Departamento de Biología Molecular Facultad de Ciencias
Doctorado en Biociencias Moleculares
CHARACTERIZATION OF THE MEMORY IMMUNE RESPONSE TO VIRUSES
Tesis Doctoral
Andrea Canto Méndez Graduada en Biotecnología
Directora: Dra. Margarita del Val Latorre
Tutor: Dr. Pablo Gómez del Arco
Centro de Biología Molecular Severo Ochoa
Madrid, 2018
Moleculares (Departamento de Biología Molecular, Facultad de Ciencias) en la Universidad Autónoma de Madrid por la graduada en Biotecnología:
Andrea Canto Méndez
Para su realización se ha contado con un contrato perteneciente a la convocatoria
“Ayudas para contratos predoctorales para la formación de doctores 2014” del Ministerio de Economía y Competitividad (BES-2014-068148) y un contrato FPI-UAM perteneciente a la convocatoria 2014.
La Directora de tesis, Margarita del Val Latorre,
Doctora en Ciencias (Químicas – Bioquímica) por la Universidad Autónoma de Madrid.
Investigadora Científica del Centro de Biología Molecular Severo Ochoa (CSIC-UAM).
Responsable del laboratorio de Inmunología Viral del departamento de Biología Celular e Inmunología, certifica que esta tesis ha sido realizada bajo su dirección en el Centro de Biología Molecular Severo Ochoa.
Firmado:
Directora de tesis, Margarita del Val
Para Alonso
de realizar esta tesis en su laboratorio, por haberme enseñado tantas cosas, por ver la solución a todos los problemas y haber estado siempre disponible para ayudar.
Gracias a todos los que han pasado por el laboratorio durante todo este tiempo: Adrián, Alfonso, Bea, Carolina C, Carolina H, Cris O, Cris R, Daniel, David, Elena, Eva, Feli, Irene, Jocy, Kayisan, María, Noemi, Rubén, Silvia, Sonsoles, Susana y Víctor. Sobre todo, gracias a Susana por su ayuda en gran parte de los experimentos de esta tesis y por haberme enseñado todas las técnicas que estuvieron a su alcance. A Eva, por su aportación fundamental en la puesta a punto de los ELISA y en los cálculos. A Bea, por tener siempre una buena respuesta para cualquier pregunta. A Cris, por toda su ayuda en los experimentos, por escuchar todas mis ideas por absurdas que puedan ser, y por compartir conmigo tantos buenos ratos con nuestro querido MCMV. Gracias también a todos los que han participado en el tipaje y mantenimiento de las líneas de ratones.
Gracias al Dr. Luis Antón y al Dr. Manuel Ramos por todas sus aportaciones a este trabajo y por toda su ayuda, y al Dr. Daniel López, por todas sus aportaciones en los seminarios. Gracias también a Barri, Carmen y Elena. A Luis Antón también le agradezco las conversaciones musicales, y el haberme escuchado tanto si hablaba de ciencia como del mundo en general.
Gracias a todo el servicio de Cultivos, Lavado y Esterilización del CBM, en especial a María Ángeles, Anuncia e Irene. Gracias al servicio de Citometría de Flujo, a Berta, Silvia y Raquel.
Gracias a todo el servicio de Animalario, en especial a María Eugenia, Macarena y Fernando, por su ayuda en el aprendizaje de las técnicas. Gracias a cualquier persona del CBM que nos haya dejado un reactivo, o contestado cualquier pregunta, que no son pocos. En especial, gracias al laboratorio del Dr. Alarcón, al de la Dra. Toribio y sobre todo al del Dr. Cobaleda, por la cesión de animales y material.
Gracias a Silvia, mi vecina de laboratorio, y a Yolanda, la chica que siempre veía en los conciertos, por hacer los días siempre más agradables. Gracias también a las dos por toda vuestra ayuda en este tramo final, que también es el vuestro. Gracias a todos los compañeros del P2 virus, el sitio donde más tiempo pasé durante la tesis.
Gracias al Dr. José Yélamos, por la cesión de los ratones deficientes en PARP, y por todas sus aportaciones. Gracias al laboratorio del Dr. Mariano Esteban, por proporcionarnos los
protocolos iniciales para la cuantificación de anticuerpos anti-VACV. Gracias a mi tutor, el Dr.
Pablo Gómez del Arco, por su ayuda y disponibilidad con los papeleos.
I would like to thank Dr. Stipan Jonjić and Dr. Silvia Vidal, for hosting me in their laboratories during my short stays and giving me the opportunity to learn from them. Thanks also to Dr.
Stipan Jonjić for providing us with the MCMVs in the beginning. Thanks also to Dr. Finn Grey, from Roslin Institute, for providing us with the MCMV-GFP-SIINFEKLie2.
Thanks to all the members of the Department of Histology and Embryology/Center for Proteomics in the University of Rijeka: Ana Lesac, Ana Marković, Ani, Branka, Božo, Danica, Daria, Dijana, Edi, Filip, Ilija, Inga, Iva, Ivana, Jelena, Josipa, Karmela, Kristina, Lea, Maja, Marko, Marta, Mia, Mijo, Olga, Paola, Sali, Sonja, Suzana, Tihana, Tina Jenus, Tina Rudančić and Vedrana. Puno hvala! Especially I would like to thank Tihana, the person who started working in the project with Nras-deficient mice and MCMV. Thanks for teaching me so many techniques and concepts, for hosting me so nicely in Rijeka and for getting involved with my experiments. Also thanks to Dijana and Edi, for showing me how to prepare MCMV stocks. To all the professors: Dr. Polić, Dr. Pernjak Pugel, Dr. Tomac, Dr. Lenac Roviš, Dr. Juranić Lisnić, Dr.
Lisnić, Dr. Wensveen and especially, to Dr. Krmpotić for showing me how to perform intravenous injections, for injecting my mice and for contributing ideas in all the meetings.
Thanks to Martina, my short stay and student dormitory companion, and to Hermina, for sharing the adventure in Rijeka.
Thanks to all the members of the Vidal Lab: Ben, Gab, Mathieu, Nat, Patricia, Priya and Salma. Merci beaucoup! Especially to Ben, for having always all the materials ready for the experiments, to Patricia for infecting all my mice and taking care of all the animal issues, to Nat for the good times in the cell culture room, and to Gab and Mathieu for their help with mice.
Gracias a mis amigos, David y Luis (Roque&Roll!), Mafalba, Lucas, Mónica, Bea y Sheila.
A tolos Méndez-Alonso-Rodríguez en xeneral (si escribo los nomes ún per ún salme más llargo que la tesis entera). A Clari, Nibarón, Ovi, Desi, Olegario y Andrea. A Migue, María, Dulce y Félix. A Norina y a Pepe. Y sobre too, a mio güelita y a mio ma.
Al mejor compañero de aventuras que podría haber encontrado. Podría escribir muchas tonterías para agradecerte todo lo que me apoyas (y me aguantas) y la suerte enorme que tengo de compartir contigo el día a día, pero lo voy a resumir en que te dedico esta tesis con my best wishes como los escritores famosos, pero escribiendo bien tu nombre. ¡Gracias Alonso!
Vaccination is the most efficient strategy to confer protection against various human pathogens. However, to this date there are still no vaccines available against some chronic pathogens of high sanitary impact. Our goal was to study immune responses against viral infections in order to obtain potentially useful information for the design of new vaccination strategies, focusing on vaccinia virus (VACV) and murine cytomegalovirus (MCMV) vectors. As VACV was the vaccination agent used for the eradication of smallpox, it is one of the best models to study host-pathogen interactions. MCMV is one of the most promising candidates in order to develop CD8+ T lymphocyte-based vaccines, which are necessary against pathogens that depend on a cellular immune response for their elimination.
