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HSV-2 may have adverse consequences in HIV. I evaluated the impact of HSV-2 co-infection on (highly active antiretroviral therapy)-untreated HIV infection in a systematic review of observational studies (study 1) and a retrospective cohort (study 2). I further evaluated whether HSV reactivation rates in co-infected persons differ by use of suppressive cART (study 3). Study 1 found modest evidence that HSV-2 seropositivity may be associated with accelerated progression to opportunistic infection or clinical AIDS, but not with increased HIV viral load. Some evidence suggests that HSV-2 disease activity is associated with increased HIV viral load and decreased CD4 counts. Study 2 compared rates of CD4 count change by HSV-2 status (Focus HerpeSelect ELISA) among 218 patients with a past period of ART-untreated follow-up using mixed linear regression models. No significant difference in the rate of CD4 count change was observed in HSV-2 seropositives at +13.6 cells/mm3/year (p=0.12) in univariate analysis, and -4.5 cells/mm3/year (p=0.68) in analysis adjusted for sex, HSV-1, oral and genital HSV symptoms, immigrant status, and immigrant*time interaction. These findings support the need for carefully designed and executed studies of HSV-2 suppression as an adjunctive management strategy for HIV disease, but raise questions regarding the exact mechanism of negative synergy between these viruses and the relative importance of HSV-2 latency and replication in driving these effects. In Study 3, 44 cART-naïve and 41 treated (HIV RNA<50 copies/mL) HIV+ adults with HSV-1 and/or 2 co-infection collected oral, genital and anal swabs daily for 28 days. Negative binomial models were used to quantify the relationship between cART and HSV shedding (Roche LightCycler HSV1/2). Overall HSV shedding was low, at a median (IQR) of 3.6% (0, 14.3%) of days. No relationship was seen between cART and HSV-1 or 2 shedding in univariate (RR=1.55, 95%CI=0.83,2.87) or multivariate analysis adjusted for sex, baseline CD4, recent immigrant status, and time since HIV diagnosis (aRR=1.05, 95%CI=0.43,2.58). Null results were also observed for HSV-1 and HSV-2 considered separately. That HSV shedding persists despite cART suggests that trials of anti-HSV drugs for improving HIV outcomes may be warranted in such patients.

HSV-2 may have adverse consequences in HIV. I evaluated the impact of HSV-2 co-infection on (highly active antiretroviral therapy)-untreated HIV infection in a systematic review of observational studies (study 1) and a retrospective cohort (study 2). I further evaluated whether HSV reactivation rates in co-infected persons differ by use of suppressive cART (study 3). Study 1 found modest evidence that HSV-2 seropositivity may be associated with accelerated progression to opportunistic infection or clinical AIDS, but not with increased HIV viral load. Some evidence suggests that HSV-2 disease activity is associated with increased HIV viral load and decreased CD4 counts. Study 2 compared rates of CD4 count change by HSV-2 status (Focus HerpeSelect ELISA) among 218 patients with a past period of ART-untreated follow-up using mixed linear regression models. No significant difference in the rate of CD4 count change was observed in HSV-2 seropositives at +13.6 cells/mm3/year (p=0.12) in univariate analysis, and -4.5 cells/mm3/year (p=0.68) in analysis adjusted for sex, HSV-1, oral and genital HSV symptoms, immigrant status, and immigrant*time interaction. These findings support the need for carefully designed and executed studies of HSV-2 suppression as an adjunctive management strategy for HIV disease, but raise questions regarding the exact mechanism of negative synergy between these viruses and the relative importance of HSV-2 latency and replication in driving these effects. In Study 3, 44 cART-naïve and 41 treated (HIV RNA<50 copies/mL) HIV+ adults with HSV-1 and/or 2 co-infection collected oral, genital and anal swabs daily for 28 days. Negative binomial models were used to quantify the relationship between cART and HSV shedding (Roche LightCycler HSV1/2). Overall HSV shedding was low, at a median (IQR) of 3.6% (0, 14.3%) of days. No relationship was seen between cART and HSV-1 or 2 shedding in univariate (RR=1.55, 95%CI=0.83,2.87) or multivariate analysis adjusted for sex, baseline CD4, recent immigrant status, and time since HIV diagnosis (aRR=1.05, 95%CI=0.43,2.58). Null results were also observed for HSV-1 and HSV-2 considered separately. That HSV shedding persists despite cART suggests that trials of anti-HSV drugs for improving HIV outcomes may be warranted in such patients.

No description available.

