteraciones homeostáticas que se producen en el sistema inmunitario durante el envejecimiento, y que afectan a la proliferación de las distintas subpoblaciones de las células T, tanto de CD4 como CD8. Sauce y cols. [11] observaron una asociación inversa entre la función tímica y la proliferación en células T naïves en sujetos ancianos, en sujetos timectomizados durante la infancia y también en sujetos infectados por VIH. Sin embargo, aunque en nuestra cohorte de ancianos observamos una mayor proliferación de células T naïves en el grupo de los no-respondedores a la vacuna, esto no se asoció aparentemente con una menor función tímica. Quizá esto podría explicarse por el hecho de que, aparentemente, nuestra cohorte mostró una función tímica relativamente preservada, en comparación con los valores de otra cohorte en la que la función tímica se determinó utilizando la misma técnica [100]. Lo que sí observamos fue una asociación entre la elevada proliferación de las subpoblaciones de células T estudiadas y diversos marcadores de inflamación, pudiendo, por tanto, esta proliferación ser un reflejo más directo del estado de inflamación. En esta línea, trabajos previos mostraron también una relación entre la inflamación en sujetos VIH y una mayor proliferación de las células T memoria [63]. Por tanto, es razonable especular que el incremento de la proliferación homeostática que se observa durante el envejecimiento pueda contribuir al iflaagig. Por otro lado, nosotros observamos una relación inversa entre los niveles de los marcadores de inflamación y la función tímica, y otros autores también 98 han descrito previamente una relación entre la involución del timo y la inflamación crónica [101]. De esta manera, en el escenario del envejecimiento, la función tímica y la inflamación parecen estar inversamente relacionadas. Además, el hecho de que los ancianos que fallecen en nuestra cohorte, durante un año de seguimiento posterior a la vacunación, presenten conjuntamente fallo tímico, elevada proliferación de las células T y mayor inflamación, refuerza la hipótesis de que estos parámetros estén estrechamente interrelacionados. Con relación a la respuesta vacunal en ancianos, además de la asociación con la mencionada proliferación de células T, observamos también una tendencia a la asociación con marcadores de inflamación. El papel deletéreo de la inflamación en este escenario es consistente con lo descrito previamente [43,44]. Por otro lado, la infección por CMV, que es uno de los principales factores asociados a inflammaging, más concretamente, el título de anticuerpos frente a CMV, se ha asociado con la respuesta vacunal en otras cohortes de ancianos [102,103]. En nuestra cohorte, no hemos observado esta asociación, pese a que sí hemos encontrado una asociación entre el título de anticuerpos anti-CMV y los niveles de marcadores de inflamación. De nuevo, quizá el hecho de que en nuestra cohorte los niveles de los marcadores de inflamación estén relativamente preservados (en comparación con otras cohortes de ancianos), puede explicar la aparente contradicción en nuestros resultados. Tampoco podemos descartar que exista asociación con marcadores de inflamación diferentes de los analizados en este estudio. En relación con el escenario de la infección por VIH, los niveles de marcadores de inflamación han sido también previamente asociados con la escasa respuesta vacunal frente a VHA, VHB y tétanos, en sujetos coinfectados por VHC y VIH [104]. Además, la proteína C reactiva ultrasensible (usPCR) se ha propuesto como predictor de la respuesta a la vacuna del herpes zoster en ancianos [105]. En nuestra cohorte de sujetos VIH hemos observado también que los niveles de usPCR tendían a asociarse inversamente con la magnitud de la respuesta a la vacuna frente a VHB (Anexo 2). Por otro lado, los niveles de usPCR se asociaron inversamente con la frecuencia de células RTE y directamente con la frecuencia de células T CD4 activadas. Dado que la 99 frecuencia de RTE depende directamente de la función tímica, esto sugiere que, en este