First, we studied the initiation of the cellular immune response and the humoral response after VACV infection in different mouse models with genetic deficiencies associated with impaired cellular immunity. Our most relevant finding was that the absence of PARP-2 in T lymphocytes in a PARP-1 deficient background causes a strong reduction of anti-VACV antibody responses. Since PARP-1 and PARP-2 are targets for cancer therapy as they are involved in DNA repair and in maintenance of genomic stability, this might be relevant for potential side effects in patients.
Then, we analyzed the capacity of MCMV vaccine vectors to induce protection in a mouse model of defective CD8+ T lymphocyte memory responses, observing that MCMV can overcome situations of deficient immune memory. In order to describe the molecular mechanism of this phenomenon, we have analyzed in detail the CD8+ T lymphocyte response induced by MCMV vectors in these animals in terms of phenotype, kinetics and function;
together with MCMV viral titers in organs and the phenotype and function of dendritic cells and NK cells.
Furthermore, we analyzed the CD8+ T lymphocyte response induced by an MCMV lacking an immune evasion protein that interferes with MHC class I-restricted antigen presentation, observing alterations in the magnitude and kinetics of the CD8+ T lymphocyte response against several epitopes. These findings might contribute to describe the mechanisms behind the generation of anti-MCMV CD8+ T lymphocyte responses, which are unique due to their strength and persistence.
La vacunación es la estrategia más eficiente para conferir protección frente a patógenos humanos. Sin embargo, actualmente no hay vacunas disponibles frente a patógenos crónicos de alto impacto sanitario. Nuestro objetivo fue el estudio de la respuesta inmunitaria frente a infecciones virales para obtener información útil en el diseño de nuevas estrategias de vacunación, centrándonos en el virus vaccinia (VACV) y el citomegalovirus murino (MCMV).
VACV es uno de los mejores modelos para estudiar las interacciones entre hospedador y patógeno, ya que fue el agente utilizado para erradicar la viruela. MCMV es uno de los candidatos más prometedores en el desarrollo de vacunas basadas en linfocitos T CD8+, útiles frente a patógenos que dependen de una respuesta inmunitaria celular para su eliminación.
Comenzamos estudiando la iniciación de la respuesta inmunitaria celular y la respuesta humoral tras la infección por VACV en modelos de ratón con deficiencias genéticas asociadas a defectos en la inmunidad celular. Nuestra observación más relevante fue el hecho de que la ausencia de PARP-2 en linfocitos T combinada con una deficiencia sistémica en PARP-1 causa una fuerte reducción de la respuesta de anticuerpos frente a VACV. Como PARP-1 y PARP-2 son dianas terapéuticas en cáncer debido a su papel en la reparación del DNA y el mantenimiento de la estabilidad genómica, esto podría ser relevante para efectos secundarios potenciales en pacientes.
Posteriormente analizamos la capacidad protectora de los vectores vacunales de MCMV en un modelo de ratón que presenta un defecto en la respuesta de linfocitos T CD8+ de memoria, observando que MCMV puede superar situaciones de deficiencia en la respuesta inmunitaria de memoria. Para describir el mecanismo molecular de este fenómeno, hemos analizado en detalle la respuesta de linfocitos T CD8+ inducida por los vectores vacunales de MCMV en estos animales centrándonos en su fenotipo, cinética y función; junto con los títulos virales de MCMV en órganos y el fenotipo y la función de las células dendríticas y de las células NK.
Además, hemos analizado la respuesta de linfocitos T CD8+ inducida por un MCMV que carece de una proteína de evasión inmunitaria que interfiere con la presentación de antígenos restringida por MHC de clase I, observando alteraciones en la magnitud y en la cinética de la respuesta de linfocitos T CD8+ frente a varios epítopos. Estas observaciones pueden contribuir a la descripción de los mecanismos de generación de respuestas de linfocitos T CD8+ frente a MCMV, que son únicas debido a su potencia y persistencia.
2- INTRODUCTION ... 27
2.1- Vaccination: achievements and remaining challenges ... 27
2.2- Antibody responses ... 28
2.3- CD8+ T lymphocyte responses ... 29
2.3.1- Mechanisms of antigen presentation to CD8+ T lymphocytes ... 30
2.3.2- Generation of memory CD8+ T lymphocytes ... 31
2.4- Poly (ADP-ribose) polymerases (PARPs) and their roles in the immune system ... 33
2.5- The role of Nras in T lymphocyte biology ... 35
2.6- Vaccinia virus (VACV) ... 37
2.6.1- Life cycle and gene expression kinetics ... 38
2.6.2- Immune response against VACV ... 39
2.7- Murine cytomegalovirus (MCMV) ... 40
2.7.1- Life cycle and gene expression kinetics ... 42
2.7.2- Immune response against MCMV ... 44
3- OBJECTIVES ... 51
4- MATERIALS AND METHODS ... 55
4.1- Chemical compounds ... 55
4.2- Mouse models ... 55
4.2.1- Congenic strains ... 55
4.2.2- Transgenic strains ... 55
4.2.3- Strains obtained by N-ethyl-N-nitrosourea (ENU) mutagenesis ... 56
4.3- Cells and culture media ... 57
4.3.1- Cell lines ... 57
4.3.2- Primary cells ... 57
4.3.3- Lymphocytes ... 59
4.4- Flow cytometry analysis ... 59
4.4.1- Antibodies, dextramers and other staining reagents ... 59
4.4.2- Surface, intracellular and intranuclear stainings ... 60
4.4.3- Acquisition and analysis ... 61
4.5- Vaccinia virus (VACV) ... 62
4.5.1- Mouse infections and organ titrations ... 62
4.5.2- Cell infections ... 63
4.5.3- Analysis of anti-VACV antibody response ... 63
4.6- Murine cytomegalovirus (MCMV) ... 64
4.6.1- Mouse infections and organ titrations ... 64
4.6.2- Cell infections ... 65
4.7- Peptides ... 66
4.8- Priming capacity of infected BMDCs to naïve CD8+ T lymphocytes ... 66
4.9- Depletion of lymphocyte subsets ... 67
4.10- Adoptive transfers of CD8+ T lymphocytes... 67
4.11- Statistical analysis ... 68
5- RESULTS ... 73
5A- ANALYSIS OF THE INITIATION OF THE CELLULAR IMMUNE RESPONSE AND THE ADAPTIVE HUMORAL RESPONSE IN ANIMAL MODELS WITH GENETIC DEFICIENCIES ASSOCIATED WITH IMPAIRED CELLULAR IMMUNITY ... 73
5.1- Basic characterization of BMDCs from different mouse models in a context of VACV infection ... 73
5.2- Humoral immune response against VACV infection in different mouse models ... 78
5B- IMPROVEMENT OF THE CD8+ T LYMPHOCYTE MEMORY RESPONSE IN NRAS-DEFICIENT MICE BY MCMV VACCINATION ... 82
5.3- Evaluation of the protective capacity of MCMV vaccine vectors against VACV infection in Nras-/- mice ... 82
5.4- Study of MCMV viral replication kinetics in Nras-/- mice ... 88
5.4.1- In vivo MCMV titers ... 88
5.4.2- Evaluation of MCMV replication in Nras-/- cells: viral production by infected macrophages ... 90
5.5- Study of anti-MCMV immune responses in Nras-/- mice ... 91
5.5.1- Characterization of DCs in a context of MCMV infection in Nras-/- mice... 91
5.5.2- Detailed study of the CD8+ T lymphocyte response after MCMV infection in Nras-/- mice ... 98
5.5.3- NK cells after MCMV infection in Nras-/- mice and their influence on CD8+ T lymphocyte response ... 115
5.6- Towards a mechanism of memory rescue: MCMV upregulates Eomes expression and restores the competitive disadvantage in antigen-specific Nras-/- CD8+ T lymphocytes ... 120
5.7- Characterization of anti-MCMV immune responses in other mouse models: comparison with Nras-/- mice as an approach to establish a molecular mechanism of memory rescue ... 125
5.7.1- CD8+ T lymphocytes ... 125
5.7.2- NK cells ... 130
