Rozprawy doktorskie na temat „Cytomegaloviruses”

The gene products produced by murine cytomegalovirus (MCMV) in infected cells prior to viral DNA synthesis are believed to control the interaction of the virus with the cells, determining whether a permissive infection results, with virus replication, or whether further virus gene expression is inhibited, resulting in a latent or abortive infection. The aim of this study was to characterize the early viral gene products that are produced in permissive and nonpermissive cells. The proteins produced in 3T3-L1 cells, permissively infected with MCMV, during the first six hours of infection (the period prior to viral DNA replication) were characterized by polyacrylamide gel electrophoresis. Ten of the proteins were classified as immediate-early (IE) and seven as early according to their time of synthesis and also according to their synthesis in the presence of actinomycin D following the reversal of a cycloheximide mediated block in protein synthesis. The estimated molecular weights ranged from 28K - 100K. The synthesis of a dominant IE protein of 100K was significantly increased, after the reversal of a cycloheximide block, compared to unenhanced conditions. The synthesis of two other major IE proteins of 96K and 89K were also significantly enhanced by this treatment. The 100K and 89K proteins partitioned with the nuclear, cytoplasmic and cytoskeletal fractions, while the 96K protein partitioned more strongly with the nuclei. These proteins were phosphorylated. The other IE proteins were synthesized in lesser amounts. The major early proteins, which had molecular weights of 39K and 36K, were also phosphorylated and were exclusively nucleus-associated. A number of the IE and early proteins had affinity for native and denatured DNA-cellulose. The same major IE and early proteins were identified in nonpermissively infected J774A.1 macrophage cells. Although 0.6% of these cells became permissively infected with MCMV and the rest appeared to be nonpermissively infected, viral DNA and late protein synthesis was not detected. The major difference between the proteins produced in 3T3-L1 cells and J774A.1 cells was the affinity of the 96K protein for denatured DNA-cellulose, which was only observed when the protein was synthesized in J774A.1 cells. The main IE and early MCMV induced proteins were also synthesized in nonpermissively infected human fibroblast cells. The only difference between the proteins produced in these cells and 3T3-L1 cells was that the 100K IE protein appeared to have a greater nuclear-affinity, when produced in the human fibroblasts, than was found when synthesized in infected 3T3-L1 cells. In conclusion, a larger number of IE and early MCMV-induced proteins were identified in infected cells than had been previously characterized. There was no evidence of restricted MCMV gene expression occurring in two different cell types that were nonpermissively infected. This appeared to indicate that, in the nonpermissive experiments described, MCMV replication was inhibited at the stage of viral DNA synthesis.
Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate

Murine cytomegalovirus (MCMV) is used as a biological model for human cytomegalovirus (HCMV). Latency, persistence and reactivation are same of the important aspects of the murine model that share analogies with human CMV infections. In order to elucidate the molecular mechanisms leading to these events, in-depth analyses of the murine model are required at the transcriptional level. During the MCMV replication cycle, there is a sequential expression of different regions of the viral genome, hence the transcripts are divided into three kinetic classes; the immediate early (IE), early (E) and late (L). This study presents the analyses of MCMV (Smith strain) transcripts of the major IE and E transcriptional units, and a more detail analysis of one of the major E regions, E10. The IE and E transcripts were studied by probing them with Ctoitplementary DNAs (cDNAs). The cDNAs were prepared from mRNA isolated from the IE and E phases of the viral replication cycle and cloned into the bacteriophage Lambda gt10. Ten E cDNAs were mapped to specific locations of the virus genome, and these represented transcripts from the major E regions in Hindlll fragments A, B, E, F, and I-J. Five E cDNAs, each representing a different major E region, and two IE cDNAs representing the major IE region, were applied as probes in one of the studies to determine the relative transcript levels during the course of infection of 3T3L1 fibroblast cells with MCMV. The major E transcriptional units were investigated further in a study where Northern blots of RNAs, isolated from different phases of the viral replication cycle, were probed with the five E cDNAs. This study revealed transcripts that were temporally regulated since they were present only during the E and usually L phases of the viral replication cycle. In addition, the quantities of these transcripts varied depending on the phase. However, all five cDNAs detected more than one transcript which indicates complex splicing events, overlapping genes, multiple initiation sites and/or the presence of gene(s) in the complementary DNA strand. One of the E cDNAs, E10, corresponding to a transcript from a major E region of Hindlll fragment I-J, was selected for further analysis. The E10 cDNA detected four transcripts of 9.5, 6.9, 4.7 and 2.1 kb in size, which were found to be transcribed from the same DNA strand. The DNA sequence of this E10 cDNA was determined and shown to contain 3223 nucleotides, however it lacked a polyadenylation signal and a poly A tract at the 3' end. The missing 3' terminus, designated as E10-A, was isolated using the polymerase chain reaction (PCJR) method and its DNA sequence of 1422 nucleotides was also determined. The combined sequence of E10 and E10-A (total of 4606 nucleotides) was designated as E10-C and is presented in this thesis. The E10-C cDNA (4.6kbp) most likely represents the 4.7 kb transcript. The E10-C cDNA sequence has one minor and one major open reading frame (ORF). The minor ORF is initiated by the first ATG triplet (nucleotide position 114) while the major ORF is initiated by the second triplet (nucleotide position 155). Since the sequence preceeding the second ATG triplet is in "good context" with regard to the translation initiation consensus sequence, it is most likely that the major ORF is translated. The major ORF (3600 bases) encodes a 1200 amino acid polypeptide, the putative E10 protein of approximately 135 kd in size. A protein close to that size was detected in one of the experiments in which RNAs, that were hybrid-selected by the E10 cDNA and eluted, were translated in vitro. The putative E10 protein lacks homology with any other protein in the data banks (SWISSPRT and GENPEPT). Portions of the viral genomic fragments Hindlll I and J were also sequenced to reveal the orientation of the gene coding for the E10 cDNA and its related transcripts.
Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate

Thesis (Ph. D.)--School of Pharmacy. University of Missouri--Kansas City, 2007.
"A dissertation in pharmaceutical sciences and pharmacology." Advisor: Ashim K. Mitra. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed May 23, 2008. Includes bibliographical references (leaves 210-225). Online version of the print edition.

Thesis (Ph.D.) - University of Glasgow, 2008.
Ph.D. thesis submitted to the Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, 2008. Includes bibliographical references. Print version also available.

Human cytomegalovirus (CMV) is a significant infectious agent causing disease in immunocompromised HIV-infected patients, transplant recipients, and neonates. The current antiviral therapeutic strategy against CMV is limited in its utility due to the inherent toxicity and lack of bioavailability of currently available anti-CMV agents, ganciclovir (GCV), cidofovir (CDV), and foscarnet (FOS). The development of the prodrug of GCV, valganciclovir (val-GCV), has vastly improved the bioavailability profile of GCV. However, val-GCV demonstrates limited effectiveness against tissue-invasive CMV diseases as side effects involved with traditional intravenously administered GCV such as haematologic and reproductive toxicities remain. In addition, the emergence of antiviral resistant CMV mutant strains due to prolonged treatment with currently available antivirals necessitates the development of novel anti-CMV agents with reduced toxicity and improved bioavailability. In this study, select groups of novel compounds were analysed for their potential for further development as anti-CMV agents. Three groups of compounds were identified based on two screening methods which included the computer simulated screening process of compounds known as in silico screening and the traditional method of random screening. The first group of compounds (CATi) were identified by in silico screening against the CMV DNA polymerase catalytic aspartate triad, resulting in the identification of 31 compounds with the potential for inhibitory activity against CMV. The second group of compounds (PRO-i) were identified through in silico screening against the CMV protease, identifying a total of 18 lead compounds exhibiting structural complementarity with CMV protease. The third and final group of compounds (TPEX) were identified through random screening and consisted of plant extracts purified from tropical plants. All three compounds were initially screened for cytotoxicity against human fibroblasts. Plaque reduction assays were performed using compounds with acceptable levels of cytotoxicity to determine the ability of the compounds to inhibit the replication of the laboratory antiviral sensitive CMV strain, Towne. Two of the PRO-i compounds demonstrated good antiviral activity against CMV. Eleven percent (2/18) of the PRO-i compounds inhibited CMV replication, with PRO-i-43 and PRO-i??-44 displaying mean 50% inhibitory concentrations (IC50) of 4.8 ?? 1.2 ??M and 8.04 ??M, respectively. PRO-i-43 and PRO-i-44 are thus good candidates for further development as novel antiviral agents against CMV. The majority of CATi and TPEX compounds displayed significant cytotoxicity against human fibroblasts and compounds with acceptable levels of cytotoxicities did not significantly inhibit CMV replication. However, the identification of compounds with low cytotoxicities provides a good foundation for further development of novel anti-CMV agents with superior antiviral activity. In silico screening against three-dimensional viral protein models is a useful strategy for the identification of novel antiviral agents with the potential for inhibitory activity against CMV. Structural modification to produce potent derivatives of the identified anti-CMV compounds (PRO-i-43 and PRO-i-44) is a good option for the further development of novel antiviral agents against CMV. Such further examination of the identified compounds with anti-CMV activity is required to investigate their activity against not only antiviral sensitive CMV strains but also resistant CMV strains. Further investigations will yield new insights into their target, allowing further identification of compounds with potential anti-CMV activity with pharmaceutical application.