escenario, también pueda existir una relación entre la inflamación y la función tímica, en la que es interesante profundizar. En este sentido, nuestro grupo ha descrito recientemente que la función tímica impacta en la progresión clínica de los sujetos VIH [106,107]. Es interesante destacar que, actualmente, se asume que la progresión clínica de los pacientes VIH tratados depende del estado inflamatorio crónico que persiste a pesar de la supresión virológica, y de la concomitante (parcial) reconstitución inmunitaria. Esta relación indirecta (timo-progresión + progresióninflamación) también refuerza la hipótesis del papel del timo en la inflamación crónica de pacientes VIH. En estos escenarios de inmunosenescencia, también hemos explorado el papel de las Treg en la respuesta vacunal, puesto que las Treg están involucradas en la supresión del sistema inmunitario, impidiendo una respuesta apropiada frente a las vacunas [46,47], entre otras funciones. Además, en ambos escenarios la frecuencia de Treg se ve aumentada [15,64]. Hemos observado una frecuencia basal de Treg más elevada en los ancianos no-respondedores a la vacuna, resultados que están en línea con los de Geest et al. [108] y con los hallados previamente en nuestro grupo en sujetos infectados por VIH [51]. Asimismo, encontramos una mayor proliferación de las Treg en los ancianos que no respondían a la vacuna, resultado que concuerda con lo observado en el escenario VIH, en el que la respuesta a la vacuna de VHB se asoció inversamente con las células Treg proliferativas (Anexo 1 del tercer trabajo de la presente Tesis Doctoral, Improved CD4 T-cell profile in HIV-infected subjects on maraviroc-containing therapy is associated with better responsiveness to HBV vaccination). El hecho de que una mayor frecuencia de Treg pudiera impedir una adecuada respuesta a la vacuna tanto en sujetos ancianos, como en sujetos infectados por VIH, refuerza el concepto de que este incremento de las Treg sea una característica común de inmunosenescencia en ambos escenarios [15], que puede comprometer la capacidad del sistema inmunitario para responder de manera apropiada [48-53]. Podemos especular que la expansión y elevada proliferación de las Treg en estos escenarios estén relacionadas con la proliferación homeostática que trata de compensar la menor función tímica en estos pacientes, como se ha observado 100 previamente en modelos animales [109] y, más recientemente en nuestro grupo, en un modelo humano de proliferación homeostática [110]. Un análisis en profundidad del papel de la inmunosenescencia en las Treg y viceversa, así como del papel de las Treg en la respuesta vacunal, podría contribuir a la búsqueda de estrategias terapéuticas que mejoren la respuesta vacunal en estos escenarios de inmunosenescencia. En esta Tesis Doctora, hemos explorado una potencial estrategia terapéutica para mejorar la respuesta vacunal atendiendo a la expansión de Treg en estos escenarios de inmunosenescencia y a su efecto deletéreo en dicha respuesta. Dado que en estudios previos, nuestro grupo había mostrado que MVC podía reducir la frecuencia de Treg en sujetos con infección por VIH [94], nos propusimos estudiar si el tratamiento con un TARc que incluyese MVC podría mejorar la respuesta a la vacuna frente a VHB, mediante un efecto reductor de la frecuencia de las Treg. Pero, además, un potencial efecto beneficioso de MVC en este escenario, también podría implicar otros mecanismos de acción como una reducción de la inflamación o una mejora de la presentación antigénica. De hecho, MVC ha presentado numerosas propiedades inmunomoduladoras, como mejorar la enfermedad de injerto frente a huésped en trasplante de células madre hematopoyéticas alogénicas [111,112], suprimir el crecimiento tumoral e inducir apoptosis en células de leucemia linfoblástica aguda [113]. En el escenario de la infección por VIH, MVC se ha asociado a una mejora de la inmunidad de la mucosa duodenal y a una reducción de la pérdida ósea [114,115]. También, parece ejercer un perfil antiinflamatorio, disminuyendo marcadores