6.1- Immune responses against VACV ... 139
6.1.1- Relevance of the study of anti-VACV immune responses ... 139
6.1.2- PARP proteins in T lymphocyte biology and anti-VACV immune responses ... 140
6.2- Achievements in the Nras-/- mouse model: MCMV can overcome situations of deficient CD8+ T lymphocyte memory ... 141
6.2.1- Searching for a mechanism of memory rescue: compared anti-MCMV immune response between Nras-/- and susceptible/resistant strains ... 142
6.2.2- Hypothesis: can enhanced TLR9 signaling in the absence of Nras be the missing piece of our puzzle? ... 143
6.3- Antigen presentation to CD8+ T lymphocytes in MCMV infection: the immune evasion paradox and the effects of m152 deletion ... 149
7- CONCLUSIONS ... 157
7- CONCLUSIONES ... 158
8- REFERENCES ... 163
SECTION 1 ABBREVIATIONS
1- ABBREVIATIONS aa Amino acid
ABC ATP-binding cassette ADP Adenosine diphosphate
AID Activation-induced cytidine deaminase Akt Protein kinase B
AP-1 Adaptor protein 1 APC Allophycocyanin
ATP Adenosine triphosphate BCL-6 B-cell lymphoma 6 protein BCR B cell receptor
BLIMP-1 B lymphocyte-induced maturation protein 1 BMDC Bone marrow derived dendritic cell
BSA Bovine serum albumin CD Cluster of differentiation cDC Conventional dendritic cell
cDNA Complementary deoxyribonucleic acid CEV Cell-associated enveloped vaccinia virus CMV Cytomegalovirus
CSR Class-switch recombination CTL CD8+ cytotoxic T lymphocyte Cy Cyanine
DC Dendritic cell
DMEM Dulbecco’s modified Eagle’s Medium DMSO Dimethyl sulphoxide
DNA Deoxyribonucleic acid dpi Days post infection
DRiP Defective ribosomal product
E Early
EDTA Ethylenediaminetetraacetic acid EE Early effector
EEV Extracellular enveloped vaccinia virion EGFP Enhanced green fluorescent protein ELISA Enzyme-linked immunosorbent assay
ENU N-ethyl-N-nitrosourea Eomes Eomesodermin
ER Endoplasmic reticulum
ERAAP Endoplasmic reticulum aminopeptidase associated with antigen processing FBS Fetal bovine serum
FITC Fluorescein isotiocyanate flOVA Full-length ovalbumin GC Germinal center
GDP Guanosine diphosphate
GDS Guanine nucleotide dissociation stimulator GFP Green fluorescent protein
GM-CSF Granulocyte-macrophage colony stimulation factor gMFI Geometric mean of fluorescence intensity
Gnl1 G protein nucleolar 1 GTP Guanosine-5'-triphosphate HCMV Human cytomegalovirus HCV Hepatitis C virus
HIV Human immunodeficiency virus HSV Herpes simplex virus
ID Inhibitor of DNA-binding/differentiation proteins IE Immediate early
IEV Intracellular enveloped vaccinia virion IFN Interferon
Ig Immunoglobulin IL Interleukin
IMV Intracellular mature vaccinia virion ip Intraperitoneal
IRAK IL-1R-associated kinase IRF Interferon response factor IV Immature vaccinia virion iv Intravenous
KLRG1 Killer cell lectin-like receptor subfamily G member 1
L Late
LMA Low-melting point agarose LPS Lipopolysaccharide
MCMV Murine cytomegalovirus MEF Mouse embryonic fibroblast MHC Major histocompatibility complex MIE Major immediate early
MOI Multiplicity of infection MPEC Memory precursor effector cell mRNA Messenger ribonucleic acid
mTORC1 Mammalian target of rapamycin complex 1 MVA Modified Vaccinia Ankara
MyD88 Myeloid differentiation factor 88 NAD Nicotinamide adenine dinucleotide NFAT Nuclear factor of activated T lymphocytes
NF-κB Nuclear factor kappa-light-chain enhancer of activated B lymphocytes NK Natural killer
NKG2D Natural killer group 2 member D NT Neutralization titer
NYVAC New York Vaccinia attenuated from Copenhagen ORF Open reading frame
OVA Ovalbumin
pAPC Antigen presenting cell PARP Poly(ADP-ribose) polymerase PBS Phosphate-buffered saline PCR Polymerase chain reaction pDC Plasmacytoid dendritic cell PE Phycoeritrin
PEC Peritoneal exudate cells
PerCP Peridinin chlorophyll protein complex PFA Paraformaldehyde
PFU Plaque-forming unit
PI3K Phosphatidylinositol-3-kinase PMA Phorbol 12-myristate 13-acetate RAE Retinoic acid early inducible RNA Ribonucleic acid
RPMI Roswell Park Memorial Institute rVACV Recombinant vaccinia virus
SEM Standard error of the mean SHM Somatic hypermutation SLEC Short lived effector cell
STAT Signal transducer and activator of transcription TAP Transporter associated with antigen processing T-bet T-box expressed in T lymphocytes
TCM Central memory T lymphocytes TCR T cell receptor
TEL Transcript expressed in latency TEM Effector memory T lymphocyte TH Helper CD4+ T lymphocyte TIR Toll-IL-1-resistance TLR Toll-like receptor TNF Tumor necrosis factor
TNP-KLH Trinitro-phenyl keyhole limpet hemocyanin TRAF TNF-receptor associated factors
TRM Tissue-resident memory T lymphocyte TSCM Stem memory T lymphocyte
VACV Vaccinia virus
vRAP Viral regulator of antigen presentation VSB Virus sucrose buffer
WR Western Reserve vaccinia virus strain YRG YlqF related GTPase
β-ME β-mercaptoethanol
SECTION 2 INTRODUCTION
2- INTRODUCTION 2.1- Vaccination: achievements and remaining challenges
The induction of specific immunity by vaccination represents the most efficient approach to confer protection against various human pathogens, as it is one of the most important contributions to the improvement of sanitary conditions and the increase of life expectancy in the Western world (Andre, 2003). In fact, vaccination is the most important contribution of immunology to human health (Sallusto et al., 2010) and the most cost-effective life saving device in history (Pulendran and Ahmed, 2011).
Most vaccines that are efficient against severe diseases caused by viruses target those producing a strong cytopathic infection, where the infected cell breaks up and large amounts of infectious viral progeny particles are released. These vaccines induce a strong humoral immune response based on neutralizing antibodies that bind and neutralize extracellular infectious viral particles. This includes vaccines against bacterial toxins, measles and poliomyelitis (Zinkernagel, 2003). However, humoral immunity is not effective enough against pathogens that rapidly gain mutations throughout their genomes or intracellular pathogens that persist within the infected cell and depend on a potent cellular immune response for their elimination (Zinkernagel, 2003; Butler et al., 2011). Among these pathogens there are some viruses with high sanitary impact such as hepatitis C virus (HCV) or human immunodeficiency virus (HIV). Therefore, there is a strong need to develop new vaccination strategies based on the induction of strong and long-lasting cellular immune responses. The induction of cytotoxic CD8+ T lymphocytes (CTLs), which possess the ability to specifically recognize cells infected with intracellular pathogens, is on the top of the most promising future vaccine strategies (Trsan et al., 2013). Although the ability to generate large numbers of quality CD8+ T lymphocytes is problematic, in the last years there have been relevant contributions such as the last studies concerning the generation of memory CD8+ T lymphocytes and new vaccination strategies based on the use of attenuated viral vectors expressing proteins from the pathogen of interest.
This introduction is focused on the description of antibody and CD8+ T lymphocyte responses, together with molecules that are a relevant target for research on antiviral immune response with available mouse models. The main characteristics of vaccinia virus (VACV) and cytomegalovirus (CMV) infection are also described: VACV represents the most successful achievement in the history of vaccination, while CMV represents some of the major challenges for future vaccine design.