Vaccination has proven a cost-effective method of managing infectious diseases, but attempts to develop an effective fetal vaccine have proven difficult due to the immaturity of the immune system and the propensity of the developing immune system to induce tolerance to immunizing antigens. This thesis is concerned with the induction of specific immunity in the second trimester ovine fetus using the oral DNA immunization method. In utero oral delivery of naked DNA plasmid was selected as the method of immunization due to previous successes in the third trimester ovine fetus and the immunostimulatory properties of the bacterial DNA backbone, which may help overcome developmental tolerance. Transfection and expression studies in the third trimester ovine fetus revealed the oral mucosal epithelium as the primary site of transgene expression and functionally active antigen was also localized to lymph nodes draining the oral cavity. Efficient transfection and expression of plasmid following oral delivery was specific to the fetus and correlated with a lesser degree of epithelial differentiation. Oral DNA delivery in the second trimester resulted in detection of transgene activity in 100% of treated fetuses and the level of transgene activity was greater than in fetuses treated in the mid-third trimester. Using a plasmid encoding the gene for bovine herpesvirus-1 truncated glycoprotein D (tgD), immunization studies were then conducted in the second trimester fetus. A new lower age limit for fetal immunization was established at 55-60 days gestation (gestation period is 148 days), which coincides with the appearance of lymphocytes in peripheral tissues. Antigen-specific antibody, interferon-× responses and/or neonatal anamnestic responses were detected in 66% of fetuses immunized between 55 and 84 days gestation. The duration of fetal primary immune responses was equivalent to that achieved in young lambs following optimized DNA vaccination, but the magnitude of fetal immune responses was limited. The persistence of immune memory from the second trimester to birth was consistent with experimental data which showed that the duration of immune memory had a stronger correlation to the duration, as compared to the magnitude, of the primary antibody response. Overall, the experiments within showed that oral DNA immunization of the early second trimester fetus is feasible and not associated with the induction of tolerance. These findings suggest that it may be possible to protect against mother-to-child transmission of infectious pathogens by targeting protection at the level of the fetus.

Vaccination has proven a cost-effective method of managing infectious diseases, but attempts to develop an effective fetal vaccine have proven difficult due to the immaturity of the immune system and the propensity of the developing immune system to induce tolerance to immunizing antigens. This thesis is concerned with the induction of specific immunity in the second trimester ovine fetus using the oral DNA immunization method. In utero oral delivery of naked DNA plasmid was selected as the method of immunization due to previous successes in the third trimester ovine fetus and the immunostimulatory properties of the bacterial DNA backbone, which may help overcome developmental tolerance. Transfection and expression studies in the third trimester ovine fetus revealed the oral mucosal epithelium as the primary site of transgene expression and functionally active antigen was also localized to lymph nodes draining the oral cavity. Efficient transfection and expression of plasmid following oral delivery was specific to the fetus and correlated with a lesser degree of epithelial differentiation. Oral DNA delivery in the second trimester resulted in detection of transgene activity in 100% of treated fetuses and the level of transgene activity was greater than in fetuses treated in the mid-third trimester. Using a plasmid encoding the gene for bovine herpesvirus-1 truncated glycoprotein D (tgD), immunization studies were then conducted in the second trimester fetus. A new lower age limit for fetal immunization was established at 55-60 days gestation (gestation period is 148 days), which coincides with the appearance of lymphocytes in peripheral tissues. Antigen-specific antibody, interferon-× responses and/or neonatal anamnestic responses were detected in 66% of fetuses immunized between 55 and 84 days gestation. The duration of fetal primary immune responses was equivalent to that achieved in young lambs following optimized DNA vaccination, but the magnitude of fetal immune responses was limited. The persistence of immune memory from the second trimester to birth was consistent with experimental data which showed that the duration of immune memory had a stronger correlation to the duration, as compared to the magnitude, of the primary antibody response. Overall, the experiments within showed that oral DNA immunization of the early second trimester fetus is feasible and not associated with the induction of tolerance. These findings suggest that it may be possible to protect against mother-to-child transmission of infectious pathogens by targeting protection at the level of the fetus.