Cytomegaloviruses (CMVs) are ubiquitous in nature, having evolved over many millenia with their hosts. While in healthy hosts most infections with CMV are asymptomatic, the virus can cause severe disease in immunocompromised hosts. Thus, the increase in organ transplantation and the HIV/AIDS pandemic have established human CMV (HCMV) as a clinically important pathogen. Indeed, HCMV infections are now the major cause of morbidity and mortality among immunocompromised patients, which has led to more research targeting CMV for effective anti-viral treatment. The discovery that cytomegaloviruses encode several genes which are involved in immune escape has prompted a new area of research, aimed at understanding immune escape mechanisms for exploitation as potential anti-viral therapeutics. By targeting the viral proteins directly, or their receptors in the host, it may be possible to treat CMV disease by agonistic/antagonistic therapy. The first part of this thesis describes the first demonstration of anti-NK1.1 staining in situ to identify NK cells using a modified in vivo perfusion/fixation method. Using this method, we have compared the acute NK1.1+ cellular response to wild-type MCMV infection in the visceral organs of genetically susceptible intra-NK complex recombinant BALB.B6-CT6 (Cmv1s, NK1.1+) mice with resistant C57B⁄J (Cmv1r, NK1.1+) and BALB.B6-Cmv1r mice (Cmv1r, NK1.1+). Expression of viral antigens and the consequences of infection on other cellular subsets, were also analyzed in this study. The data show that in susceptible mice (Cmv1s) MCMV infection is predominent in the marginal zone of splenic white pulp, resulting in local changes in various cellular constituents, including macrophages, NK cells and DC. In the liver, distinct foci of infection were comprised of large numbers of macrophages and NK1.1+ cells surrounding infected cytomegalic cells. In resistant mice (Cmv1r), 6 MCMV infection predominantly affected the red-pulp of the spleen and was associated with increased accumulation of NK1.1+ cells and macrophages at sites of viral infection

[Truncated abstract] The design and development of effective anti-viral immunotherapies requires a comprehensive understanding of the cellular and molecular processes that are involved in the generation and regulation of immune responses. The fundamental objective of the immune system is to successfully complete the task of eliminating/controlling the invading pathogen without causing overt pathology. Cytomegaloviruses (CMVs) are large DNA viruses that are able to evade immune attack and persist lifelong within the host. In a healthy host, CMV causes an asymptomatic infection, but in instances of decreased immune functions, such as in newborns, acquired immunodeficiency syndrome (AIDS) patients and transplant recipients, the infection can result in serious morbidity and mortality. Thus, human CMV (HCMV) is a clinically important pathogen and an understanding of the pathogenesis, mechanisms of immune subversion and, importantly the cascade of immune events that ensue following infection is highly relevant. The studies presented in this thesis have provided useful insight into various aspects of viral immunity and it is hoped that they will assist in the design of more effective therapies against viruses of clinical importance. Genetic variability in humans can greatly influence anti-viral immune responses and the outcome of viral infection. ... Furthermore, these studies provide novel evidence that NK cells are also crucial for the control of virus in some organs of susceptible mice during early acute infection. The data reveals that both NK cells and CD8+ T cells utilise perforin- and IFN-? dependent control of MCMV. Furthermore, these studies provide novel evidence that protection mediated by Ly49H+ NK cells in resistant mice is dependent on perforin. Chapter 3 focuses on the biological relevance of Grz during MCMV infection. These studies found that GrzA and GrzB are essential components of the machinery involved in limiting MCMV during acute infection. These analyses also provide the first evidence suggesting that GrzM plays a role, albeit minor, in controlling MCMV replication. Furthermore, the current studies suggest that Grz can mediate direct antiviral activities independent of the induction of cell death in conjunction with perforin. Interestingly, in the absence of both GrzA and GrzB (GrzAB), mice were as susceptible to MCMV infection as perforin-deficient mice. However, unlike perforin-deficient mice, GrzAB-deficient mice controlled and survived the infection. In Chapter 4 the roles of perforin, GrzA and GrzB in anti-viral immunity and immunopathology during MCMV infection were examined. These studies show that NK cell-derived perforin is required to eliminate infected targets as well as activated effector cells, suggesting that NK cells are crucial not only in defensive immunity but also in limiting the immune activation that follows MCMV infection. In summary, the studies presented in this thesis define the significant role played by specific effector molecules in limiting MCMV replication during different stages of this viral infection. Furthermore, these studies provide novel evidence that perforin, GrzA and GrzB play distinct roles in defensive immunity and limiting immunopathology during MCMV infection.

Thrombocytopenia is one of the most common hematologic presentations of active human cytomegalovirus (HCMV) infection, especially in recipients of allogeneic hematopoietic stem cell transplantations and newborns of congenital HCMV infection. However, mechanisms of HCMV-induced thrombocytopenia have not been well understood. The precursor of circulating platelets – megakaryocyte, is derived from hematopoietic stem/progenitor cell in bone marrow. We postulate that inhibition to megakaryocytic development is the major pathogenesis of HCMV-induced thrombocytopenia. Megakaryocytic cells as well as supportive microenvironment in bone marrow are major targets of HCMV infection. Presented study mainly focused on the impacts of HCMV to megakaryocytic cells and multipotent mesenchymal stromal cells (MSCs) - the precursor of bone marrow stromal cells. Based on a megakaryocytic cell model challenged by HCMV in vitro, inhibited megakaryocytic endomitosis, proliferation, and cellular expression were respectively demonstrated as decreased polyploidy population, decreased colony formation, and reduced c-Mpl (thrombopoietin receptor) expressing cells. Evoked apoptosis of megakaryocytic cells was also evidenced with increased phosphatidylserine exposure on cell surface and intracellular caspase-3 activation after HCMV infection. Involvement of mitochondrial-mediated intrinsic apoptosis was further shown as losing JC-1 fluorescent signal in infected megakaryocytic cells. These results suggest that inhibition induced by HCMV is exerted through multiple processes directly affecting the megakaryopoietic development. Functional failure of bone marrow microenvironment was demonstrated in bone marrow derived MSCs infected by HCMV in vitro. Suppressed cytokine production, impaired cellular migration, and hindered differentiation of HCMV-infected MSCs were respectively demonstrated by lowered level of stromal cell-derived factor 1 in culture medium, decreased number of cells passed through a porous membrane in a transwell culture, and reduced differentiated cells in either adipogenic or osteogenic induction cultures. Alongside with these changes, HCMV-induced programmed cell death further contributed to the supportive failure. Autophagic cell death in infected MSCs was demonstrated as massive accumulation of vacuoles with double membrane structure and LC-3b II molecules followed by viability loss. De novo apoptosis was also observed as another process of programmed cell death, shown as increased phosphatidylserine exposure on cell surface and intracellular caspase-3 activation of infected MSCs. Increased programmed cell death appeared to be associated with extensive HCMV replication in MSCs, which was featured with typical cytopathic morphology, expression of viral tegument protein pp65, and massive accumulation of various viral particles including mature virions. Sustained activation of extracellular signal-regulated kinases likely represented a signal transduction network connecting viral expression or replication with programmed cell death. In a “MSCs-dependent” megakaryopoiesis model, HCMV-infected MSCs failed to support survival and maintenance of megakaryocytic cells. Taken together, these results suggest that active HCMV expression or replication inhibits multiple cellular functions and induces multiple processes of programmed cell death of MSCs. Such inhibition compromises supportive functions of bone marrow microenvironment, and subsequently reduces platelet production in an indirect manner. In summary, HCMV suppresses cellular function and induced apoptosis on both megakaryocytic cells and their supportive cells, MSCs. Therefore, the inhibitory effects of HCMV on megakaryopoiesis are operated via both direct and indirect mechanisms.
published_or_final_version
Paediatrics and Adolescent Medicine
Doctoral
Doctor of Philosophy

This thesis presents a comparative analysis of the type I Interferon (IFN) subtypes and an evaluation of their potential as DNA vaccines in a model of murine cytomegalovirus (MCMV) infection and disease. MCMV induces acute and chronic phases of myocarditis, a heart disease characterised by an inflammatory cell infiltrate, in susceptible BALB/c mice. The type I IFNs comprise 14 IFN alpha genes in the human and >10 IFN alpha genes in the mouse with a single IFN beta gene in both species, however, the purpose of their multiplicity has remained unclear to date. An extensive panel of murine type I IFN subtype genes, including IFNA1, A2, A4, A5, A6, A9 and B, were sub-cloned into the mammalian expression vector pkCMVint (Vical, Inc.) for expression in BALB/c mice. These DNA constructs express biologically active IFN both in vitro and in vivo with systemic, low level expression persisting in the mouse for up to 4 weeks. The individual type I IFN subtypes differentially affect the immune response to MCMV challenge. IFNA6 proved most efficacious, reducing viral replication and inflammation in the acute and chronic phase of disease. Data suggests this occurs via induction of a Th1-like cytokine and antibody response. Furthermore, IFNA6 inoculation after the acute phase was shown to protect mice from the onset of chronic myocarditis. Characterisation of the immune cell response in IFN-treated, MCMV-infected mice demonstrated that type I IFN subtypes modulate the type of immune cells infiltrating the myocardium during myocarditis. Notably, reduced CD8+ and CD4+ T cells and B cell numbers in the heart was associated with reduced chronic myocarditis. Finally, coadministration of type I IFNs, in particular, IFNA6 and IFNB, synergistically improved immunotherapy against MCMV infection and myocarditis. The findings detailed here highlight the potential for the type I IFNs as DNA vaccines and most importantly, demonstrate that the type I IFNs have differential antiviral and immunomodulatory efficacies in the MCMV model of infection and myocarditis.

This thesis presents a comparative analysis of the type I Interferon (IFN) subtypes and an evaluation of their potential as DNA vaccines in a model of murine cytomegalovirus (MCMV) infection and disease. MCMV induces acute and chronic phases of myocarditis, a heart disease characterised by an inflammatory cell infiltrate, in susceptible BALB/c mice. The type I IFNs comprise 14 IFN alpha genes in the human and >10 IFN alpha genes in the mouse with a single IFN beta gene in both species, however, the purpose of their multiplicity has remained unclear to date. An extensive panel of murine type I IFN subtype genes, including IFNA1, A2, A4, A5, A6, A9 and B, were sub-cloned into the mammalian expression vector pkCMVint (Vical, Inc.) for expression in BALB/c mice. These DNA constructs express biologically active IFN both in vitro and in vivo with systemic, low level expression persisting in the mouse for up to 4 weeks. The individual type I IFN subtypes differentially affect the immune response to MCMV challenge. IFNA6 proved most efficacious, reducing viral replication and inflammation in the acute and chronic phase of disease. Data suggests this occurs via induction of a Th1-like cytokine and antibody response. Furthermore, IFNA6 inoculation after the acute phase was shown to protect mice from the onset of chronic myocarditis. Characterisation of the immune cell response in IFN-treated, MCMV-infected mice demonstrated that type I IFN subtypes modulate the type of immune cells infiltrating the myocardium during myocarditis. Notably, reduced CD8+ and CD4+ T cells and B cell numbers in the heart was associated with reduced chronic myocarditis. Finally, coadministration of type I IFNs, in particular, IFNA6 and IFNB, synergistically improved immunotherapy against MCMV infection and myocarditis. The findings detailed here highlight the potential for the type I IFNs as DNA vaccines and most importantly, demonstrate that the type I IFNs have differential antiviral and immunomodulatory efficacies in the MCMV model of infection and myocarditis.