2.2- Antibody responses
The role of the humoral immune response is the protection of the extracellular spaces through the production of antibodies (also called immunoglobulins (Igs); Figure 2.1) by terminally differentiated B lymphocytes which are denominated plasma cells. Antibodies have three main functions in the immune response against a pathogen: neutralization of the toxic effects or infectivity by direct binding to the pathogen (neutralizing antibodies), induction of the internalization and destruction of pathogens by cells through binding to Fc receptors (opsonization) and activation of the complement system (Janeway et al., 2001).
The activation of B lymphocytes for further antibody production is initiated by the binding of a specific antigen to the B cell receptor (BCR). The BCR is composed of two parts: a membrane- bound Ig molecule of one isotype, which is identical to a monomeric version of the secreted Ig form with the exception of the presence of an integral membrane domain, and an intracellular signal transduction moiety (Dal Porto et al., 2004). Although B lymphocytes can recognize soluble antigens, the recognition of antigens bound to the membranes of cells such as dendritic cells (DCs; Huang et al. (2005)) and macrophages (Koppel et al., 2005) seems to be the predominant form of activation in vivo (Carrasco and Batista, 2006; Depoil et al., 2008) and the most efficient (Batista and Neuberger, 2000; Batista et al., 2001). These cells might use a combination of lectin receptors, complement receptors and/or Fc receptors, and might internalize antigens into non-degradative endosomes prior to their recycling to the cell surface, as B lymphocytes recognize antigen in its unprocessed native state (Batista and Harwood, 2009).
After binding of the antigen, the BCR induces signaling cascades for the proliferation and differentiation of the B lymphocyte and, at the same time, induces the internalization of the antigen by receptor-mediated endocytosis. Endocyted antigens are then partially degraded and recycled to the cell surface in the form of peptides bound to major histocompatibility complex (MHC) class II molecules, and are presented to helper CD4+ T lymphocytes (TH) that
Figure 2.1. Structure of an Ig molecule. Igs have a Y shape with two arms, the Fab fragments, containing identical antigen binding sites.
Each Ig molecule has two identical heavy and light chains, and each chain has an N-terminal variable domain. Light chains have one constant domain, while heavy chains can have three or four.
Depending on the isotype two or three of these constant domains will comprise the Fc region, which interacts with Fc receptors or complement proteins. In mice, there are five types of heavy chains that give rise to five antibody isotypes each with a different function:
α (IgA), γ (IgG), δ (IgD), ε (IgE) and μ (IgM). IgG isotype is represented.
CHL= constant domain on heavy/light chain. VHL=variable domain on heavy/light chain. Modified from Beck et al. (2010).
were previously activated by the recognition of a peptide (derived from the same antigen previously recognized by the B lymphocyte) presented by professional antigen-presenting cells (pAPCs). CD4+ TH lymphocytes differentiate mainly into TH1 or TH2 effector lymphocytes. TH1 lymphocytes activate macrophages, CTLs and B lymphocytes, while TH2 lymphocytes principally activate B lymphocytes. This helper action of CD4+ T lymphocytes facilitates the complete activation of B lymphocytes (Harwood and Batista, 2010). In addition, B lymphocytes are also capable of recognizing and responding to antigens independently of T lymphocytes (Mosier and Subbarao, 1982). While T-dependent antigens are mostly soluble proteins, T-independent antigens comprise mitogenic stimuli (such as lipopolysaccharide or DNA) that elicit polyclonal activation via Toll-like receptors (TLRs), or polysaccharides that directly engage the BCR inducing specific responses (Obukhanych and Nussenzweig, 2006).
The production of antibody-secreting plasma cells against T-dependent antigens is a two- step process, with the first and second steps providing immediate and persistent protection, respectively. In the first step, B lymphocytes differentiate into short-lived plasmablasts that secrete low and moderate-affinity antibodies. This is the source of the majority of the early protective antibodies that are produced. In the second step, some of the activated B lymphocytes enter the B follicles in secondary lymphoid organs and proliferate to form a germinal center (GC). Dividing B lymphocytes undergo class-switch recombination (CSR), a process that modulates antibody effector functions by replacing the expressed isotype while retaining the antigen-binding specificity (Stavnezer et al., 2008). Then, the B lymphocyte undergoes affinity maturation, which presents two phases: somatic hypermutation (SHM) and clonal selection. SHM introduces point mutations in the variable region of the Ig genes that encode the antibody molecules, increasing antibody affinity for antigen (Di Noia and Neuberger, 2007). B lymphocytes that have undergone SHM are then selected by their ability to bind antigens with a high affinity in the clonal selection phase. Then, B lymphocytes with high-affinity antigen receptors exit the GC and differentiate into either memory B lymphocytes or long-lived plasma cells (Nutt et al., 2015). Memory B lymphocytes can differentiate into antibody secreting plasma cells after re-exposure to antigen (Kometani et al., 2013), and long- lived plasma cells are capable of sustaining a high level of antibody secretion (Shlomchik and Weisel, 2012). This GC phase of the response provides effective protection against a future reinfection, even though it occurs in parallel with the rapid first phase (Nutt et al., 2015).
2.3- CD8+ T lymphocyte responses
CTLs screen the surface of all the cells in the organism to eliminate pathogen-infected cells, mutated or cancer cells by the recognition of peptides presented by MHC class I molecules via
the T cell receptor (TCR). To first activate naïve CD8+ T lymphocytes, a process termed priming, peptides are presented by pAPCs, which also express a combination of costimulatory cell surface and secreted molecules that are required for the initiation of a functional CD8+ T lymphocyte response. In the context of a viral infection, the recognized antigen can be originated from a viral protein synthesized and processed endogenously in the infected pAPC and presented by direct presentation, or from an endocyted exogenous viral protein or virus- infected cell and presented through cross-presentation. The most common size of the peptides recognized by CD8+ T lymphocytes is 9 amino acids (aa), and they are frequently derived from longer precursors that require a proteolytic trimming previous to MHC class I loading. The recognition of MHC/peptide complex by the TCR triggers CD8+ T lymphocyte activation and proliferation, promoting the elimination of the infected or tumor cell by cytotoxic activity or blocking infection by cytokine release.
DCs are the predominant pAPC population driving the induction of primary CD8+ T lymphocyte responses to viral pathogens in vivo (Jung et al., 2002; Zammit et al., 2005), influencing three essential elements of T lymphocyte biology: repertoire, recognition and response (Steinman, 2007). DCs bear specialized antigen-processing machinery together with costimulatory molecules that enable them to efficiently present endogenous and exogenous antigens to T lymphocytes and provide them with the necessary accessory signals to trigger their effective activation (Masson et al., 2008). Mouse DCs are divided in two broad groups, defined as plasmacytoid (pDCs) and conventional DCs (cDCs), which in turn can be divided in two subsets named type 1 and 2 (cDC1 and cDC2) (Guilliams et al., 2014). All these groups can be further subdivided into distinct subpopulations with different markers, diverse functions, distinct ontogeny, and differential turnover in vivo. pDCs produce type I interferons (type I IFNs, IFN-α/β) in response to pathogens (Asselin-Paturel et al., 2001). CD8α+ cDC1s have been recognized as the most efficient cell type in cross-presentation of exogenous antigens to CD8+ T lymphocytes (den Haan et al., 2000). On the other hand, CD11b+ cDC2s are superior in priming CD4+ T lymphocytes than cDC1s, potentially because of their prominent expression of MHC class II presentation machinery (Dudziak et al., 2007; Lewis et al., 2011).
2.3.1- Mechanisms of antigen presentation to CD8+ T lymphocytes
In the classical antigen presentation pathway, antigenic peptides derive from cytosolic degradation of endogenous proteins or defective ribosomal products (DRiPs) through the activity of the proteasome. The peptides generated in the cytosol are transported to the endoplasmic reticulum (ER) through the transporter associated with antigen processing (TAP), and must be trimmed in order to get the accurate size to bind a MHC class I molecule. Then,
peptides are loaded onto a complex formed by the nascent MHC class I heavy chains and β2 microglobulin. The stable MHC/peptide complex is further transported through the constitutive secretory pathway to the plasma membrane allowing the recognition of the cell by CD8+ T lymphocytes.
