Impaired immune evasion in HIV through intracellular delays and multiple infection of cells

With its high mutation rate, HIV is capable of escape from recognition, suppression and/or killing by CD8(+) cytotoxic T lymphocytes (CTLs). The rate at which escape variants replace each other can give insights into the selective pressure imposed by single CTL clones. We investigate the effects of specific characteristics of the HIV life cycle on the dynamics of immune escape. First, it has been found that cells in HIV-infected patients can carry multiple copies of proviruses. To investigate how this process affects the emergence of immune escape, we develop a mathematical model of HIV dynamics with multiple infections of cells. Increasing the frequency of multiple-infected cells delays the appearance of immune escape variants, slows down the rate at which they replace the wild-type variant and can even prevent escape variants from taking over the quasi-species. Second, we study the effect of the intracellular eclipse phase on the rate of escape and show that escape rates are expected to be slower than previously anticipated. In summary, slow escape rates do not necessarily imply inefficient CTL-mediated killing of HIV-infected cells, but are at least partly a result of the specific characteristics of the viral life cycle.

Impaired CD19 expression and signaling, enhanced antibody response to type II T independent antigen and reduction of B-1 cells in CD81-deficient mice

The tetraspanin CD81 is ubiquitously expressed and associated with CD19 on B lymphocytes and with CD4 and CD8 on T lymphocytes. Analysis of mice with disrupted CD81 gene reveals normal T cells but a distinct abnormality in B cells consisting of decreased expression of CD19 and severe reduction in peritoneal B-1 cells. CD81-deficient B cells responded normally to surface IgM crosslinking, but had severely impaired calcium influx following CD19 engagement. CD81-deficient mice had increased serum IgM and IgA and an exaggerated antibody response to the type II T independent antigen TNP-Ficoll. These results suggest that CD81 is important for CD19 signaling and B cell function.

Targeting HIV proteins to the major histocompatibility complex class I processing pathway with a novel gp120-anthrax toxin fusion protein

A challenge for subunit vaccines whose goal is to elicit CD8+ cytotoxic T lymphocytes (CTLs) is to deliver the antigen to the cytosol of the living cell, where it can be processed for presentation by major histocompatibility complex (MHC) class I molecules. Several bacterial toxins have evolved to efficiently deliver catalytic protein moieties to the cytosol of eukaryotic cells. Anthrax lethal toxin consists of two distinct proteins that combine to form the active toxin. Protective antigen (PA) binds to cells and is instrumental in delivering lethal factor (LF) to the cell cytosol. To test whether the lethal factor protein could be exploited for delivery of exogenous proteins to the MHC class I processing pathway, we constructed a genetic fusion between the amino-terminal 254 aa of LF and the gp120 portion of the HIV-1 envelope protein. Cells treated with this fusion protein (LF254-gp120) in the presence of PA effectively processed gp120 and presented an epitope recognized by HIV-1 gp120 V3-specific CTL. In contrast, when cells were treated with the LF254-gp120 fusion protein and a mutant PA protein defective for translocation, the cells were not able to present the epitope and were not lysed by the specific CTL. The entry into the cytosol and dependence on the classical cytosolic MHC class I pathway were confirmed by showing that antigen presentation by PA + LF254-gp120 was blocked by the proteasome inhibitor lactacystin. These data demonstrate the ability of the LF amino-terminal fragment to deliver antigens to the MHC class I pathway and provide the basis for the development of novel T cell vaccines.

Heat shock fusion proteins as vehicles for antigen delivery into the major histocompatibility complex class I presentation pathway

Mice immunized with heat shock proteins (hsps) isolated from mouse tumor cells (donor cells) produce CD8 cytotoxic T lymphocytes (CTL) that recognize donor cell peptides in association with the major histocompatibility complex (MHC) class I proteins of the responding mouse. The CTL are induced apparently because peptides noncovalently associated with the isolated hsp molecules can enter the MHC class I antigen processing pathway of professional antigen-presenting cells. Using a recombinant heat shock fusion protein with a large fragment of ovalbumin covalently linked to mycobacterial hsp70, we show here that when the soluble fusion protein was injected without adjuvant into H-2b mice, CTL were produced that recognized an ovalbumin-derived peptide, SIINFEKL, in association with Kb. The peptide is known to arise from natural processing of ovalbumin in H-2b mouse cells, and CTL from the ovalbumin-hsp70-immunized mice and a highly effective CTL clone (4G3) raised against ovalbumin-expressing EL4 tumor cells (EG7-OVA) were equally effective in terms of the concentration of SIINFEKL required for half-maximal lysis in a CTL assay. The mice were also protected against lethal challenge with ovalbumin-expressing melanoma tumor cells. Because large protein fragments or whole proteins serving as fusion partners can be cleaved into short peptides in the MHC class I processing pathway, hsp fusion proteins of the type described here are promising candidates for vaccines aimed at eliciting CD8 CTL in populations of MHC-disparate individuals.