de inflamación como la 2-microglobulina, el CD40L soluble y el CD14 soluble [93], lo que podría mejorar la respuesta inmunitaria a las vacunas. De hecho, se había descrito previamente que MVC ejercía un efecto beneficioso en otros escenarios distintos de respuesta vacunal, como era la respuesta a la inmunización frente a tétanos, meningoco o cólera [97]. Por todo ello, en el segundo trabajo de la presente Tesis Doctoral, Association between a suppressive combined antiretroviral therapy containing maraviroc and the hepatitis B virus vaccine response. Antimicrob Agents Chemother, quisimos explorar el posible efecto beneficioso de un TARc que incluyese MVC en la respuesta vacunal 101 frente al VHB. Observamos que este tratamiento efectivamente se asociaba con una mayor magnitud de la respuesta a la vacuna, aunque principalmente en sujetos VIH menores de 50 años. Estos resultados llevan a recomendar una estrategia de vacunación temprana, en sujetos menores de 50 años, y acompañada de un TARc que incluya MVC. Con la edad, el sistema inmunitario se altera debido a la inmunosenescencia y la respuesta a las vacunas disminuye [59]. En los individuos con infección por VIH, este proceso está acentuado además por la inmunosenescencia prematura que propicia el propio virus [56,57]. Es probable que esta inmunosenescencia prematura esté interfiriendo con el efecto beneficioso de MVC en los sujetos mayores de 50 años. Nos propusimos profundizar también en los posibles mecanismos inmunológicos implicados en este efecto beneficioso de un TARc que incluye MVC, concretamente, en el tercer trabajo de la presente Tesis Doctoral, Improved CD4 T-cell profile in HIV- infected subjects on maraviroc-containing therapy is associated with better responsiveness to HBV vaccination. Así, hemos explorado el potencial efecto de un TARc que incluía MVC en diferentes parámetros asociados con la inmunosenescencia, la inflamación, la función de las células T (incluidas las Treg), y células presentadoras de antígenos (incluidas las DC). Lo hemos analizado en el contexto de la eficacia de la vacuna VHB en la misma población VIH menor de 50 años estudiada en el trabajo anterior. Hemos encontrado que los pacientes tratados con un TARc incluyendo MVC mostraban un perfil inmunológico menos activado, con mayor contribución de las RTE y una menor frecuencia de mDC y de Treg. Estos factores habían sido previamente asociados con la respuesta vacunal en otras cohortes [116-118]. La elevada frecuencia de RTE en sujetos tratados con un TARc que incluía MVC, junto con la reducción de sus células T CD4 proliferativas, hace pensar que dicho régimen posee capacidad regenerativa del sistema inmune. La involución tímica que sufren los sujetos VIH [11] promueve la proliferación de células T por compensación homeostática, limitando la competencia inmunitaria al reducir el repertorio de los TCR [119]. Por tanto, si MVC mejora la función tímica, uno de los mecanismos por los cuales MVC podría estar mejorando la respuesta a la vacuna, sería aumentando la diversidad de los TCR. Por otro lado, un potencial efecto sobre la función tímica podría 102 repercutir a su vez en el grado de inflamación sistémica asociada a la infección por VIH. Además, el tiempo de exposición al TARc que incluía MVC también se asoció a una reducción en la frecuencia de células T CD4 activadas. Al respecto, resulta interesante que la menor frecuencia de células T CD4 proliferativas y células T CD4 activadas, correlaciona inversamente con la magnitud de la respuesta a la vacuna de VHB. En esta línea, como ya se ha comentado anteriormente, en el escenario de ancianos pudimos observar que, tanto las Treg proliferativas, como las subpoblaciones de maduración CD4 y CD8 proliferativas, correlacionaban inversamente con la respuesta a la vacuna frente a influenza. Respecto al posible efecto inmunomodulador de un TARc que incluye MVC sobre la inflamación en la cohorte de sujetos con infección por VIH, no observamos un efecto directo de dicho tratamiento sobre el marcador de inflamación usPCR. Sin