TAP is one of the central players of the classical antigen presentation pathway. It is a heterodimer formed by the gene products TAP1 and TAP2, and belongs to the family of ATP- binding cassette (ABC) transporters. TAP translocates peptides unidirectionally from the cytosol to the ER lumen in a range of 8-16 aa, although the most efficient transport is restricted to 8-12 aa (Androlewicz and Cresswell, 1994; van Endert et al., 1994). Due to its relevance, TAP appears to be one of the prime targets for viral immune evasion, as shutting down peptide supply to the ER prevents efficient MHC class I loading and subsequent antigen presentation to CD8+ T lymphocytes. In fact, TAP is the only known ABC protein that is inhibited by several viral proteins at various steps within its translocation cycle (Mayerhofer and Tampe, 2015). In addition, low to undetectable levels of TAP have been reported in primary cells and cell lines from several tumors (Leone et al., 2013), thus blocking the recognition of the tumor cells by CD8+ T lymphocytes.
Despite all these mechanisms dedicated to TAP blockade, the existence of epitopes which are presented by MHC class I molecules in TAP-deficient cells has been previously demonstrated. TAP-deficient individuals are susceptible to chronification of infections by extracellular bacteria but are capable of controlling viral infections and generating viral-specific CD8+ T lymphocytes (Gadola et al., 2000; Cerundolo and de la Salle, 2006). This indicates an efficient TAP-independent presentation of viral antigens in these patients. In addition, specific CD8+ T lymphocyte responses against several antigens have been described in TAP-deficient mouse models (Sigal and Rock, 2000; Norbury et al., 2001; Medina et al., 2009). Specifically, 12 epitopes from VACV that are presented in the absence of TAP have been identified using TAP deficient mice in Dr. Del Val’s laboratory (Lázaro, Gamarra et al., unpublished results). All these facts highlight the relevance of the study of TAP-independent antigen presentation pathways and, in general, antiviral immune response in the absence of TAP.
2.3.2- Generation of memory CD8+ T lymphocytes
A CD8+ T lymphocyte response to an acute infection can be characterized by three distinguishable phases: clonal expansion, contraction of the CD8+ T lymphocyte population and persistence of memory (Figure 2.2). As the antigen-specific CD8+ T lymphocytes clonally expand after activation, they differentiate into effector cells, many of which migrate to sites of
infection. CD8+ T lymphocytes differentiate into CTLs that kill cells infected with intracellular viruses or bacteria (through granzyme B and perforin or Fas-FasL interaction) and secrete cytokines such as IFN-γ and tumor necrosis factor (TNF). After the elimination of the infecting pathogen, specific effector CD8+ T lymphocytes undergo a contraction phase wherein the majority of them die by apoptosis (Kaech and Cui, 2012). However, memory CD8+ T lymphocytes generated in response to the initial pathogen encounter survive, providing protection against re-infection with the same pathogen and contributing to long-term immune control of chronic infection if the pathogen cannot be eliminated. After an acute infection, memory CD8+ T lymphocytes are maintained in an antigen-independent, cytokine-dependent manner mainly through the actions of IL-7 and IL-15, which promote memory CD8+ T lymphocyte survival and self-renewal through homeostatic proliferation (Surh and Sprent, 2008). Memory CD8+ T lymphocytes can also survive in situations of continuous antigen challenge (Utzschneider et al., 2013).
Figure 2.2. Kinetics of a CD8+ T lymphocyte response against a viral infection. During an acute viral infection, antigen-specific CD8+ T lymphocytes rapidly proliferate during the expansion phase and differentiate into effector cells that mediate viral clearance. Most of these cells die the next weeks during the contraction phase. Only a small percentage of cells survive: memory CD8+ T lymphocytes. Effector CD8+ T lymphocytes can be divided into at least two subsets, memory precursor effector cells (MPECs) that can become long-lived memory CD8+ T lymphocytes and short-lived effector cells (SLECs) that do not. TEM: effector memory cells. TCM: central memory cells. Taken from Lazarevic et al. (2013).
Many different mechanisms can influence effector-memory cell fate specification in the first 24-72 hours after activation: TCR signal strength, co-stimulation, inflammatory cytokines, tissue microenvironment, metabolic regulators, asymmetry of cell division and transcription factors. These mechanisms are not mutually exclusive and act simultaneously in concert, yielding heterogeneous progeny (Chang et al., 2014). Several transcription factors that regulate effector and memory potential have been identified, most of them functioning in pairs to form counter-regulatory axes. T-bet and Eomesodermin (Eomes), two T-box transcription factors, form one of the most relevant pairs in the regulation of effector-memory balance. CD8+ T lymphocytes lacking T-bet are impaired in terminal effector differentiation (Joshi et al., 2007b), whilethe deletion of Eomes has a modest effect on the effector pool but
results in impaired CD8+ T lymphocyte memory (Banerjee et al., 2010) and failure to generate self-renewing central memory CD8+ T lymphocytes (Paley et al., 2013). The phenotype, function and fate of CD8+ T lymphocytes are acutely sensitive to the relative ratio of T-bet and Eomes (Intlekofer et al., 2005; Joshi et al., 2007b; Banerjee et al., 2010; Joshi et al., 2011): the ratio of T-bet to Eomes is highest at effector cell stages and lowest at memory cell stages.
Other pairs of transcription factors are involved in the effector/memory balance, such as BLIMP1/BCL-6 (Crotty et al., 2010), ID2/ID3 (Yang et al., 2011) or STAT4/STAT3 (Nguyen et al., 2002; Siegel et al., 2011).
Heterogeneity among memory T lymphocytes has been long recognized (Hamann et al., 1997; Sallusto et al., 1999), with the description of at least four subsets of memory CD8+ T lymphocytes whose properties are described on Table 2.1: central memory (TCM), effector memory (TEM), tissue-resident memory (TRM) and stem memory (TSCM) lymphocytes.
Interestingly, different memory subsets can have distinct transcription factor profiles: for example, differentiation towards CD8+ TRM lymphocytes requires the extinction of Eomes and a low level of T-bet expression (Mackay et al., 2015).
Cell type Location Functional properties Central memory
(TCM) Lymph nodes, spleen,
blood ↑ Proliferative potential
↑ IL-2 production
↑ Migration
↓ Effector functions and cytotoxicity Effector memory
(TEM) Spleen, blood, non-
lymphoid tissues ↓ Proliferative potential
↓ IL-2 production
↑ Migration
↑ Effector functions and cytotoxicity Tissue-resident
memory (TRM) Non-lymphoid tissues (mucosal tissues, brain)
↓ Proliferative potential
↓ IL‑2 production
↓ Migration
↑ Effector functions and cytotoxicity Stem memory
(TSCM) Lymph nodes, spleen,
blood, bone marrow Non-recirculating Capacity for self-renewal
Multipotent ability to derive TCM, TEM and effector cells Table 2.1. Memory CD8+ T lymphocyte subsets. Information extracted from Kaech and Cui (2012); Chang et al.
(2014); Gattinoni et al. (2017).
2.4- Poly (ADP-ribose) polymerases (PARPs) and their roles in the immune system PARP proteins belong to a family of enzymes that catalytically cleave β-nicotinamide adenine dinucleotide (β-NAD+) and transfer an adenosine diphosphate ribose (ADP-ribose) moiety onto residues of acceptor proteins, modifying their functional properties through (ADP ribosyl)ation (Yelamos et al., 2011; Daniels et al., 2015). This post-translational modification regulates fundamental biological functions such as cell death and survival,
telomere cohesion, mitotic spindle formation during cell division, intracellular trafficking and energy metabolism. PARP superfamily is composed of 17 members, and the most extensively studied are PARP-1 and PARP-2 (Schreiber et al., 2006).