Les primates non-humains (PNH) sont un important réservoir de pathogènes et notamment de rétrovirus. Plusieurs agents infectieux ont émergé dans la population humaine à partir de ce réservoir animal, comme par exemple le virus de l’immunodéficience humaine ou le virus T lymphotrope humain (HTLV) de type 1 qui se sont répandus mondialement et causent de graves pathologies. L’émergence de rétrovirus chez l’Homme nécessite plusieurs étapes passant par la transmission primaire du virus du PNH à l’Homme, la persistance du virus dans l’organisme,la transmission secondaire inter-humaine et enfin sa diffusion dans la population. Mes deux projets de thèse ont porté sur l’étude de deux rétrovirus qui ont un potentiel d’émergence chez l’Homme, le virus foamy simien (SFV) et le virus T-lymphotrope de type 4, dans des cohortes d’individus à risque vivant au Cameroun et au Gabon. Les SFV sont des rétrovirus ubiquitaires chez de nombreux PNH. Plus d’une centaine de cas d’infection d’humains par des SFV ont été observés à ce jour, l’origine étant un contact(principalement par morsure) avec un PNH. L’infection est chronique et semble asymptomatique.De plus, aucune transmission secondaire n’a été détectée à ce jour. Le laboratoire a pu isoler, chez deux individus camerounais, deux souches de SFV de gorille et a constaté une forte variabilité génétique au niveau du gène d’enveloppe. Nous avons donc étudié la variabilité du gène d’enveloppe de SFV de gorille mais également de chimpanzé infectant une soixantaine de chasseurs camerounais et gabonais et une trentaine de PNH. Nous avons pu mettre en évidence la co-circulation de souches de SFV présentant des variants moléculaires du gène d’enveloppe différents chez les gorilles et les chimpanzés. Ces mêmes souches peuvent être transmises à l’Homme à l’occasion de morsures, les deux variants pouvant être transmis simultanément. Ces variants diffèrent au niveau d’une région de 753 pb située dans la région codant la glycoprotéine de surface, au niveau du domaine de liaison au récepteur. Ils pourraient ainsi avoir des propriétés fonctionnelles différentes, notamment au niveau de l’élicitation de la réponse immunitaire de l’hôte. Ces variants sont potentiellement issus d’événements de recombinaison.HTLV-4 a été détecté chez un unique individu, un chasseur camerounais, aujourd’hui décédé.En 2014, le réservoir simien a été identifié comme étant les gorilles. Nous avons recherché la présence de HTLV-4 chez 300 individus camerounais et gabonais qui avaient été mordus par un PNH. Nous avons identifié deux chasseurs gabonais infectés par HTLV-4, qui avaient été mordus par des gorilles 9 à 15 ans avant d’être prélevés. Nous confirmons donc la présence de HTLV-4 infectant de manière persistante des humains et étendons sa répartition au Gabon. Nous suggérons très fortement une origine zoonotique de ces infections. L’une des souches isolées est divergente par rapport aux souches déjà connues et permet donc de définir le sous-type B deHTLV-4.Ces travaux confortent la notion que les PNH, et notamment les gorilles, sont une source importante d’agents infectieux pour l’Homme. Des études supplémentaires seront nécessaires pour mieux caractériser l’infection chez l’Homme et notamment l’éventuelle pathogénicité et transmissibilité de ces deux virus. / Non-human primates (NHPs) are an important reservoir of pathogens, including retroviruses. Several retroviruses have emerged in human population from NHP reservoir, like the human immunodeficiency virus and the human T-lymphotropic virus type 1 who have spread globally and are the causative agents of serious pathologies. During my PhD, I interested in two retroviruses who have an emerging potential in human population, the simian foamy virus (SFV) and the human T-lymphotropic virus type 4 (HTLV-4), in cohorts of individuals at risk in Central Africa. SFV are retroviruses ubiquituous in NHPs. A hundred of human infections with SFV are known, originating from contacts with NHP. The infection is chronic, asymptomatic although no secondary transmission has been observed yet. We showed that two envelope molecular variants of SFV are co-circulating in gorilla and chimpanzee populations. These strains can be transmitted to humans through bites. The variants differ in the receptor binding domain on the envelope and could have different functional properties. HTLV-4 had been detected in a single individual (a cameroonese hunter) and the simian reservoir idenfied as gorillas. We have detected two gabonese hunters infected with HTLV-4, who had been bitten by gorillas. Then we confirm the presence of HTLV-4 in humans in Central Africa. One of the strains is divergent and defines the prototype of a new subtype of HTLV-4

In order to facilitate a biophysical analysis of the carboxy terminal acidic transactivation domain (AAD) of Vmw65 from Herpes Simplex Virus Type 1 (HSV-1), an overexpression system in Escherichia coli was constructed and optimized to produce milligram quantities of this polypeptide. Purification of the polypeptide was facilitated by creating a fusion protein to glutathione S-transferase (GST) from Schizosoma japonicum using a commercially available vector. Upon thrombin digestion of the fusion protein, the carrier and AAD products were resolved by anion-exchange chromatography. With typically 15 mg of AAD available from a 12 litre culture, several biophysical studies were initiated. Circular dichroism and fluorescence spectroscopy both described a polypeptide with an extended structure reminicent of a random-coil; that is, it did not possess substantial quantities of known elements of secondary structure such as a-helicies and β-sheets under physiological conditions. A new structure high in α-helical content was induced upon addition of trifluoroethanol to mimic a hydrophobic milieu. Ultracentrifugation data supported the spectroscopic observations by describing an extended, monomeric polypeptide. The ultimate goal of the study, a teritiary structure, was sought by attempting to crystallize AAD with popular salts and organic solvents. Biologically, the described random-coil structure of AAD could be relevant to its role as a promoter and stablizer of the transcriptional pre-initation complex, the determining step in gene expression. A structurally labile domain would support AAD’s ability to interact with several targets including TFIID and TFIIB, though not necessarily by similar mechanisms. The requirement for a drastic conformational change such as a random-coil to α-helical transition currently remains unclear though observations made in this study of AAD in trifluoroethanol have shown that a conformational change is indeed possible. With a means of producing large quantities of AAD, the opportunity now arises to study its interaction with available cloned targets. The ensuing biophysical studies will then provide a greater understanding of AAD’s important role in gene expression. / Thesis / Master of Science (MSc)

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