embargo, la usPCR, como hemos comentado previamente, se asoció inversamente con la frecuencia de RTE y directamente con la frecuencia de células T CD4 activadas, variables que sí se vieron impactadas por el TARc con MVC. Esto sugiere un posible efecto indirecto del TARc con MVC en el estado inflamatorio. Además, no podemos descartar que el TARc con MVC ejerza su efecto en otras citoquinas inflamatorias, como ya ha sido descrito [93]. Curiosamente, tampoco observamos una asociación directa entre estar bajo un TARc incluyendo MVC y una reducción de Treg. Esta aparente discrepancia con nuestro trabajo previo podría explicarse en base a las notables diferencias en las cohortes estudiadas, sobre todo en relación con el hecho de ser pacientes no tratados previamente con antirretrovirales, pero también en cuanto al periodo de infección calculado desde la fecha del diagnóstico [94]. Sin embargo, el tiempo de exposición a dicho TARc con MVC sí se asoció con menores frecuencias de Treg. Lo que indica que, probablemente en esta cohorte, al haber experimentado los pacientes una mayor progresión clínica, sea necesaria una prolongada exposición a MVC para que su efecto reductor sobre la frecuencia delas Treg sea patente. Creemos, por tanto, que nuestros resultados refuerzan la hipótesis de que el efecto inmunomodulador que ejerce MVC sobre las Treg puede ser otro mecanismo por el cual MVC mejora la respuesta vacunal. 103 En resumen, el efecto beneficioso de un TARc que incluye MVC en la respuesta a la vacuna en sujetos VIH menores de 50 años, se debe en parte a la reducción que ejerce sobre la proliferación y la activación de las células T CD4, al efecto reductor de la frecuencia de células Treg, incluso a su potencial regeneración del sistema inmunitario y todo ello además parece impactar positivamente en el grado de inflamación. Por tanto, MVC, gracias a su efecto inmunomodulador, mejora diferentes funciones del sistema inmunológico necesarias para ejercer una respuesta adecuada tras la vacunación frente a VHB. REFLEXIÓN FINAL La originalidad de esta Tesis Doctoral recae en que es la primera vez que se asocia la respuesta vacunal con alteraciones homeostáticas proliferativas propias de la inmunosenescencia. Además, hemos encontrado asociaciones interesantes entre la función tímica, la proliferación homeostática, la inflamación y la respuesta vacunal, en las que sería interesante profundizar en el futuro, puesto que una mejor comprensión de estas podría ayudar a entender mejor cómo la inmunosenescencia limita las capacidades del sistema inmunitario de manera global. Además, ayudaría a encontrar mejores estrategias para mejorar la respuesta vacunal en estas poblaciones inmunosenescentes. En este sentido, los resultados obtenidos en los sujetos infectados por VIH son de gran relevancia clínica y terapéutica y nos llevan a recomendar que sea considerada la prescripción de un régimen antirretroviral incluyendo MVC en los protocolos de vacunación frente a VHB. Asimismo, dado que la pérdida de la función tímica asociada al envejecimiento podría desempeñar un papel en el origen de la inflamación y de las alteraciones proliferativas de células T, pensamos que alternativas terapéuticas encaminadas a mejorar la función tímica también podrían tener repercusión en mejorar la respuesta vacunal en estos escenarios. Por ello, sería interesante desarrollar ensayos clínicos en este contexto, como los realizados con la hormona del crecimiento en sujetos infectados por VIH, en los que se muestra una mejora de la respuesta inmunitaria frente al VIH, debido a su efecto beneficioso sobre la función tímica [120,121]. 104 7. CONCLUSIONES 105 CONCLUSIONES 1. Las alteraciones homeostáticas proliferativas que se dan en las subpoblaciones de maduración de las células T CD4 y CD8, incluyendo las Treg, asociadas al fenómeno de inmunosenescencia, repercuten en una menor respuesta a la vacuna frente a influenza en ancianos. Estas alteraciones homeostáticas podrían estar íntimamente relacionadas con el aumento de inflamación sistémica y con la disminución de la función tímica que se producen durante el envejecimiento. 