Together with PARP-3, PARP-1 and 2 are the only known DNA damage-dependent PARPs, as they are activated by DNA breaks (Ame et al., 1999; Huber et al., 2004; Boehler et al., 2011).
PARP-1 and PARP-2 play a critical role in the response against DNA damage, causing chromatin decondensation around sites of damage, recruitment of the repair machinery and accelerated DNA repair (Yelamos et al., 2011; Bai, 2015). Specifically, PARP-1 has been demonstrated to be a survival factor that functions on the surveillance and maintenance of genome integrity (de Murcia et al., 1997; Wang et al., 1997). PARP-2 has a 69 % similarity in the catalytic domain with PARP-1 (Oliver et al., 2004), and it is required for the maintenance of genomic stability and for the efficient repair of single-stranded DNA lesions (Schreiber et al., 2002; Ménissier de Murcia et al., 2003). PARP-1 and PARP-2 can heterodimerize, and share common nuclear binding partners (Schreiber et al., 2006). Furthermore, PARP-1 and PARP-2 display partially redundant functions, as double knockout mice die at the onset of gastrulation showing that the expression of both PARPs is essential during early embryogenesis (Ménissier de Murcia et al., 2003).
Due to their roles in DNA repair and maintenance of genomic stability, PARP inhibitors that compete with β-NAD+ at the catalytic domain have gained attention as new therapeutic drugs for cancer treatment. PARP inhibitors have been applied for cancer therapy with two different strategies: as single therapy in tumors that are defective in homologous recombination and thus dependent on PARP-mediated DNA repair, or combined with DNA-damaging chemo- or radiotherapy. In both cases, the inhibition of PARP would cause chromosome instability and apoptosis (O'Connor, 2015; Sonnenblick et al., 2015). However, PARP inhibitors currently in clinical trials or approved for clinical use are still unable to discriminate between individual PARP isoforms, despite evidence that PARP proteins play specific roles in different cellular processes. PARP-1 and PARP-2 can become selectively activated by specific stimuli, have different targets and interact with specific protein partners (Isabelle et al., 2010; Troiani et al., 2011; Daniels et al., 2015; Riccio et al., 2016), suggesting distinct biological functions. For instance, PARP-2 but not PARP-1 has a role in processes that entail high levels of proliferation, such as spermatogenesis (Dantzer et al., 2006), hematopoiesis under stress conditions (Farres et al., 2013), and erythropoiesis (Farres et al., 2015).
PARP-1 and PARP-2 also seem to have differential roles in the immune system. Although peripheral T lymphocyte homeostasis seems to be normal in PARP-1 deficient mice, PARP-2 deficiency produces a significant reduction in CD4+ CD8+ double-positive thymocytes that is associated with decreased cell survival (Yelamos et al., 2006). PARP-1 is involved in the regulation of nuclear factor of activated T lymphocytes (NFAT), probably by poly(ADP-ribosyl)ation of NFAT facilitating its nuclear export during physiological T lymphocyte stimulation (Valdor et al., 2008). PARP-1 is also a negative regulator of the suppressive function of regulatory T lymphocytes (Treg) by the poly(ADP-ribosyl)ation of forkhead box protein 3 (Foxp3; Nasta et al. (2010); Rosado et al. (2013); Zhang et al. (2013); Luo et al.
(2015)). Furthermore, PARP-1 deficiency biases CD4+ T lymphocyte responses to a TH1 phenotype (Sambucci et al., 2013). In addition, PARP-1 can also indirectly activate nuclear factor kappa-light-chain-enhancer of activated B lymphocytes (NF-κB) and activator protein 1 (AP-1), both transcription factors involved in inflammatory responses (Bai and Virag, 2012;
Rosado et al., 2013).
2.5- The role of Nras in T lymphocyte biology
The members of the Ras family are small, membrane-localized guanine nucleotide binding proteins that act as a molecular switch to transduce extracellularly derived signals inside the cell. Ras proteins are regulated by a guanosine diphosphate-guanosine-5'-triphosphate (GDP- GTP) cycle, and are active when bound to GTP. In their active form, Ras proteins interact with their effectors to stimulate signaling cascades that are involved in key functions of the cell such as Golgi trafficking, proliferation and cell survival. The best characterized molecular effectors of Ras are Ral guanine nucleotide dissociation stimulators (Ral GDS), Raf kinases and phosphatidylinositol-3-kinases (PI3K) (Scheele et al., 2007). In mammals, there are at least three Ras genes that give rise to four Ras isoforms: Hras, Nras, Kras4A and Kras4B (Barbacid, 1987). These isoforms have conserved effector binding domains but differ substantially in their carboxyl-terminal region, which is important for selective membrane association, compartmentalization (Mor and Philips, 2006) and activation (Ibiza et al., 2008).
All Ras isoforms are expressed in T lymphocytes and have been collectively implicated in signaling downstream of the TCR to control T lymphocyte development and function, as they are rapidly activated after TCR engagement. This was demonstrated by using T lymphocyte cell lines (Bivona et al., 2003) or transgenic mice expressing a dominant-negative Ras protein that inhibits all four Ras isoforms (Swan et al., 1995; Fields et al., 1996). However, a preferential role of Nras in lymphocyte signal transduction was suspected since it is the main Ras isoform activated in human myeloid and lymphoid oncogenic disorders (Bos, 1989). In order to analyze
the contribution of each specific Ras isoform, single knockout mouse models were developed.
Concerning Nras knockout models (Nras-/-) it was observed that the absence of Nras did not affect mouse development, growth or fertility. In addition, these mice presented normal numbers and frequencies of immune cells such as T and B lymphocytes and macrophages (Umanoff et al., 1995). The same results were obtained for Hras-/- mice (Esteban et al., 2001).
However, deletion of Kras resulted in embryonic lethality (Johnson et al., 1997; Koera et al., 1997).
Considering all the previous information, further investigations were performed in order to assess the specific role of Nras in the immune response, using the Nras-/- mouse models and focusing on T lymphocytes. Nras-/- mice were found to be more susceptible to influenza virus infection, and this was associated to reduced numbers of NK cells, macrophages and CD8+ T lymphocytes after infection (Perez de Castro et al., 2003). Moreover, a marked upregulation of immunity-related genes such as signal transducer and activator of transcription 1 (STAT1) was detected in Nras-/- fibroblasts (Castellano et al., 2007), in addition to an upregulation of several loci related to apoptosis. Taken together, these results indicated that Nras could be involved in immune and apoptotic responses. In fact, Nras was found to be essential for the generation of CD4+ TH1 responses: in the absence of Nras, IFN-γ production by CD4+ T lymphocytes after TCR ligation was impaired, and this was associated to a reduced expression of T-bet (Iborra et al., 2011); the two essential hallmarks of TH1 CD4+ T lymphocyte responses (Szabo et al., 2003).
This defect led to an increased susceptibility of Nras-/- mice to the intracellular pathogen Leishmania major (Iborra et al., 2011). Although Nras does not play a role in normal thymocyte development and mature T lymphocyte activation (Iborra et al., 2011), its involvement in fine- tuning thymocyte selection should not be excluded, particularly in that mediated by low affinity ligands (Perez de Castro et al., 2003; Daniels et al., 2006).
Concerning CD8+ T lymphocyte responses, Dr. Del Val’s group discovered that Nras is crucial for early determination of the CD8+ T lymphocyte memory fate, using the VACV infection model (Iborra et al., 2013). It was first observed that, while the anti-VACV primary CD8+ T lymphocyte response was not impaired by the absence of Nras, the survival of anti-VACV CD8+ T lymphocytes in Nras-/- mice was reduced during the contraction phase. This observation led to a further analysis of the CD8+ T lymphocyte memory response. To do this, mice were vaccinated with DCs loaded with viral peptides prior to a VACV challenge, analyzing the secondary CD8+ T lymphocyte protective response. DC vaccination selectively induces the CD8+ compartment while providing reduced inflammatory environment, accelerating memory differentiation and secondary response (Badovinac et al., 2005). Nras-/- mice developed a
normal CD8+ T lymphocyte primary response after DC vaccination, but the memory response was deficient. After VACV challenge, Nras-/- mice failed to mount a protective CD8+ T lymphocyte secondary response, leading to a worse viral control. Both observations were linked to an impaired Eomes expression in CD8+ T lymphocytes, which is critical in determining memory commitment (section 2.3.2). Cause-effect relationship was shown by the rescue of the memory defect after Eomes overexpression in Nras-/- CD8+ T lymphocytes. Thus, it was concluded that upon antigen encounter, the active form of Nras mediates TCR signals required for Eomes upregulation through PI3Kγ and protein kinase B (Akt). Thus, Nras is a key mediator of early signals downstream the TCR that control Eomes expression, and thereby the generation of functional protective memory CD8+ T lymphocytes (Figure 2.3).