Cytomegalovirus (CMV) is a ubiquitous pathogen affecting over 95% of the world’s population. While infection is typically asymptomatic in healthy individuals, the virus persists life-long in its host and can be reactivated following withdrawal of immune control. As such, it remains a serious clinical concern in individuals who are immunocompromised, such as newborns and neonates, transplant and/or chemotherapy recipients, and HIV/AIDS patients. CMV also has the ability to cause immunosuppression, the mechanisms of which include defective antigen presentation to T cells and interference with haematopoiesis in the bone marrow (BM). Due to strict species specificity, murine CMV (MCMV) provides a relevant model for the study of CMV modulation of the immune system in vivo in its natural host. The type I interferons (IFNs) represent a major family of cytokines involved in the early response to MCMV infection. Their anti-viral activity and regulation of NK cell activation and cytotoxicity are of significant interest in the context of MCMV infection, as genetic resistance to MCMV is mediated by the ability of Ly49H+ NK cells to directly recognise and lyse infected cells. Chapter 2 comprises an analysis of acute MCMV infection in the absence of type I IFN activity. These studies were conducted in IFNAR1 and IFNAR2 deficient mice, which lack components of the type I IFN receptor. Data obtained from these studies confirmed the essential requirement for type I IFN in controlling viral titres, promoting expansion of splenic Ly49H+ NK cells, and inducing early activation of NK cell cytotoxicity. In addition, our data depicted an accumulation of infected myeloid cells in the absence of effective NK cell-mediated control. This was paralleled by a significant increase in the level of serum TNF-a and IFN-¿, an effect which in some cases has been linked to serious pathological disease. Thus, the data described in this chapter provide an insight into the consequences arising from delayed NK cell responses to MCMV infection in the absence of type I IFN. vii Type I IFN can also potentially affect BM haematopoiesis. BM atrophy and impairment of myelopoiesis are serious consequences of CMV infection. During acute MCMV infection we consistently observed a profound loss of splenic dendritic cells (DCs) in BALB/c mice. Since all DC subsets are derived from BM haematopoietic progenitor cells, the possibility that MCMV might interfere with BM haematopoiesis and DC differentiation was explored. Chapters 3 and 4 describe the impact of acute MCMV infection on BM progenitors, with particular emphasis on the differentiation capabilities of these cells in ex vivo culture systems. Chapter 3 focuses on the effect of MCMV infection on BM cellularity and frequency of specific BM progenitor populations. A thorough analysis of contributing factors, such as viral infection of BM cells, involvement of type I and II IFNs, progenitor cell trafficking and NK cell activity in the BM compartment, was conducted. Our results showed that a severe loss of BM cellularity occurs in MCMV-infected mice. Furthermore, when BM cells from MCMV-infected mice were cultured ex vivo in granulocyte macrophage-colony stimulating factor (GM-CSF), there was an impairment in their ability to differentiate into DCs.

The most promising therapy for cytomegalovirus (CMV) disease focuses on enhancing the patient’s immunity to viral pathogens. In this investigation, the therapeutic efficacy of bulk-cultured cytotoxic T lymphocytes was examined in primary murine cytomegalovirus infection. Virus-specific cytotoxic T cells (CTL( were generasted using immune spleen cells stimulated with irradiated murine CMV-infected cells. These CTL demonstrated significant cytolytic activity in a chromium-release assay. They were administered to mice infected with a lethal dose of murine CMV. The group receiving 107 CTL had a 50% reduction of mortality and lost 10% less weight. Virus titer of mice who received 107 CTL decreased 10 fold in the lungs and spleen and decreased 100 fold in the salivary gland. However, the therapy failed to decrease serum aminotransferase levels or restore lymphocyte blastogenic responsiveness. Although the mechanism is unclear, this therapy was effective in reducing mortality, morbidity, and virus titer in select organs.
Center for Medical Education

Thesis (Ph.D.) - University of Glasgow, 2007.
Ph.D. thesis submitted to the Division of Virology, Institute of Biomedical and Life Sciences, University of Glasgow, 2007. Includes bibliographical references. Print version also available.

Cytomegaloviruses (CMV), the prototypical β-herpesviruses, have co-evolved with their hosts and thus acquired multiple strategies for modulation of the immune response. Viral engagement of pattern recognition receptors (PRR), such as toll-like receptors (TLRs) and cytosolic nucleic acids sensors, initiates the host immune response through activation of elaborate signalling programs. The ensuing inflammatory response is further sustained and amplified through cytokines, such as IL-1β, activating signalling pathways greatly overlapping those utilized by TLRs. The central hypothesis of this thesis is that a viral counter-measure by murine CMV (MCMV) involves specific targeting of TLR- and IL-1β-induced signalling along the MyD88 to NF-κB pathway. To test this hypothesis MCMV inhibition of IL-1β signalling was initially investigated in a fibroblast cell line. It was demonstrated that in MCMV infected cells IL-1β-induced IκBα degradation is largely inhibited. Comparison of productive and non-productive infection showed this modulation requires de-novo viral gene expression beyond the immediate early region. Further investigations utilising a ORF M45 deletion mutant identified viral gene M45 as necessary for mediating the observed modulation of IL-1β- induced IκBα degradation. To further test the hypothesis, studies were extended to include TLR stimulation in the context of bone marrow-derived macrophages (BMDM) infection. It was found that TLR7/9-induced NF-κB activation is inhibited in MCMV infected BMDM. Overall, data presented in this study demonstrate a previously unrecognised MCMV inhibition of IL-1β- and TLR7/9-induced NF-κB activation, and indicate a role for viral gene M45 in mediating this effect.

Les nouveau-nés et les jeunes enfants ont une susceptibilité plus élevée aux infections par rapport aux enfants plus âgés et aux adultes. Cette caractéristique est en partie attribuée à l’immaturité de leur système immunitaire qui est associée à une capacité limitée à développer des réponses immunitaires à médiation cellulaire. L’infection par le cytomégalovirus (HCMV) est la cause la plus fréquente d’infection congénitale chez l’Homme et une cause majeure de surdité et de retard mental. En Belgique, le dépistage anténatal de l’infection primaire par le HCMV chez les femmes enceintes offre l’opportunité d’étudier les réponses immunitaires du foetus à ce virus et de les comparer à celles de leur maman.

Les lymphocytes T CD4+ Th1 et les lymphocytes T CD8+ cytotoxiques jouent un rôle crucial dans le contrôle des pathogènes intracellulaires dont le HCMV fait partie. La littérature montre une capacité limitée des enfants congénitalement infectés par le HCMV à développer des réponses T CD4+ spécifiques du HCMV. En contraste, des réponses de lymphocytes T CD8+ spécifiques du HCMV ont été rapportées chez des enfants infectés in utero, mais ces réponses n’ont pas été comparées en détails à celles de l’adulte. De plus, notre connaissance des réponses T spécifiques du HCMV durant l’infection primaire par ce virus est limitée. Des études antérieures ont rapporté un défaut de prolifération et de production d’IL-2 des lymphocytes T spécifiques du HCMV chez des adultes avec durant la phase primaire de l’infection, mais les mécanismes restent non-élucidés.

Nous avons caractérisé les réponses de lymphocytes T CD4+ et CD8+ spécifiques du HCMV provenant du sang de cordon de nouveau-nés congénitalement infectés par le HCMV, et nous avons comparé ces réponses à celles de leurs mamans diagnostiquées avec une infection primaire par le HCMV durant la grossesse. En plus, nous avons comparé les réponses T CD4+ et CD8+ de ces mamans à celles d’adultes infectés chroniquement par le virus. Chez les nouveau-nés, nous avons démontré que des lymphocytes T CD4+ de sang de cordon exprimant un phénotype de différentiation spécifique du HCMV (CD27-CD28-) ainsi qu’un phénotype Th1 similaire à celui des cellules maternelles étaient induits in utero lors de l’infection congénitale par le HCMV. De plus, la détection d’expansions oligoclonales suggérait fortement une expansion antigène-spécifique de ces cellules. Cependant, les T CD4+ de nouveau-nés présentaient une capacité fortement réduite à produire des cytokines anti-virales (IFN-γ, TNF-α et MIP-1β) en réponse à une stimulation ex vivo avec les antigènes du HCMV, par rapport aux cellules maternelles. Les lymphocytes T (CD27-CD28-) CD4+ de nouveau-nés produisaient également des niveaux plus bas de cytokines antivirales en réponse à des stimulations polyclonales avec l’anti-CD3 et la PMA/ionomycine, suggérant des altérations en amont et en aval de la voie de signalisation du TCR. Nos résultats suggèrent que ces altérations pourraient impliquer la diminution de l’expression de molécules impliquées dans cette voie de signalisation. De la même manière, nous

avons montré que chez le nouveau-né, la fonction des T CD8+ spécifiques du HCMV était altérée par rapport à celle de l’adulte. Nous avons observé des proportions similaires de T CD8+ (CD27-CD28-) chez les nouveau-nés et les adultes. De plus, l’analyse du répertoire du TCR Vβ de ces cellules par séquençage haut-débit a révélé une capacité similaire à générer un répertoire T diversifié dans les deux groupes. Comme rapporté précédemment, nous avons détecté des fréquences similaires de lymphocytes T CD8+ spécifiques pour l’antigène immunodominant pp65. Cependant, lorsque les stimulations ont été étendues à d’autres antigènes du HCMV, nous avons observé que le répertoire antigénique reconnu par ces cellules était significativement réduit chez les nouveau-nés, en association avec une diminution de la polyfonctionalité et de la production de cytokines par cellule.

Nous avons également montré que, dans une moindre mesure, la fonction des lymphocytes T spécifiques du HCMV était diminuée durant l’infection primaire chez l’adulte. Comme reporté précédemment, les T CD4+ spécifiques du HCMV proliféraient moins et produisaient moins d’IL-2 par rapport à des individus dans la phase chronique de l’infection. Ce défaut de production d’IL-2 affectait à la fois les populations de cellules CD28+ et CD28-, montrant que l’accumulation de lymphocytes T CD4+ ayant perdu l’expression de la molécule CD28 (un signal de co-stimulation important pour la production d’IL-2) est seulement un des facteurs contribuant à la diminution de la production d’IL-2 par les cellules spécifiques du HCMV. En accord avec cette observation, nous avons montré une diminution de la production par cellule d’IFN-γ et de TNF-α touchant également à la fois les populations de T CD4+ CD28+ et CD28- durant la phase primaire de l’infection, un défaut associé avec une avidité fonctionnelle diminuée de ces cellules. De la même manière, la polyfonctionalité et la production de cytokines par cellule des lymphocytes T CD8+ spécifiques du HCMV étaient également diminuées chez les adultes durant la phase d’infection primaire.

En résumé, nos résultats montrent que la fonction des lymphocytes T spécifiques du HCMV de nouveau-nés et d’adultes est altérée durant l’infection primaire par rapport à des individus infectés chroniquement par le virus. Nous montrons que cette régulation fonctionnelle ressemble à l’exhaustion fonctionnelle des lymphocytes T observée durant les infections virales chroniques associées à des charges virales élevées. L’infection primaire par le HCMV est caractérisée par une réplication virale intense qui dure pendant plusieurs mois suivant l’infection. Nous émettons l’hypothèse que les hauts taux de réplication virale observés durant l’infection congénitale et chez l’adulte durant l’infection primaire par le HCMV pourraient interférer avec certaines fonctions des lymphocytes T./Neonates and young infants have a higher susceptibility to infections compared to older infants or adults. This feature is in part attributed to the immaturity of their immune system associated with a limited capacity to mount cellular-mediated immune responses. Congenital human cytomegalovirus (HCMV) infection is the most common cause of congenital infection worldwide and a major cause of hearing loss and mental retardation. In Belgium, antenatal screening of pregnant women for primary HCMV infection offers an opportunity to study neonatal immune responses to the virus and to compare them to those of their mother.