2. Un tratamiento antirretroviral incluyendo Maraviroc se asocia con una mayor magnitud de la respuesta a la vacuna frente a Virus de la Hepatitis B en sujetos con infección por VIH menores de 50 años. Por tanto, un régimen conteniendo este fármaco durante el protocolo de inmunización podría ser una estrategia terapéutica adecuada para mejorar la respuesta vacunal en estos sujetos. 3. Los pacientes tratados con un tratamiento antirretroviral incluyendo Maraviroc muestran un perfil inmunológico menos activado, con mayor contribución de las células T recientemente emigradas del timo y una menor frecuencia de células T reguladoras, perfil que se asocia con una mejor respuesta a la vacuna frente al Virus de la Hepatitis B. 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FINANCIACIÓN 118 FINANCIACIÓN Los estudios llevados a cabo en la presente tesis doctoral han sido financiados por las siguientes entidades:  Fondo de Investigación Sanitaria [FIS; PI11/02014; PI14/01693], co-financiado por Fondos Europeos para el Desarrollo Regional (FEDER)  Junta de Andalucía, Consejería de Economía, Innovación, Ciencia y Empleo [Proyecto de Investigación de Excelencia; CTS2593]  ViiV-Pfizer (proyecto competitivo a nivel internacional) “Role of a combined antiretroviral treatment including maraviroc in the response to hepatitis B and A virus vaccine” [Ref. 205644]. Finalmente, Yolanda María Pacheco López en calidad de directora de la presente tesis ha sido la Investigadora Principal de los proyectos mencionados. Su contrato ha sido fiaiado po los Fodos de Iestigaió Saitaia ediate los pogaas Miguel Seet [CP/; CPII/] y po la Cosejeía de Salud y Bieesta Soial de la Juta de Adaluía, ediate el pogaa Niolás Moades [C-0010/13; C-0013- 2017]. 119 10. OTRAS PUBLICACIONES 120 OTRAS PUBLICACIONES  Rosado-Sánchez I, Herrero-Fernández I, Sobrino S, Carvajal AE, Genebat M, Tarancón-Díez L, Fontillón-Alberdi M, de Pablo RM, Muñoz-Fernández MA, Leal M, Pacheco YM. Thymic function impacts the colonic mucosal integrity of successfully-treated HIV-infected subjects. (En revisión)  Rosado-Sánchez I, Herrero-Fernández I, Genebat M, Del Romero J, Riera M, Podzamczer D, Olalla J, Vidal F, Muñoz-Fernández MA, Leal M, Pacheco YM. HIV-Infected Subjects With Poor CD4 T-Cell Recovery Despite Effective Therapy Express High Levels of OX40 and α4β7 on CD4 T-Cells Prior Therapy Initiation. Front Immunol. 2018; 9:1673.  Rosado-Sánchez I, González-Magaña A, Pozo-Balado MM, Herrero-Fernández I, Polaino MJ, Rodríguez-Méndez MM, González-Escribano MF, Leal M, Pacheco YM. An in vitro system of autologous lymphocytes culture that allows the study of homeostatic proliferation mechanisms in human naive CD4 T-cells. Lab Invest. 2018; 98:500-511.  Rosado-Sánchez I, Herrero-Fernández I, Tarancon-Diez L, Moreno S, Iribarren JA, Dalmau D, Vera-Méndez F, Leal M, Pacheco YM. Increased frequencies of Th17 cells and IL17a-producing regulatory T-cells preceding the immunodiscordant response to antiretroviral treatment. J Infect. 2018; 76:86- 92.  Rosado-Sánchez I, Herrero-Fernández I, Álvarez-Ríos AI, Genebat M, Abad- Carrillo MA, Ruiz-Mateos E, Pulido F, González-García J, Montero M, Bernal- Morell E, Vidal F, Leal M, Pacheco YM. A Lower Baseline CD4/CD8 T-Cell Ratio Is Independently Associated with Immunodiscordant Response to Antiretroviral Therapy in HIV-Infected Subjects. Antimicrob Agents Chemother. 2017; 61:e00605-17.  Rosado-Sánchez I, Jarrín I, Pozo-Balado MM, de Pablo-Bernal RS, Herrero- Fernández I, Alvarez-Ríos AI, Rodríguez-Gallego E, Genebat M, Vera M, 121 Berenguer J, Martín ML, Bernal E, Vidal F, Blanco J, Leal M, Pacheco YM. Higher levels of IL-6, CD4 turnover and Treg frequency are already present before cART in HIV-infected subjects with later low CD4 recovery. Antiviral Res. 2017; 142:76-82.  Rosado-Sánchez I, Herrero-Fernández I, Genebat M, Ruiz-Mateos E, Leal M, Pacheco YM. Thymic Function Impacts the Peripheral CD4/CD8 Ratio of HIV- Infected Subjects. Clin Infect Dis. 2017; 64:152-158.