2.6- Vaccinia virus (VACV)
The greatest achievement of vaccination remains the worldwide eradication of smallpox, a disease caused by variola virus which was responsible for 8–20 % of all deaths in Europe before the introduction of vaccination (Andre, 2003). This was achieved by the use of VACV as a vaccination agent. VACV is a large and enveloped double-stranded DNA virus which, as variola virus, belongs to the Poxviridae family (genus Orthopoxvirus). The success of VACV as a vaccine against variola virus infection suggests a strong identity of antigenic peptides between the two viruses. The origin of VACV is controversial, but it is thought to derive from the cowpox or horsepox viruses (Wilkinson, 1982; Baxby, 1996; Tulman et al., 2006). Since VACV is the only vaccine that has been successful in the eradication of a human disease, it has become the best studied poxvirus and an excellent model to study virus-host interactions (Smith et al., 2013).
Nevertheless, the study of anti-VACV immune response is not only relevant because of its past success on the eradication of smallpox: VACV has experienced a renaissance of interest as a viral vector for the development of recombinant vaccines. This is due to the extensive
Figure 2.3. Nras plays a critical role as a TCR-proximal regulator of Eomes for early determination of the CD8+ T lymphocyte memory fate. Early after TCR engagement, the active form of Nras mediates signals for Eomes upregulation through PI3K and Akt.
Particularly, PI3Kγ is involved in this signaling pathway, although the involvement of other PI3K family members in Eomes regulation cannot be excluded (Delgado et al., 2009). The inhibition of mammalian target of rapamycin complex 1 (mTORC1) can partially rescue Eomes expression in the absence of Nras but only at late time points after antigen exposure. Figure based on Iborra et al. (2013).
characterization of VACV as a vaccination agent, its ability to elicit strong humoral and cell- mediated immune responses, its thermal stability and the capacity of genetically manipulate its large DNA while retaining infectivity and immunogenicity (Gomez et al., 2011a; Verardi et al., 2012). However, as VACV can cause complications in young children and immune- compromised individuals (Lane et al., 1969; Redfield et al., 1987), highly attenuated strains such as Modified Vaccinia Ankara (MVA, Sutter and Staib (2003)) and New York Vaccinia attenuated from Copenhagen (NYVAC, Tartaglia et al. (1992)) have been developed. MVA was successfully used in the last decades of smallpox eradication campaign with no reported adverse side effects (Mayr et al., 1978). In the last years, attenuated MVA and NYVAC vectors have been developed as vaccine candidates against influenza (Sutter et al., 1994; Kreijtz et al., 2009), tuberculosis (Feng et al., 2001) and Ebola virus (Lazaro-Frias et al., 2018). Remarkably, several MVA (Garcia et al., 2011; Gomez et al., 2011b; Kreijtz et al., 2014; Guardo et al., 2017;
de Vries et al., 2018) and NYVAC-based (Bart et al., 2008; Harari et al., 2008; McCormack et al., 2008; Harari et al., 2012; Bart et al., 2014) recombinant viruses have been evaluated in clinical studies as vaccine candidates against HIV and influenza A, with encouraging results.
2.6.1- Life cycle and gene expression kinetics
The infection starts when the virus enters the target cell. This step shows variation among VACV strains and cell types; and VACV Western Reserve (WR) strain, which was the only strain used in this thesis, uses a low-pH endocytic pathway to enter the target cell. Once the extracellular enveloped virion (EEV) is internalized, the nucleoprotein core is released to the cytoplasm, where the viral DNA is accessible for transcription of viral early genes. After virus entry the viral factories are formed, which are concentrated in infection-specific cytoplasmic domains and are believed to be the sites for viral assembly and viral DNA replication (Tolonen et al., 2001). The most characteristic structures of the viral factories are the crescent membranes, and after their formation, viral DNA replication and virion assembly take place. As the crescent enlarges to form the spherical immature virion (IV), the material containing core proteins and DNA is engulfed (Dales and Siminovitch, 1961). Subsequently, the IV undergoes several changes culminating in the brick-shaped infectious intracellular mature virion (IMV) (Condit et al., 2006). Most IMVs are released after lysis of the infected cell. However, some IMVs are wrapped with a double membrane from the trans-Golgi network or endosomal cisternae to form the intracellular enveloped virion (IEV), with three lipid bilayers. Then, IEVs are transported via microtubules to the periphery of the cell, where exocytosis and loss of the outer membrane occur to liberate EEVs. Alternatively, if the enveloped virion remains attached to the cell surface it is called cell-associated enveloped virus (CEV) and is propelled into
surrounding cells by growing actin tails beneath the plasma membrane (Smith and Law, 2004).
EEVs are considered to be primarily involved in dissemination within the same host, while IMVs are more suited to transmit infection between hosts (Smith et al., 2002a).
Concerning gene expression kinetics, VACV open reading frames (ORFs) are temporally regulated. Early-expressed genes do not require DNA replication to be expressed, while intermediate and late-expressed genes have a post-replicative expression. There is a cascade mechanism of regulation by which early genes are transcribed by late transcription factors present in the infecting virions, intermediate genes are transcribed by newly-expressed early transcription factors and late genes are transcribed by intermediate transcription factors (Keck et al., 1990; Baldick and Moss, 1993).
2.6.2- Immune response against VACV
As mentioned at the beginning of the present section, immunization with VACV results in long-lasting protection against smallpox. However, this was achieved without a detailed understanding of the underlying immune response (Xu et al., 2004). Both humoral and cellular immunity have been thought to play a role in protection against orthopoxviruses, as there had been individuals bearing humoral or cellular immune defects who were unable to control VACV infection (Lane et al., 1969). After many studies in different animal models, especially in mice, a detailed description of the anti-VACV immune response is now available, involving both innate and adaptive immunity. Since the immune response against extracellular virions is not sufficient to eliminate the infection as VACV can spread from cell to cell (Moss, 2006), anti-VACV responses target both extracellular virions and infected cells.
The innate immune response against VACV includes macrophages and monocytes (Hickman et al., 2013; Byrd et al., 2014), neutrophils (Jones, 1982; West et al., 1987) and natural killer (NK) cells (Brutkiewicz et al., 1992; Chisholm and Reyburn, 2006), which infiltrate the affected tissue after infection and can be observed in the infection site at early days post infection.
Regarding adaptive anti-VACV response, CD4+ T lymphocyte-dependent antibody response plays an important role on viral clearance following acute infection (Xu et al., 2004; Mota et al., 2011). Antibody response against VACV is strong in mice, although low levels of antibodies are detected 7 days after infection (Xu et al., 2004). At 14 days post infection, strong IgM and IgG responses are clearly present (Spriggs et al., 1992). One month after infection, antibody titers are even higher (Xu et al., 2004) and are maintained for more than 3 months (Coulibaly et al., 2005). The anti-VACV antibody response is almost completely CD4+ T lymphocyte-dependent, with a minor T lymphocyte-independent IgM response (Xu et al., 2004). Neutralizing antibodies
develop with a similar kinetics to overall IgG response, and are long-term maintained (Wyatt et al., 2004). By using a proteome array of VACV, antibodies against 21 different VACV proteins have been identified in serum of infected mice, which constitutes 11 % of the total VACV proteome (Davies et al., 2005a).