T lymphocytes are major players of the immune system. In particular, Th1 CD4+ T cells and CD8+ cytotoxic T cells play a crucial role in the control of intracellular pathogens, including HCMV infection. Previous literature has reported a limited capacity of infants born with congenital HCMV infection to mount HCMV-specific CD4+ T cell responses. In contrast, fetal antigen-specific CD8+ T cell responses have been reported following in utero HCMV infection, but these responses have not been compared in detail to those of adults with primary infection. In addition, our knowledge regarding adult HCMV-specific T cell responses during primary HCMV infection is limited. Previous studies have reported defective T cell proliferation and IL-2 production in adults with primary HCMV infection, showing that some of the T cell functions are altered during primary infection.

In this study, we have characterized neonatal HCMV-specific CD4+ and CD8+ T cell responses from the cord blood of newborns with congenital HCMV infection, and we have compared these responses to that of their mothers diagnosed with primary HCMV infection during pregnancy. Also, we compared CD4+ and CD8+ T cell responses of adults with primary HCMV infection to that of adults with chronic infection.

In newborns, it was not known if the defective CD4+ T cell responses could be attributed to the absence of HCMV-specific cells or to the induction of dysfunctional cells. We demonstrate that neonatal CD4+ T cells with a differentiation phenotype typical of HCMV infection (CD27-CD28-) and expressing a Th1 phenotype similar to that of maternal cells can differentiate in utero following HCMV infection. In addition, the detection of oligoclonal expansions by spectratyping and flow cytometry analyses strongly suggests antigen-specific responses. However, neonatal CD4+ T cells were markedly less able to produce antiviral cytokines (IFN-γ, TNF-α and MIP-1β) following ex vivo stimulation with HCMV antigens, compared to maternal cells. Also, neonatal CD27-CD28- CD4+ T cells produce lower levels of antiviral cytokines in response to polyclonal stimulations with anti-CD3 and PMA/ionomycin, suggesting alterations up-stream and down-stream of the TCR signaling pathway. Our results suggest that these alterations could involve the down-regulation of the expression of molecules that are part of the TCR signaling pathway. Similarly, we show that the function of

neonatal HCMV-specific CD8+ T cells is impaired compared to adults. Similar proportions of (CD27-CD28-) CD8+ T cells, typical of HCMV infection, were detected in newborns and adults. Analysis of the TCR Vβ repertoire of neonatal and maternal (CD27-CD28-) CD8+ T cells by high-throughput sequencing revealed a similar capacity to generate a diverse clonal repertoire. As previously reported, we detected similar frequencies of HCMV-specific CD8+ T cells specific for the immunodominant viral antigen pp65. However, when extending ex vivo stimulations to other HCMV antigens, we observed that the antigenic repertoire recognized by these cells was significantly reduced in newborns. In addition, neonatal CD8+ T cells had a reduced polyfunctionality and per cell cytokine production.

To a lower extent, the function of adult HCMV-specific T cells was also impaired during primary infection. As previously reported, maternal HCMV-specific CD4+ T cells were markedly less able to produce IL-2 and to proliferate compared to individuals in the chronic stage of the disease. Both CD28+ and CD28- T cell subsets produced decreased levels of IL-2. This observation shows that the accumulation of HCMV-specific CD4+ T cells having lost the expression of the CD28 molecule (an important co-stimulatory signal for IL-2 production) during primary infection is only one of the factors contributing to the decreased IL-2 production. Accordingly, both CD28+ and CD28- CD4+ T cell subsets had a decreased per cell production of IFN-γ and TNF-α during primary HCMV infection. This defect was associated with a lower functional avidity of these cells. Similarly, the polyfunctionality and per cell cytokine production of adult HCMV-specific CD8+ T cells was also impaired compared to adults with chronic infection.

Altogether, our results show that adult and neonatal HCMV-specific T cell responses are impaired during primary infection, compared to individuals with chronic infection. We show that this functional regulation resembles that of functional T cell exhaustion observed during chronic viral infections that are associated with high levels of viral replication. Primary HCMV infection is characterized by an intense viral replication lasting for several months post-infection. We hypothesize that the high levels of viral replication observed during congenital and adult primary HCMV infection could interfere with some of the T cell functions.
Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

Orientador: Sandra Cecilia Botelho Costa
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: O Citomegalovírus Humano (HCMV) continua sendo uma causa significante de morbidade em pacientes imunocomprometidos, especialmente em transplantados de medula óssea, e pode manifestar diversas complicações que incluem hepatite, doença gastrointestinal e pneumonia intersticial ou a denominada "Síndrome Viral por HCMV"caracterizada por febre, leucopenia e trombocitopenia. O HCMV pode também ter um efeito imuno-modulador, fazendo da infecção por esse vírus um fator de risco importante para o desenvolvimento de rejeição ao enxerto aguda e crônica e para co-infecção com outras herpesviroses. A detecção do genoma do HCMV pela PCR (Reação em Cadeia da Polimerase) é específica e sensível, e pode ser usada como uma poderosa ferramenta para o diagnóstico precoce da infecção causada por este vírus. Variações em regiões funcionalmente relevantes do genoma do HCMV têm sido utilizadas como marcadores genéticos em diversos estudos clínicos para diferenciar as linhagens do vírus e associá-las com a patogênese viral e com as manifestações clínicas no paciente. A glicoproteína B (gB) é a maior glicoproteína do envelope do HCMV e tem sido relacionada à entrada na célula hospedeira, transmissão célula-a-célula, e conseqüentemente à fusão das células infectadas. A amplificação do gene gB pela PCR combinada com análise de restrição por RFLP em regiões polimórficas deste gene são eficientes para a identificação dos genótipos do HCMV, tornando possível a distinção de pelo menos 4 padrões eletroforéticos. Por outro lado, a determinação da carga viral em pacientes imunologicamente afetados tem sido associada como marcador ou preditor do desenvolvimento de doença por HCMV órgão-específica. Sendo assim, a determinação da carga viral, especificamente nestes pacientes, é fundamental para a supervisão da terapia antiviral. Além disso, os valores da carga viral estão relacionados aos níveis de imunossupressão, à patogênese do HCMV e ao grupo de pacientes e/ou ao tipo de transplante e podem indicar o início da administração da terapia antiviral.O método de real-time PCR (RT-PCR) foi aplicado para a quantificação do genoma do HCMV em amostras clínicas e a detecção e posterior quantificação do DNA do HCMV em amostras de soro por esta técnica é capaz de distinguir entre pacientes com infecções sintomáticas daqueles com infecções inativas ou latente.Avanços têm sido feitos na prevenção da doença por HCMV após o transplante de medula óssea, inclusive a administração profilática, por períodos prolongados, de antivirais como o Acyclovir e o Ganciclovir e como conseqüência, pode originar linhagens resistentes relacionadas principalmente a dois genes virais: a fosfotransferase viral (UL97) e a DNA polimerase viral (UL54). Sabendo-se da importância da identificação das linhagens do HCMV em pacientes transplantados de medula óssea e da possível relação com a infecção e apresentação clínica; da relevância em determinar a carga viral como preditor de doença; e finalmente, da detecção de linhagens resistentes aos agentes antivirais disponíveis, este estudo avaliou, prospectivamente, pacientes transplantados de medula óssea em seguimento no Hemocentro/UNICAMP. Além disso, teve como principais objetivos: determinar a prevalência dos genótipos do HCMV e avaliar uma possível associação com a apresentação clínica nesses pacientes; determinar a carga viral para o monitoramento da terapia antiviral; e identificar e correlacionar mutações que conferem resistência ao Ganciclovir com carga viral e apresentação clínica. Foram incluídas na casuística, 169 amostras de DNA de sangue periférico e 187 amostras de DNA de soro de 22 pacientes transplantados de medula óssea. Dentre as 47 amostras de DNA de sangue periférico HCMV positivas, 42 foram genotipadas e observamos a prevalência do genótipo gB1 (47%) como descrito em literatura, e embora sem comprovação estatística, notamos a tendência deste genótipo com melhor prognóstico. Aplicamos a RT-PCR em 96 amostras de DNA de soro de 12 pacientes transplantados de medula óssea seguidos no Ambulatório de Hematologia, e observamos que o método é adequado para a avaliação da carga viral neste grupo de pacientes. No entanto, é necessário estabelecer um valor de corte a fim de se utilizar esta metodologia para obtenção de um valor que seja preditivo de doença e para o monitoramento do tratamento dos pacientes. Este método mostrou-se mais preciso que a ?nested?-PCR no mesmo tipo de amostra. Além disso, identificamos 8 novas mutações no gene UL97, uma delas pode estar relacionada à resistência viral ao Ganciclovir. Dentre os polimorfismos identificados, 3 parecem estar relacionados ao genótipo gB1 e possivelmente podem ser utilizadas como marcadores genéticos para a genotipagem do HCMV. Para o gene UL54 foram identificadas 5 novas mutações na região IV do gene e que geralmente é relacionada à resistência ao Ganciclovir. Nós concluímos que a determinação da carga viral é importante, mas não é o único modo de avaliar a eficiência do tratamento antiviral. Dessa forma, a avaliação de outros parâmetros moleculares, como a genotipagem e mutações relacionadas à resistência aos antivirais, são informações complementares e devem ser consideradas para o monitoramento da evolução clínica em pacientes transplantados de medula óssea
Abstract: Human Cytomegalovirus (HCMV) remains a significant cause of morbidity in immunocompromised patients, especially in bone marrow transplant recipients. It may manifest severe complications including hepatitis, gastrointestinal disease, and interstitial pneumonitis or as so-called ?HCMV viral syndrome? with fever, leukopenia, and thrombocytopenia. The HCMV may also has an immunomodulatory effect, potentially making HCMV infection an important risk factor for the development of an acute and chronic allograft rejection and for coinfection with other herpesviruses. The detection of the HCMV genome by PCR (Polymerase Chain Reaction) is specific and sensitive. Besides this, it can be used as a powerful tool for the early diagnoses of the infection caused by this virus. Variations in functionally relevant areas of the HCMV genome have been used as genetic markers in numerous clinical studies to differentiate the HCMV strains and to associate them with the viral pathogenesis further with the patients? clinical manifestations. The glycoprotein B (gB) is the major glycoprotein of HCMV?s envelope and it has been implicated in host cell entry, cell-to-cell virus transmission, consequently in the fusion of infected cells. The gB amplification by PCR combined with the restriction analysis by RFLP in polymorphic areas are effective for the identification of the HCMV genotypes, becoming possible the distinction of at least 4 electrophoretic patterns. On the other hand, the determination of the viral load in the immunologically affected patients has been associated as marker or predictor for the development of the organ specific disease by the HCMV. Hence, the determination of the viral load in these specific patients is fundamental for the management of the antiviral therapy. In addition, the viral load values are related to levels of the immune-suppression, the pathogenesis of the HCMV and the group of patients and/or the type of transplant. Furthermore, the viral load values can indicate the beginning of the antiviral therapy administration. A real-time PCR (RT-PCR) assay was applied for quantifying the HCMV genome load in clinical samples and the detection and quantification of HCMV DNA in blood serum through RT-PCR are able to distinguish patients with symptomatic infections among those with latent or inactive infections. Advances have been made in the prevention of HCMV disease after bone marrow transplantation, including prophylactic administration of antivirals such as Acyclovir and Ganciclovir. The HCMV prophylaxis with antiviral in this patients? group is administered for prolonged periods of therapy, consequently it can originate resistant viruses related mainly to two genes: the viral phosphotransferase (UL97) and the viral DNA polymerase (UL54). Ahead the importance of the identification of HCMV strains in bone marrow transplant patients, the HCMV strains performance in the patients? infection and clinical presentation, the relevance of determinating the viral load as a disease predictor, and finally, the detection of the resistant strains to the available antivirals, this study prospectively evaluated bone marrow transplant recipients followed at Hemocentro/UNICAMP. Moreover, it had as main goals: to determine the prevalence of the HCMV gB genotypes, to evaluate a possible gB genotype association with the patients? clinical presentation; to determinate the viral load for monitoring the antiviral therapy, and to correlate Ganciclovir resistant mutations in UL97 and UL54 gene with the viral load and patients? clinical presentation. From 22 bone marrow transplant recipients, DNA samples of peripheral blood (169) and DNA samples of blood serum (187) were included in this casuistic. Among 47 HCMV positive samples, 42 were genotyped. We observed the prevalence of gB1 genotype (47%), as described in the specific literature, however without statistical analysis, the raw data exhibited that gB1 genotype can be related to patients? better prognostics. From 12 followed bone marrow transplant recipients, we applied the RT-PCR in 96 DNA blood serum samples and we observed that the method was accurate for the viral load evaluation in this patients? group. However, it is necessary to establish a crucial cutoff to consider whether a specific value of viral load is a predictive value to cause HCMV disease and to monitor the patients? treatment. This method was more precise than the nested-PCR for blood serum samples. Additionally, we identified 8 new mutations in UL97 gene, one of them can be related to Ganciclovir HCMV resistance. Among all of identified polymorphisms, 3 of them can be related to gB1 genotype and may be used as genetic marker to HCMV genotyping. In the region IV of the UL54 gene, 5 new mutations were identified, and can possibly be related to Ganciclovir HCMV resistance. We concluded that the determination of the patients? viral load is crucial, even so it is not the only way to evaluate the antiviral treatment efficacy. Then, the evaluation of other molecular parameters as genotyping and mutations related to the HCMV antiviral resistance, are complementary information and must be considered to monitor the clinical evolution of bone marrow transplant recipients
Doutorado
Ciencias Basicas
Doutor em Clínica Médica

L’infection par le cytomégalovirus est le plus souvent asymptomatique chez les sujets immunocompétents mais entraine une morbidité et une mortalité importantes chez les patients immunocompromis et en cas d’infection congénitale.

Après l’infection primaire, le virus persiste tout au long de la vie à l’état latent mais peut se réactiver de manière intermittente. Ceci est associé à l’expansion de lymphocytes T CD4+ fortement différenciés ayant des fonctions auxiliaires et cytolytiques. L’infection primaire est, par contre, caractérisée par une réplication virale intense qui dure plusieurs mois. Il a été montré que l’exposition prolongée à des concentrations élevées d’antigènes entraine une perte progressive de fonction par les lymphocytes T appelée épuisement et caractérisée par l’expression de récepteurs inhibiteurs. L’impact de la réplication virale intense observée au cours de l’infection primaire par le CMV sur la fonction des lymphocytes T CD4+ n’est pas bien connu.

La fonctionnalité des lymphocytes T CD4+ a été explorée chez l’humain et le singe rhésus au cours de l’infection primaire et comparée à celle de sujets porteurs chroniques du virus.

Les résultats montrent que l’infection primaire par le CMV est associée à la détection de lymphocytes T CD4+ circulants ayant une faible capacité de prolifération et de production de cytokines et d’IL-2 en particulier.

L’impact de la différenciation sur la fonction des lymphocytes a été exploré en détail chez l’humain. Il a été observé qu’un degré de différenciation plus élevé des lymphocytes T CD4+ spécifiques du CMV joue un rôle dans la production réduite d’IL-2. Toutefois, la fraction moins différenciée (exprimant la molécule CD28) présente également une sécrétion d’IL-2 moindre au cours de l’infection primaire. Ceci fait partie d’une diminution globale de la production de cytokines au cours de l’infection primaire qui affecte également la sécrétion d’IFNγ et TNFα, entraine une polyfonctionnalité réduite et est indépendante de la différenciation. L’épuisement des lymphocytes T CD4+ spécifiques du CMV contribue à leur fonctionnalité moindre comme l’indique l’expression accrue du récepteur inhibiteur PD-1 et l’augmentation des réponses prolifératives en présence d’anticorps bloquant PD-1.

Le lien entre excrétion virale et fonction lymphocytaire a été étudié chez le macaque rhésus. L’infection par le CMV est observée chez les singes juvéniles et adultes mais pas chez les nourrissons. L’excrétion urinaire et salivaire est significativement plus fréquente et intense chez les singes juvéniles par rapport aux adultes. Comme chez l’humain au cours de l’infection primaire, les lymphocytes T CD4+ spécifiques du virus sont moins

polyfonctionnels et prolifèrent moins efficacement chez les singes juvéniles par rapport aux singes adultes. Ceci est associé à l’expression accrue du récepteur inhibiteur PD-1 chez les singes juvéniles. La réponse proliférative des lymphocytes T CD4+ est accrue en présence d’anticorps bloquant PD-1 ou d’IL-2 exogène. Enfin, une association inverse entre fonction lymphocytaire et excrétion urinaire a été mise en évidence chez les macaques adultes.

Ces résultats indiquent que l’infection par le CMV présente des caractéristiques semblables chez l’humain et le singe rhésus. L’infection primaire est associée à la détection de lymphocytes T CD4+ ayant une fonctionnalité moindre qu’au cours de l’infection chronique. L’expression du récepteur inhibiteur PD-1 typique des cellules épuisées est l’un des mécanismes impliqués et pourrait être la cible de stratégies immunomodulatrices visant à améliorer les fonctions lymphocytaires et le contrôle de la réplication virale. Les résultats présentés indiquent que l’infection naturelle chez le singe rhésus constitue un modèle potentiellement utile à l’étude de la réponse immune au CMV humain et à l’évaluation de stratégies immunomodulatrices.

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Cytomegalovirus infection is mostly asymptomatic in immunocompetent hosts but leads to severe morbidity and mortality in immunocompromised subjects and foetuses.

After primary infection, CMV establishes lifelong persistence but can reactivate intermittently. This is associated with the expansion of highly differentiated CD4+ T lymphocytes exhibiting helper functions and cytolytic activity.

Primary infection is characterised by an intense viral replication lasting several months. It has been shown that prolonged exposure to elevated antigen concentrations induces a progressive loss of function by T lymphocytes called exhaustion. This state of functional impairment is associated to the expression of inhibitory receptors. The consequence of the intense viral replication seen in primary CMV infection on CD4+ T cell function is unknown.

CD4+ T cell function has been studied in human and rhesus macaque during primary CMV infection. Chronic CMV carriers have been used as controls.

The results show that primary CMV infection is associated to the detection of circulating CD4+ T lymphocytes exhibiting weak proliferative capacities and reduced cytokine production affecting IL-2 in particular.

The impact of differentiation on lymphocyte function has been explored in detail in human. An increased proportion of terminally differentiated CD4+ T cells (CD28-) is observed during primary infection. These lymphocytes are unable to secrete IL-2 in response to CMV antigens. Interestingly, CD28+ CMV-specific CD4+ T cells also exhibit reduced IL-2 production during primary infection. This is part of a global reduction of cytokine production affecting IFNγ and TNFα as well. The impaired cytokine production is associated to reduced polyfunctionality and is independent of differentiation. Exhaustion of CMV-specific CD4+ T lymphocytes contributes to the reduced functionality as shown by an increased expression of the inhibitory receptor PD-1 and improved proliferative responses in the presence of PD-1 blocking antibodies.

The relationship between viral replication and lymphocyte function has been explored in rhesus macaques. CMV infection is observed in juvenile and adult monkeys but not in newborns. Excretion in urine and saliva is significantly more frequent and intense in juvenile monkeys than adults. As in primary infection in human, CMV-specific CD4+ T lymphocytes are less polyfunctional and have lower proliferative capacities in juveniles as compared to adults. This is associated with an increased expression of PD-1 in juvenile monkeys. CD4+ T cell proliferative responses are increased when PD-1 blocking antibodies or exogenous IL-2 are added to the culture medium. Finally, an inverse association between lymphocyte function and urinary excretion has been observed in adult macaques.