Anti-VACV neutralizing antibodies mainly confer protection through the recognition of structures on the surface of virus particles. Most of them are directed against IMVs, which are abundantly released when infected cells die. Neutralizing antibodies against EEVs have also been reported; however, EEVs are more fragile and less abundant than IMVs. Neutralizing antibodies against five IMV proteins and one EEV protein have been reported, but the fragility of EEVs makes difficult to measure neutralizing antibodies accurately (Amanna et al., 2006).
Antibodies against either IMV or EEV particles can be protective (Galmiche et al., 1999;
Ramirez et al., 2002; Davies et al., 2005b; Lustig et al., 2005), although in the context of prevention of host-to-host transmission antibodies against IMV are expected to play a major role in protection. The exact contributions of antibodies against IMV versus EEV are still not clearly defined in vivo, although it has been suggested that anti-IMV antibodies might protect primarily by neutralizing the virus inoculum, while anti-EEV antibodies would limit viral spread after infection (Law et al., 2005; Amanna et al., 2006).
VACV-specific CD4+ and CD8+ T lymphocytes peak 7 days after infection and stable numbers can be still detected from 1 to 7 months after infection (Harrington et al., 2002). In addition to their role as helpers in B lymphocyte activation, CD4+ T lymphocytes also produce cytokines such as IFN-γ, TNF-α and IL-2. CD8+ T lymphocytes can eliminate infected cells and have an important role in protection against VACV-induced disease in mice and after a secondary infection, and could be crucial when humoral response is deficient (Xu et al., 2004). Direct antigen presentation by infected pAPCs is sufficient for the activation of VACV-specific CD8+ T lymphocytes in mice infected with WR strain (Xu et al., 2010), although cross-presentation plays an important role when using the non-replicative VACV strain MVA (Gasteiger et al., 2007). There is a large expansion of VACV-specific CD8+ T lymphocytes following infection, which exhibit high levels of cytotoxicity ex vivo (Harrington et al., 2002).
2.7- Murine cytomegalovirus (MCMV)
Cytomegalovirus is a genus of double-stranded DNA viruses of the subfamily Betaherpesvirinae in the family Herpesviridae. MCMV was first isolated from salivary glands of mice and propagated in cell culture of murine cells by Dr. Margaret G. Smith in 1954. The prototype strain is named after her and is still in use as Smith strain (Smith, 1954). Two years
later, Dr. Smith and others reported on the isolation of human cytomegalovirus (HCMV) and its propagation in cell culture (Rowe et al., 1956; Smith, 1956; Craig et al., 1957). It was early observed that MCMV could not be propagated in human tissue, as well as HCMV did not replicate in murine cells (Weller, 1970). This was confirmed over the years: CMVs are highly species-specific and only replicate in cells of their own host or closely related species (Ostermann et al., 2015), excluding the study of HCMV in animal models. This makes MCMV the most used CMV in research, as it is a convenient virus-host model for studying biology and pathogenesis of CMV infection, as well as the principles of its immune control (Koszinowski et al., 1990). The primary infection of an immunocompetent host is usually asymptomatic, and the virus replicates to a low level in infected tissues. Immune control mechanisms eliminate the infectious virus from tissues but not the viral genome, and this leads to a non-infectious, replicatively dormant state known as viral latency (Seckert et al., 2012).
The majority of the world’s population is infected with HCMV. In Western countries the prevalence of HCMV infection is on average 60 %, and increases to higher than 90 % in those aged over 80 years; in developing countries the infection rate is greater than 90 % at a young age (Gandhi and Khanna, 2004; Cannon et al., 2010; Arens et al., 2015). HCMV establishes a latent infection that generally remains asymptomatic in immune-competent hosts for decades.
However, HCMV is an important cause of morbidity and mortality in congenitally infected (Stagno et al., 1986; Dollard et al., 2007) and immunosuppressed individuals such as transplant recipients, both of solid organ (Limaye et al., 2006) or hematopoietic cell transplantation (Ljungman et al., 2011). In fact, HCMV is the leading non-genetic cause of congenital malformation in developed countries (Cannon and Davis, 2005; Kenneson and Cannon, 2007;
Hamilton et al., 2014). HCMV is also suspected to be involved in several medical conditions such as deterioration while in intensive care (Kalil and Florescu, 2009; Osawa and Singh, 2009).
Moreover, even in apparently healthy hosts, the persistence of HCMV can impact the modulation of innate and adaptive immune responses. Although no causal relationship has been found up to date, HCMV infection has been linked to immunosenescence, which is the age-associated dysregulation and dysfunction of the immune system (Koch et al., 2007;
Sansoni et al., 2014; Weltevrede et al., 2016; Redeker et al., 2017). Furthermore, the presence of high titers of anti-HCMV antibodies in elderly cohorts (over 70 years old) has been associated with a higher risk of mortality (Strandberg et al., 2009; Wang et al., 2010).
The development of a vaccine against HCMV is an immense public health challenge (Arvin et al., 2004). Despite efforts, no HCMV vaccine is currently available (Plotkin, 2015). Interestingly, CMV is also relevant as a vaccine vector against other pathogens due to its ability to induce
CD8+ T lymphocyte responses (section 2.7.2) and its large genome, which allows the insertion of multiple foreign genes. CMV-based vaccine vectors have been successfully tested in animal models against bacterial (Trsan et al., 2013; Beverley et al., 2014) and tumor targets (Klyushnenkova et al., 2012; Trsan et al., 2017); and against viral infections such as herpes simplex virus 1 (HSV-1; Dekhtiarenko et al. (2013)), simian immunodeficiency virus (Hansen et al., 2009; Hansen et al., 2011; Hansen et al., 2013) and Ebola virus (Tsuda et al., 2011; Tsuda et al., 2015). All these vectors induce a protective response based on CD8+ T lymphocytes, and most of them express a single MHC class I-restricted epitope.
2.7.1- Life cycle and gene expression kinetics
In general, β-herpesviruses have a long replication cycle and slow cell to cell spread in culture. The infected cell usually becomes enlarged (cytomegalia) and production of infectious progeny is always accompanied by the destruction of the infected cell. MCMV and HCMV have essentially the same replication cycle, and cause similar effects in the respective host cells (Mocarski et al., 2006). CMV infects cells by a serial process including attachment, fusion of the viral envelope with the cell membrane, penetration, dismantling and transportation of the capsid to the nuclear membrane and entrance of the viral DNA to the cell nucleus where it becomes transcriptionally active (Lucin and Jonjic, 1995). Independently of the infected cell type or virus strain, CMV gene expression is carried out in a coordinated regulated manner during productive infection (Mocarski et al., 2006). According to expression kinetics, CMV genes can be divided into immediate-early (IE), early (E) and late (L) (Honess and Roizman, 1974; Emery and Griffiths, 1990). Particularly, in MCMV the genes ie1/3 and ie2 comprise the major IE locus (MIE), which is transcriptionally regulated by the MIE promoter and the MIE enhancer. The IE phase starts directly after nuclear entry of the viral DNA. The E phase is induced by the IE gene products, which act as transcription factors (Keil et al., 1984). The corresponding E gene products are necessary for viral DNA replication, which is carried out according to the principle of rolling circle, producing concatenates that are later cleaved (Marks and Spector, 1988). The synthesis of progeny DNA is the requisite for the start of the L phase (Keil et al., 1984; Lucin et al., 1994), with the generation of structural proteins that are required for the assembly of virus particles (Mocarski et al., 2006).
In the nucleus of the infected cell, a nuclear inclusion body composed of granular and fibrilar material develops through the E phase, representing a matrix for nucleocapsid assembly (Cavallo et al., 1981), which is the earliest step in CMV morphogenesis. Once the DNA is packed into the capsid shell, completely formed nucleocapsids migrate through the nuclear membrane, acquiring a transient envelope of inner nuclear membrane that fuses with