These results indicate that CMV infection shares common features in human and rhesus macaque. Primary infection is associated to the detection of CD4+ T lymphocyte displaying lower functional capacities as compared to chronic infection. Exhaustion contributes to the functional impairment and the inhibitory receptor PD-1 could be targeted by immunomodulatory strategies aiming at improving lymphocyte functions and controlling viral replication. Natural CMV infection in rhesus macaque might be useful as a model to evaluate the efficacy and safety of immunomodulatory approaches.
Doctorat en Sciences médicales
info:eu-repo/semantics/nonPublished

Thesis (MScMedSc)--University of Stellenbosch, 2011.
ENGLISH ABSTRACT: Introduction: Reactivation of human cytomegalovirus (HCMV) infection in individuals infected with human immunodeficiency virus (HIV) may lead to life-threatening end-organ diseases (EOD). The EOD becomes clinically apparent when a critical number of cells in the affected organs become damaged as a consequence of HCMV-infection. Treatment of the HCMV-associated disease at this point may not be effective. Therefore, early detection of HCMV reactivation may be useful to guide pre-emptive therapy. Aim: The aim of this study was to determine whether there is a point at which the HCMV-specific cellular immune response breaks down, as determined by the interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISPOT) assay, and HCMV reactivation occurs in HIV-positive, antiretroviral therapy (ART)-naïve individuals in a South African setting. This was done in relation to the CD4+ T cell count and the HCMV viral load as determined by real-time polymerase chain reaction (qPCR). Materials and methods: Fifty-two (52) HIV-infected, ART-naïve subjects were recruited from primary healthcare centres that they attended for the management of their HIV infection. Heparinised blood samples were collected to quantify the HCMV-specific cellular immune response using the IFN-γ-ELISPOT assay and to determine the HCMV IgG serostatus. Ethylenediaminetetraacetic acid (EDTA) blood samples were collected for the determination of the CD4+ T cell counts and the HCMV viral loads. Results: All 52 subjects recruited were confirmed to be HIV-HCMV co-infected based on their HCMV IgG serostatus. The results of 34 subjects with completed data sets were analysed. The CD4+ T cell counts of these subjects ranged from 10 to 784 cells/μl. Twenty-two (22) (65%) subjects had positive HCMV IFN-γ-ELISPOT results with 94% having no detectable HCMV viral loads. All subjects (28) with a CD4+ T cell count above 100 cells/μl had undetectable HCMV viral loads. Two of the six subjects with CD4+ T cell counts <100 cells/μl had detectable HCMV viral loads. There was no statistically significant association between the CD4+ T cell counts and the HCMV IFN-γ-ELISPOT results. Conclusion: No specific point could be determined when there is loss of integrity of the HCMV-specific cellular immune response in HIV-positive individuals. Low CD4+ T cell counts did not correlate with HCMV IFN-γ-ELISPOT results suggesting that the HCMV-specific cellular immunity did not necessarily break down at low CD4+ T cell counts. Nevertheless, a CD4+ T cell count above 100 cells/μl appeared to be protective against viraemia as determined by the HCMV viral load qPCR. The IFN-γ-ELISPOT assay was employed as a tool to determine the integrity of the HCMV-specific cellular immune response in HIV-positive individuals. However, the IFN-γ-ELISPOT assay should be used in conjunction with the CD4+ T cell count and the HCMV viral load qPCR to determine when there is loss of integrity of the HCMV-specific cellular immune response and HCMV reactivation occurs. This may assist clinicians in their choice of management and appropriate pre-emptive treatment in the HIV-HCMV co-infected individual at a risk for HCMV reactivation.
AFRIKAANSE OPSOMMING: Inleiding: Heraktivering van menslike sitomegaalvirus (MSMV) in menslike immuniteitsgebreksvirus (MIV)-MSMV ko-geïnfekteerde individue kan lei tot dodelike end-orgaan siektes (EOS). Die EOS word klinies duidelik wanneer 'n kritieke aantal selle in die organe beskadig raak as gevolg van die MSMV-infeksie. Behandeling van die MSMV-verwante siekte op hierdie punt mag moontlik nie meer effektief wees nie. Daarom kan die vroeë opsporing van MSMV heraktivering nuttig wees in die gebruik van voorkomende terapie. Doel: Die doel van hierdie studie is om die punt te bepaal wanneer die MSMV-spesifieke sellulêre immuun reaksie afgebreek word met behulp van die interferon gamma (IFN-γ) ensiem-gekoppelde immunospot (ELISPOT) toets en MSMV heraktivering voorkom in MIV-positiewe, antiretrovirale terapie (ART)-naïewe individue in' n Suid-Afrikaanse instelling. Dit word gedoen in verhouding met die CD4+ T sel telling en die MSMV virale lading. Materiale en metodes: Twee-en-vyftig (52) MIV-geïnfekteerde, ART-naïewe pasiënte is vanaf primêre gesondheidsentrums, wat hul bywoon vir die behandeling van hul MIV infeksie, genader. Gehepariniseerde bloedmonsters is gebruik om die MSMV-spesifieke sellulêre immuun reaksie met behulp van die IFN-γ-ELISPOT toets en die MSMV IgG serostatus te bepaal. Etileendiamientetra-asynsuur (EDTA) bloed monsters is versamel vir die bepaling van hul CD4+ T sel telling en hul MSMV virale lading met behulp van die ―real-time‖ polimerase kettingreaksie (qPKR) waardes. Resultate: Al 52 pasiënte is bevestigde MIV-MSMV ko-infeksies, gebasseer op hul serologiese status. Die resultate van 34 pasiënte met voltooide data is ontleed. Die CD4+ T sel tellings van hierdie pasiënte het gewissel 10-784 selle/μl. Twee-en-twintig (22) (65%) pasiënte het positiewe MSMV IFN-γ-ELISPOT resultate met 94% wat ‗n negatiewe qPKR resultate. Alle pasiënte (28) met 'n CD4+ T-seltelling bo 100 selle/μl het' n negatiewe qPKR resultate. Twee van die ses pasiënte met 'n CD4+ T-seltelling <100 selle/μl het waarneembare MSMV virale ladings oor die qPKR. Daar was geen statisties beduidende assosiasie tussen die CD4+ T sel tellings en die MSMV IFN-γ-ELISPOT resultate nie. Gevolgtrekking: Geen spesifieke punt wanneer die MSMV-spesifieke sellulêre immuun reaksie afgebreek word kon in MIV-positiewe individue bepaal word nie. Lae CD4+ T sel tellings het nie ooreengestem met die MSMV IFN-γ-ELISPOT resultate nie en dui daarop dat die MSMV-spesifieke sellulêre immuniteit nie noodwendig afgebreek word teen 'n lae CD4+ T sel tellings nie. Tog blyk 'n CD4+ T-seltelling bo 100 selle/μl om beskerming teen viremie te bied. Die meriete van die gebruik van die IFN-γ-ELISPOT toets die integriteit van die MSMV-spesifieke sellulêre immuun response in MIV-positiewe individue te bepaal, is waargeneem in die opgehoopte data. Tog kan die gebruik van die IFN-γ-ELISPOT toets in samewerking met die CD4+ T sel telling en die MSMV virale lading meer voordelig in die bepaling van 'n punt wanneer die MSMV-spesifieke sellulêre immuun reaksie afbreek en herstel plaasvind. Dit kan help om klinici in hul keuse van bestuur en gepaste voorkomende behandeling in die MIV-MSMV mede-geïnfekteerde individu op 'n risiko vir herstel.

Els valors d’exposició sistèmica (àrea sota la corba de concentracions plasmàtiques=AUC) terapèutica a ganciclovir són 40-50 μg·h/mL. L’avaluació de l’exposició assolida amb el règim de dosificació recomanat en fitxa tècnica va mostrar que només un 16% dels pacients assoleixen l’objectiu terapèutic. La optimització de la posologia en base a un model predictiu ajustant la dosi segons les característiques individuals de cada pacient, per assolir l’exposició adequada a ganciclovir, seria de gran utilitat. Per aquest motiu es va dissenyar un assaig clínic de superioritat (marge de superioritat del 40%), randomitzat en dos grups segons l’estratègia de dosificació en pacients transplantats d’òrgan sòlid sota tractament profilàctic o de la infecció per citomegalovirus. La dosificació en el grup A fou segons fitxa tècnica i en el grup B per predicció bayesiana utilitzant el model poblacional prèviament desenvolupat i en base a les concentracions assolides. L’objectiu principal de la tesi va ser optimitzar la posologia de ganciclovir i valganciclovir, assessorada per l’exposició al fàrmac segons l’AUC. Els objectius secundaris van incloure 1) la validació de la metòdica analítica, 2) establir un disseny d’estratègia limitada d’extracció de mostres per poder estimar paràmetres farmacocinètics, entre ells valors d’AUC de GCV i 3) avaluar l’impacte de l’optimització de la posologia en la resposta i tolerància clínica. La metòdica analítica per a la determinació de GCV mitjançant UHPLC/UV i UHPLC/MS/MS va resultar precisa i exacte complint les guies de la FDA i EMA respectivament. El valor del límit de quantificació (LLOQ) va ser de 0.5 µg/mL. Es va reduir el temps de retenció (5 minuts amb HPLC vs 0.7 min amb UHPLC) i el volum de mostra injectat (200 µL). Amb el detector MS/MS el temps de retenció va ser de 0.51 min i el LLOQ de 0.06 µg/mL. L’especificitat i correlació d’ambdós mètodes es va confirmar demostrant la intercanviabilitat de les dues tècniques. Es següent pas va ser el desenvolupament d’una estratègia de mostreig limitat. Les finestres de mostreig que es van seleccionar foren el disseny amb 3 punts (0.5-1.5, 4-5h i 6-8h post-dosi) seguit del de 2 punts (2-3h i 6-8h post-dosi) perquè presentaven la major exactitud i precisió per a l’estimació d’aclariment (CL) i volum de distribució (V2). La població per intenció de tractar inclosos en l’assaig van ser de 53 pacients (27 grup A i 26 grup B) i no es van observar diferències estadísticament significatives en els característiques basals. L’objectiu principal de percentatge d’assoliment de l’AUC diana es va avaluar mitjançant les medianes d’AUC per pacient (18.5% Grup A vs 88.4% Grup B) i analitzant totes les determinació d’AUC aïllades (18.7% Grup A vs 65.9% in Grup B). Es va demostrar la superioritat de l’estratègia de dosificació per predicció bayesiana vs la dosificació segons la fitxa tècnica, assolint el criteri de superioritat del 40%. Considerant els diferents talls de CLCR, les diferències van ser significatives pels pacients amb CLCR<30mL/min o >60 mL/min. Els resultats clínics van mostrar que els pacients tractats per la infecció per CMV i dosificats segons predicció bayesiana tenien una tendència a un eradicació viral més precoç (17.6 dies Grup A i 12.5 dies Grup B). El nombre de recidives i primoinfeccions va ser major en el grup A vs el grup B (66.67% vs 9.01% i 33.3% vs 8.3%, respectivament). La potencia del l’estratègia de dosificació per predir la probabilitat de recidiva o primoinfecció va mostrar ser significativa per a la recidiva (p=0.007). No es van observar diferències en la incidencia d’efectes adversos hematològics. La present tesi demostra l’efecte de l’ajust de dosi de GCV/VGCV per predicció bayesiana en base al model poblacional en l’assoliment de la diana terapèutica.
A previous study of our group showed that solid organ transplant (SOT) patients treated with ganciclovir/valganciclovir following dosage manufacturer’s recommendations were either over or underexposed. The main objective of the doctoral thesis was dose adjustment by Bayesian prediction based on population pharmacokinetic model to optimize treatment according to a target value (area under the curve [AUC]=40-50µg·h/mL). Secondary objectives were: 1) developed and validate a chromatographic method coupled with ultraviolet (UV) and mass spectrometry (MS/MS) detection, 2) develop a clinically applicable limited sampling strategy (LSS) and 3) evaluate the impact of dose optimization in clinical efficacy and safety. The method UHPLC/UV and UHPLC/MS/MS were precise and accurate and the retention time was 0.7 and 0.51 min, respectively. The limit of quantification was 0.5 mg/L for UV and 0.06 mg/L for MSMS detection method. There was a good correlation between both techniques. An LSS was developed and sampling windows were designed. Predicted exposure using 3 sampling times (0.5–1.5, 4–5, and 6–8 hours) showed the best predictive performance. Finally a two arm, randomized and open-label, superiority trial (margin of 40%) in adult SOT recipients treated with ganciclovir/valganciclovir, either as prophylaxis or treatment of CMV infection, was carried out. (Group A: manufacturer’s recommended doses. Group B: dose adjustment based on target exposure by Bayesian prediction). Results of 53 SOT patients were analyzed. CGV dose adjustment by Bayesian prediction optimized exposure as the 88.4% of patients were within the therapeutic AUC range vs the 18.7% in the group following the manufacturer's recommendations, fulfilling the 40% of superiority margin (p=1.7x10-6). The time required to reach AUC was delayed in Group A (p=0.000029). A trend towards shorter time to viral clearance was seen for Group B vs group A (17.6 vs 12.5 days, p=0.125). The incidence of relapse (66.67% vs 9.01%) and the late CMV disease (33.3% vs 8.3%) were both higher in Group A vs B. No relevant differences in toxicity were observed. Results showed that dose adjustment based on target exposure by Bayesian prediction contributes to optimize the ganciclovir/valganciclovir treatment achieving the therapeutic AUC from the early stages of the treatment

Cytomegalovirus (CMV) is a widespread infection affecting 50-90% of the human population. A typical silent primary infection is followed by life-long persistence in the host under control by virus-specific CD8 (“killer”) and CD4 (“helper”) T cells. Although harmless in most people, CMV may cause disease and sequelae in patients with deficient cellular immunity, such as AIDS patients, recipients of organ transplants and children who have acquired the virus before birth. In this thesis we have characterized the cellular immunity to CMV in immunocompetent subjects, in patients receiving transplants and in infants.

In healthy individuals with latent CMV, the frequencies of CMV-specific CD8 T cells varied considerably between the donors. Within the same individual, the changes over time were usually small. In patients with primary, symptomatic CMV infection, the frequencies of CMV-specific CD8 T cells peaked within the first month after the appearance of symptoms. The frequencies then declined to levels similar to those in latently infected CMV carriers. The CD4 T-cell function followed the same pattern, but with lower peak values.

Immunosuppressed renal transplant patients with latent CMV had CMV-specific CD4 cell function similar to healthy controls. The frequencies of CMV-specific CD8 T cells were also comparable, but their function was impaired. When renal transplant recipients were investigated longitudinally, we found that their CMV-specific T cells decreased rapidly after transplantation. Whereas the frequencies and function of CD8 T cells rebounded within 3 months, CD4 T-cell recovery was impaired during the entire first year after transplantation.

Finally, the frequencies and function of CMV-specific T-cells were investigated in children with congenital and postnatal CMV. CMV-specific CD8 T cells could be detected in even the youngest children, suggesting that these cells can develop early in life. In contrast, CMV specific CD4 T cells were low or absent in the youngest children but increased slowly with age.

Human cytomegalovirus (HCMV) is a highly prevalent opportunistic infection and a major pathogen in immune-compromised patients. The virus exhibits a wide cell tropism and is able to lytically infect virtually any cell type, with detectable gene expression and release of new virions, but not the neutrophil. This cell is the first immune cell to engage most pathogens, engulfing and killing them before undergoing apoptosis and clearance by macrophages. However certain viruses and bacteria are able to evade host defences and use the neutrophil as a “Trojan horse” for replication and dissemination. In this context, enhanced neutrophil survival may promote infection. This work describes a profound neutrophil survival phenotype elicited by contact with live or UV-inactivated HCMV, in the absence of lytic viral gene expression. The effect does not involve signalling through candidate Toll-like receptors, but is dependent on activation of the ERK MAPK and NFκB signalling pathways, and is viral strain-dependent, restricted to clinical strains of the virus. Furthermore, HCMV triggers the secretion of a bioactive secretome that induces a similar paracrine anti-apoptotic effect in fresh neutrophils, and stimulates monocyte chemotaxis and differentiation to a phenotype that is permissive for HCMV infection. This “transferrable” effect is not due to residual virus or the presence of well-known neutrophil survival factors such as IL-6 or IL-8, but is mediated by a heat-stable protein or lipid, secreted late in culture. These results are supported by data in neutrophils isolated from patients with CMV viraemia and pneumonitis which show increased survival ex vivo, and will be further investigated using plasma membrane profiling by amino-oxybiotinylation and tandem mass tag mass spectrometry. This technique, used for the first time here in a primary cell type, allows quantitative proteomics to be performed for the first time in the neutrophil. This work demonstrates that the technique provides a comprehensive readout of all neutrophil plasma membrane proteins in a sample, with high plasma membrane purity and minimal neutrophil activation and necrosis, validated by flow cytometry. Furthermore, this has been applied to generate plasma membrane profiles for the resting, inflammatory and apoptotic neutrophil, revealing a number of neutrophil cell surface molecules not found by previous membrane proteomic methods. This technique has the potential to analyse the effect of HCMV and other pathogens on the expression profile of the neutrophil surface membrane and to examine how neutrophil signalling and function is modulated. These data shed light on the role of neutrophil apoptosis as a potential promoter of HCMV infection, and have the potential to increase our understanding of both the neutrophil’s response to pathogen invasion and to generate future approaches to combating HCMV dissemination and pathogenesis.

Les virus appartenant à la famille Herpesviridae (ordre Herpesvirales) sont répartis au sein de trois sous-familles : Alpha-, Beta- et Gamma-herpesvirinae. Ils ont été identifiés à partir d’un large spectre d’espèces hôtes, allant des mammifères aux reptiles en passant par les oiseaux et ont la capacité de persister toute au long de la vie de l’hôte. La plupart de ces virus sont par ailleurs spécifiques d’une espèce hôte. La large distribution des herpèsvirus, associée à une infection généralement asymptomatique chez l’hôte naturel suggèrent que ces virus auraient co-évolué avec leurs hôtes.Mon premier axe de travail a porté sur l’identification de cytomégalovirus (CMV) chez les primates non-humains du Nouveau Monde (PNHNM) afin de voir si la diversification de ces virus suivait celle de leurs hôtes. De fait, une étude préalable conduite sur les primates de l’Ancien Monde avait démontré que les CMV les infectant présentaient un fort signal de co-divergence avec leurs hôtes. Or, parmi les différentes espèces de primates testées, seules quelques-unes provenaient du Nouveau Monde. Afin de répondre à cette question, nous avons effectué un criblage moléculaire de 244 échantillons d'ADN sanguin provenant de 20 espèces d'Amérique centrale et du sud. Par une approche de PCR utilisant des amorces consensus dégénérées ciblant des motifs hautement conservés du gène de l'ADN polymérase des herpèsvirus, nous avons caractérisé de nouvelles séquences virales de sept genres représentatifs des trois familles de PNHNM. Ces résultats démontrent ainsi que la plupart des espèces de PNHNM peuvent être infectées par un virus appartenant au genre Cytomegalovirus. Par ailleurs, les analyses phylogénétiques effectuées, couplées à la datation moléculaire des séquences obtenues, soutiennent cette hypothèse co-évolutive.Mon second axe visait à identifier des herpèsvirus de chauves-souris du Nouveau Monde afin d’en étudier la distribution et la diversité. La recherche d’herpèsvirus chez les chauves-souris est plus récente. Elle a bénéficié de l’intérêt porté à ces mammifères au début des années 2000 du fait de leur rôle de réservoirs de virus potentiellement zoonotiques. C’est à partir de 2007 que les premières séquences d’herpèsvirus de chauves-souris ont été décrites. Il s’en est suivi la description de dizaines de nouvelles séquences identifiées de différentes espèces d’Afrique, d’Asie, d’Océanie, d’Europe et d’Amérique. Néanmoins, la distribution des espèces testées était géographiquement inégale et seules quelques-unes provenaient du Nouveau Monde. Nous avons effectué un criblage moléculaire de 195 échantillons d’ADN sanguin provenant de 11 espèces appartenant à trois familles (Phyllostomidae, Mormoopidae et Molossidae). En utilisant la même approche que celle appliquée au PNHNM, nous avons obtenu des séquences virales (ADN polymérase et/ou Glycoprotéine B) de toutes les espèces testées. Celles-ci se répartissent au sein des sous familles Beta- et Gamma-herpesvirinae. Quatorze séquences partielles du gène de l'ADN polymérase, correspondant à trois beta- et onze gamma-herpèsvirus, ont été identifiées. Douze séquences partielles du gène de la glycoprotéine B, toutes de gamma-herpèsvirus, ont été caractérisées. Chaque séquence était spécifique à une espèce de chauve-souris et, chez certaines espèces, de multiples virus ont été identifiés. Les analyses phylogénétiques de ces séquences ont permis d'identifier des clades spécifiques aux virus de chauve-souris. Ceux composés de séquences obtenues à partir de différentes espèces appartenant à des sous-familles distinctes suivent la taxonomie des chauves-souris. Cette étude confirme l'étonnante diversité des herpèsvirus de chauve-souris et élargit nos connaissances sur leur spectre d'hôtes
Viruses belonging to the Herpesviridae family (order Herpesvirales) are enveloped double-stranded DNA viruses distributed into three subfamilies: Alpha-, Beta- and Gamma-herpesvirinae. These viruses have been identified from a wide range of host species, ranging from mammals to reptiles to birds. They have the ability to persist throughout the life of the host in a latent form and to reactivate. Most of these viruses are specific to a host. The wide distribution of herpesviruses, generally associated with an asymptomatic infection in their natural host, suggests that these viruses have co-evolved with their hosts.The first part of this work was dedicated to the identification of cytomegaloviruses (CMV, genus Cytomegalovirus) in New World non-human primates (NWNHP) to see if the diversification of these viruses followed that of their hosts. A previous study on Old World primates had demonstrated that CMVs infecting them showed a strong signal of co-divergence with their hosts. Nevertheless, among the different species of primates tested, only a few were from the New World. To test this hypothesis, we performed a molecular screening of 244 blood DNA samples from 20 Central and South American species. Through a PCR approach using degenerate consensus primers targeting highly conserved motifs of the DNA polymerase gene of herpesviruses, we characterized new viral sequences from 12 species belonging to seven representative genera of the three families of NWNHP. These results demonstrate that most species of NWNHP can be infected with a virus belonging to the Cytomegalovirus genus. In addition, phylogenetic analyzes of the obtained sequences combined with their molecular dating support a co-evolutionary scenario. This study led us to propose that CMVs sequences of NWNHP could serve as a molecular marker with which to infer the not yet fully resolved systematics of these primates.The second part of this work was on identifying herpesviruses from New World bats to study their distribution and diversity. The search for herpesviruses in bats is more recent. In the early 2000s it benefited from studies dedicated to other viral families given their role as reservoirs of potentially zoonotic viruses. The first description of bat herpesvirus sequences dated back from 2007. Over the past decade, dozens of herpesvirus sequences have been described from different bat species on every continent. Nevertheless, the distribution of the tested species was geographically uneven and only a few were from the New World. Molecular screening of 195 blood DNA samples from 11 species belonging to three families (Phyllostomidae, Mormoopidae and Molossidae) was performed. Using the same approach as applied to NWNHP, we obtained viral sequences (DNA polymerase and/or glycoprotein B) from all tested species. These are distributed within the Beta- and Gamma-herpesvirinae subfamilies. Fourteen partial sequences of the DNA polymerase gene, corresponding to three beta- and eleven gamma-herpesviruses, have been identified. Twelve partial sequences of the glycoprotein B gene, all gamma-herpesviruses, have been characterized. Each sequence was specific to a bat species and in some species multiple viruses were identified. Phylogenetic analyzes of these sequences have identified clades specific to bat viruses. Those composed of sequences obtained from different species belonging to distinct subfamilies follow the taxonomy of bats. This study confirms the astonishing diversity of bat herpesviruses and broadens our knowledge on their host spectrum.This work is the largest conducted to date in terms of species diversity of non-human primates and bats from the Neotropical realm. Nevertheless, the samples tested represent only a tiny part of this diversity. Further analyzes, on a broader panel of representative species from other geographic areas will increase our understanding of the evolutionary history of these viruses

Cytomegalovirus (CMV) is a highly immunogenic beta-herpesvirus that establishes asymptomatic infection in immune competent individuals. The ability of CMV to induce high frequencies of functional effector memory CD8+ T cells that increase over time (termed ‘memory inflation’) makes this virus an attractive vaccine vector. The primary focus of this thesis is centred on the hypothesis that engineering a CMV-based vector expressing a tumour-associated antigen will be able to induce a robust and long-lived anti-tumour CD8+ T cell response. Using the murine CMV (MCMV) infection model, several MCMV-based vectors were constructed to express the tumour-associated antigens 5T4 and NY-ESO-1, from different locations within the m123-m128 locus of the MCMV genome, which has been associated with the induction of memory inflation. Systemic immunisation with these vectors revealed that induction of tumour-specific CD8+ T cells correlated with high tumour antigen expression during MCMV replication in vitro and in turn, depended on the site of insertion within the MCMV genome. MCMV-based vectors were also examined in prime-boost strategies that incorporated recombinant adenovirus expressing the corresponding tumour-associated antigen. Prime-boost immunisation resulted in the generation of polyfunctional tumour-specific CD8+ T cells that delayed tumour onset. Interestingly, immunisation with the MCMV vector led to an increased influx of lymphocytes infiltrating the tumour. Finally, the immunogenicity of a spread-defective MCMV vector, DgL-MCMV, was investigated upon subcutaneous challenge. Encouragingly, although the magnitude of the response was reduced compared to wild-type MCMV, DgL-MCMV was able to drive memory inflation. Collectively, these data show the importance of vector construction in an effective CMV-based vaccine and highlight the potential of CMV in influencing lymphocyte infiltration into tumours. Ultimately, it provides further evidence of the potential use of CMV as a vaccine vector for cancer therapy.

Human Cytomegalovirus (HCMV) is the most common cause of viral congenital infection in the world. Around 5-10% of HCMV infected children are symptomatic at birth, and 50-90% of these develop severe manifestations with a 30% mortality rate. Among the asymptomatic children at birth, 10-15% develop late sequelae. The major cell entry glycoproteins of HCMV form three complexes: gC-I containing gB; gC-II containing gM & gN; and gC-III containing gH, gL, and gO (or UL128-131). These entry glycoproteins are polymorphic, producing different glycoprotein genotypes. The polymorphic nature of the glycoproteins as well as their ability to elicit neutralizing antibodies made them of interest in correlating them with the severity and outcome of the disease. This study aimed to develop a robust system to identify clusters of glycoprotein genotypes and to correlate them with disease manifestation. PCR assays of high sensitivity were used to identify all six glycoproteins. The PCR products were digested using restriction enzymes (RFLP) to identify the glycoprotein genotypes. Available laboratory strains (AD169, Towne, Davis, Toledo, and Merlin) as well as 112 clinical samples were amplified and genotyped using the assay, and their glycoprotein genotype profile was determined. A reliable sensitive assay was successfully developed to identify all glycoprotein genotypes including a novel gM assay using PCR/RFLP. The clinical samples were grouped according to disease manifestation (Group 1: congenital/postnatal patients (subgroup 1A: confirmed congenital patients & subgroup 1B: patients with either congenital or postnatal infection), Group 2: immunocompetent patients, Group 3: immunocompromised patients (subgroup 3A: immunocompromised patients with primary infection, subgroup 3B: immunocompromised patients with recurrent infection & subgroup 3C: immunocompromised patients with unconfirmed primary or recurrent infection)). Genotype gB1 was found predominantly prevalent in congenital/postnatal and immunocompromised patients, while gB3 was the most common genotype in immunocompetent patients. This result along with the phylogenetic analysis performed in this study suggest a relationship between gB genotypes and the immune response of the patients, where gB3 may be positively selected by host immune pressure. The novel gM assay genotyped the highly conserved gene (UL100) into three distinct genotypes; gM3 genotype associated with the congenital/postnatal group; which may provide an insight into understanding viral attachment and spread into the host cell. In congenital/postnatal infection, gH1 (72.7%) and gL4 (65.1%) were the most prevalent genotypes (gH1= 32/44, gL4= 28/43; P=0.000). In immunocompetent patients, mixed gH and mixed gL genotypes significantly correlated with the group, and in the immunocompromised group gH2 and mixed gL genotype were the most common genotypes (51.1% and 46.9% respectively). Glycoproteins gO, gH and gL are components of gC-III complex and gO1 was found to be the most prevalent gO genotype in all infection types (Group 1= 32.1%, Group 2= 85.7%, Group 3= 18.8%; P < 0.05). Also, in congenital/postnatal infection gN and gO were found to significantly link with each other and this is expected since both glycoproteins are highly polymorphic and are located on adjacent gene loci in HCMV genome (gN1+gO1a (P=0.000), gN3a+gO4 (P= 0.000)). The specific gN-gO linkages found here could be potential indicators for congenital/postnatal infection. In congenital/postnatal infection group, gH had significant linkages to gN and gO (gH1+gN1 (P=0.023, gH1+gO1a (P=0.013)) suggesting that interlinked selection of glycoprotein genotypes in the gC-II and gC-III complexes is involved in the development of congenital infection. High viral loads were found trending with immunocompromised patients, while low viral loads were significantly associated with mixed infected patients. This study has shown significant associations between a number of glycoproteins and congenital infection. Previously ignored glycoproteins gM and gL have been shown to be potentially of significant interest in this study and a larger study to confirm this is needed. In most cases the pattern of glycoprotein genotypes in congenital infection is more similar to that of immunocompromised than immunocompetent patients and it is possible that immune pressure is selecting for or against particular glycoprotein genotypes. The relationship between mixed infection and sample type may offer opportunities for development of prognostic biomarkers for congenital disease and further work is warranted.

As doenças respiratórias estão entre as maiores causas de prejuízo para a indústria suinícola, seja pelo retardo no crescimento e ganho de peso, mortalidade de animais ou pelos gastos com vacinas, medicamentos e assistência veterinária. Neste contexto os quadros de rinite têm apresentado uma contribuição significativa. O presente estudo propõe a determinação dos perfis de eliminação de agentes envolvidos em rinite nos suínos avaliando diferentes faixas etárias em nove propriedades de ciclo completo com histórico de lesão em cornetos e que utilizem diferentes formas de prevenção e controle destas manifestações. Foram avaliados suabes de tonsilas de 12 animais, nas seguintes faixas etárias: matrizes, leitões de 20, 40 e 60 dias, suínos de 90, 110 e 140 dias, totalizando 84 animais por propriedade e 756 amostras em todo o estudo. As amostras foram submetidas à pesquisa de P. multocida tipo capsular A e D, gene codificador de toxina dermonecrótica de P. multocida, B. bronchiseptica e cytomegalovirus suíno através da reação em cadeia pela polimerase (PCR). Apesar do histórico de lesões em corneto em todas as propriedades apenas um animal foi positivo para presença de P. multocida tipo A e todos foram negativos para a presença do gene codificador da toxina dermonecrótica. Dentre os 756 animais 22 (2,9%) foram positivos para presença de B. bronchiseptica e 198 (26,1%) para detecção cytomegalovirus suíno. A presença B. bronchiseptica apresentou associação estatisticamente significativa com as fases de maternidade e terminação. A maior freqüência de cytomegalovirus suíno apresentou associação estatisticamente significativa com a fase de creche. Observaram-se matrizes eliminando B. bronchiseptica nos três tipos de granjas avaliadas, indicando que a fêmeas tem participação ativa na infecção dos leitões pelo agente. O mesmo não foi detectado na disseminação do cytomegalovirus suíno. Maiores estudos devem ser realizados para esclarecer a baixa eliminação de P. multocida e o verdadeiro impacto do cytomegalovirus nos rebanhos suínos.
Respiratory diseases are one of the largest cause of economic losses in swine industry, it is related with grown and weight gain reduction, mortality, vaccines and medicaments costs, veterinary assistance. In that context, rinithis cases have been a major contribution. The present study propose the determination of elimination profile of agents related with rhinitis evaluating different ages in nine swine herds with history of cornet lesions and that uses different ways to control and prevent this problem. There were examined tonsils swabs from 12 animals in the following ages: sows, piglets of 20, 40 60 days and pigs of 90, 110 and 140 days, totalizing 84 pigs for farm. The swabs were searched to P. multocida capsular type A and D, dermonecrotic toxin gene from P. multocida, B. bronchiseptica and porcine cytomegalovirus through polymerase chain reaction (PCR). Despite de turbinate bones lesions present in all herds P. multocida type A was detected in only one pig and none were positive to dermonecrotic toxin gene. From 756 animals, 22 (2.9%) were positive to B. bronchiseptica and 198 (26.1%) to porcine cytomegalovirus detection. The presence of B. bronchiseptica presented statistical association with the farrowing and finishing times. Larger number of animals positive to cytomegalovirus show statistical association with the post weaning pigs. Sows carrying B. bronchiseptica in the three types of herds examined, suggesting that sows have an active participation in piglet infection by this agent. The same was not observed in porcine cytomegalovirus spread. More projects were need to clarify the low detection of P. multocida and to understand the impact of cytomegalovirus in swine production.