Identification of functional domains on human interleukin 10

Interleukin 10 (IL-10) is a recently described natural endogenous immunosuppressive cytokine that has been identified in human, murine, and other organisms. Human IL-10 (hIL-10) has high homology with murine IL-10 (mIL-10) as well as with an Epstein–Barr virus genome product BCRFI. This viral IL-10 (vIL-10) shares a number of activities with hIL-10. IL-10 significantly affects chemokine biology, because human IL-10 inhibits chemokine production and is a specific chemotactic factor for CD8+ T cells. It suppresses the ability of CD4+ T cells, but not CD8+ T cells, to migrate in response to IL-8. A nonapeptide (IT9302) with complete homology to a sequence of hIL-10 located in the C-terminal portion (residues 152–160) of the cytokine was found to possess activities that mimic some of those of hIL-10. These are: (i) inhibition of IL-1β-induced IL-8 production by peripheral blood mononuclear cell, (ii) inhibition of spontaneous IL-8 production by cultured human monocytes, (iii) induction of IL-1 receptor antagonistic protein production by human monocytes, (iv) induction of chemotactic migration of CD8+ human T lymphocytes in vitro, (v) desensitization of human CD8+ T cells resulting in an unresponsiveness toward rhIL-10-induced chemotaxis, (vi) suppression of the chemotactic response of CD4+ T human lymphocytes toward IL-8, (vii) induction of IL-4 production by cultured normal human CD4+ T cells, (viii) down-regulation of tumor necrosis factor-α production by CD8+ T cells, and (ix) inhibition of class II major histocompatibility complex antigen expression on IFN-γ-stimulated human monocytes. Another nonapeptide (IT9403) close to the NH2-terminal part of hIL-10 did not reveal cytokine synthesis inhibitory properties, but proved to be a regulator of mast cell proliferation. In conclusion, we have identified two functional domains of IL-10 exerting different IL-10 like activities, an observation that suggests that relatively small segments of these signal proteins are responsible for particular biological functions.

Long-term kinetics of T cell production in HIV-infected subjects treated with highly active antiretroviral therapy

The long-term kinetics of T cell production following highly active antiretroviral therapy (HAART) were investigated in blood and lymph node in a group of HIV-infected subjects at early stage of established infection and prospectively studied for 72 wk. Before HAART, CD4 and CD8 T cell turnover was increased. However, the total number of proliferating CD4+ T lymphocytes, i.e., CD4+Ki67+ T lymphocytes, was not significantly different in HIV-infected (n = 73) and HIV-negative (n = 15) subjects, whereas proliferating CD8+Ki67+ T lymphocytes were significantly higher in HIV-infected subjects. After HAART, the total body number of proliferating CD4+Ki67+ T lymphocytes increased over time and was associated with an increase of both naive and memory CD4+ T cells. The maximal increase (2-fold) was observed at week 36, whereas at week 72 the number of proliferating CD4+ T cells dropped to baseline levels, i.e., before HAART. The kinetics of the fraction of proliferating CD4 and CD8 T cells were significantly correlated with the changes in the total body number of these T cell subsets. These results demonstrate a direct relationship between ex vivo measures of T cell production and quantitative changes in total body T lymphocyte populations. This study provides advances in the delineation of the kinetics of T cell production in HIV infection in the presence and/or in the absence of HAART.

Melan-A/MART-151–73 represents an immunogenic HLA-DR4-restricted epitope recognized by melanoma-reactive CD4+ T cells

The human Melan-A/MART-1 gene encodes an HLA-A2-restricted peptide epitope recognized by melanoma-reactive CD8+ cytotoxic T lymphocytes. Here we report that this gene also encodes at least one HLA-DR4-presented peptide recognized by CD4+ T cells. The Melan-A/MART-151–73 peptide was able to induce the in vitro expansion of specific CD4+ T cells derived from normal DR4+ donors or from DR4+ patients with melanoma when pulsed onto autologous dendritic cells. CD4+ responder T cells specifically produced IFN-γ in response to, and also lysed, T2.DR4 cells pulsed with the Melan-A/MART-151–73 peptide and DR4+ melanoma target cells naturally expressing the Melan-A/MART-1 gene product. Interestingly, CD4+ T cell immunoreactivity against the Melan-A/MART-151–73 peptide typically coexisted with a high frequency of anti-Melan-A/MART-127–35 reactive CD8+ T cells in freshly isolated blood harvested from HLA-A2+/DR4+ patients with melanoma. Taken together, these data support the use of this Melan-A/MART-1 DR4-restricted melanoma epitope in future immunotherapeutic trials designed to generate, augment, and quantitate specific CD4+ T cell responses against melanoma in vivo.

Inhibition of MHC class I-restricted antigen presentation by γ2-herpesviruses

The γ-herpesviruses, in contrast to the α- and β-herpesviruses, are not known to inhibit antigen presentation to CD8+ cytotoxic T lymphocytes (CTLs) during lytic cycle replication. However, murine γ-herpesvirus 68 causes a chronic lytic infection in CD4+ T cell-deficient mice despite the persistence of a substantial CTL response, suggesting that CTL evasion occurs. Here we show that, distinct from host protein synthesis shutoff, γ-herpesvirus 68 down-regulates surface MHC class I expression on lytically infected fibroblasts and inhibits their recognition by antigen-specific CTLs. The viral K3 gene, encoding a zinc-finger-containing protein, dramatically reduced the half-life of nascent class I molecules and the level of surface MHC class I expression and was by itself sufficient to block antigen presentation. The homologous K3 and K5 genes of the related Kaposi's sarcoma-associated virus also inhibited antigen presentation and decreased cell surface expression of HLA class I antigens. Thus it appears that an immune evasion strategy shared by at least two γ-herpesviruses allows continued lytic infection in the face of strong CTL immunity.

Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy

Most tumor-associated antigens represent self-proteins and as a result are poorly immunogenic due to immune tolerance. Here we show that tolerance to carcinoembryonic antigen (CEA), which is overexpressed by the majority of lethal malignancies, can be reversed by immunization with a CEA-derived peptide. This peptide was altered to make it a more potent T cell antigen and loaded onto dendritic cells (DCs) for delivery as a cellular vaccine. Although DCs are rare in the blood, we found that treatment of advanced cancer patients with Flt3 ligand, a hematopoietic growth factor, expanded DCs 20-fold in vivo. Immunization with these antigen-loaded DCs induced CD8 cytotoxic T lymphocytes that recognized tumor cells expressing endogenous CEA. Staining with peptide-MHC tetramers demonstrated the expansion of CD8 T cells that recognize both the native and altered epitopes and possess an effector cytotoxic T lymphocyte phenotype (CD45RA+CD27−CCR7−). After vaccination, two of 12 patients experienced dramatic tumor regression, one patient had a mixed response, and two had stable disease. Clinical response correlated with the expansion of CD8 tetramer+ T cells, confirming the role of CD8 T cells in this treatment strategy.

Oxidative/reductive conjugation of mannan to antigen selects for T1 or T2 immune responses.

The induction of CD8+ cytotoxic T lymphocytes (CTLs) is desirable for immunization against many diseases, and recombinant-synthetic peptide antigens are now favored agents to use. However, a major problem is how to induce CTLs, which requires a T1-type response to such synthetic antigens. We report that T1-type (generating high CTL, low antibody) or T2-type (the reciprocal) responses can be induced by conjugation of the antigen to the carbohydrate polymer mannan: T1 responses are selected by using oxidizing conditions; T2 responses are selected by using reducing conditions for the conjugation. Using human MUC1 as a model antigen in mice, immunization with oxidized mannan-MUC1 fusion protein (ox-M-FP) led to complete tumor protection (challenge up to 5 x 10(7) MUC1+ tumor cells), CTLs, and a high CTL precursor (CTLp) frequency (1/6900), whereas immunization with reduced mannan-MUC1 FP (red-M-FP) led to poor protection after challenge with only 10(6) MUC1+ tumor cells, no CTLs, and a low CTLp frequency (1/87,800). Ox-M-FP selects for a T1 response (mediated here by CD8+ cells) with high interferon gamma (IFN-gamma) secretion, no interleukin 4 (IL-4), and a predominant IgG2a antibody response; red-M-FP selects for a T2-type response with IL-4 production and a high predominant IgG1 antibody response but no IFN-gamma.

Selection of conserved epitopes from hepatitis C virus for pan-populational stimulation of T-cell responses

The hepatitis C virus (HCV) is able to persist as a chronic infection, which can lead to cirrhosis and liver cancer. There is evidence that clearance of HCV is linked to strong responses by CD8 cytotoxic T lymphocytes (CTLs), suggesting that eliciting CTL responses against HCV through an epitope-based vaccine could prove an effective means of immunization. However, HCV genomic plasticity as well as the polymorphisms of HLA I molecules restricting CD8 T-cell responses challenges the selection of epitopes for a widely protective vaccine. Here, we devised an approach to overcome these limitations. From available databases, we first collected a set of 245 HCV-specific CD8 T-cell epitopes, all known to be targeted in the course of a natural infection in humans. After a sequence variability analysis, we next identified 17 highly invariant epitopes. Subsequently, we predicted the epitope HLA I binding profiles that determine their potential presentation and recognition. Finally, using the relevant HLA I-genetic frequencies, we identified various epitope subsets encompassing 6 conserved HCV-specific CTL epitopes each predicted to elicit an effective T-cell response in any individual regardless of their HLA I background. We implemented this epitope selection approach for free public use at the EPISOPT web server. © 2013 Magdalena Molero-Abraham et al.

Original Research Article Humoral and cellular immune responses to modified hepatitis B plasmid DNA vaccine in mice

Purpose: To evaluate the immunogenicity and types of immune response of a quality-controlled modified recombinant hepatitis B surface antigen (HBsAg) plasmid encoding HBsAg in mice. Methods: The characterized plasmid DNA was used in the immunization of Balb/c mice. Three groups of mice were intramuscularly injected with three different concentrations (50, 25 and 10 μg/100 μL) of the modified plasmid. Humoral immune response was monitored by enzyme-linked immunosorbent assay (ELISA), while cellular immune response was investigated by analysis of spleen cytokine profile (TNFα, IFN γ and IL2) as well as CD69 expression level in CD4 and CD8 positive cells. Results: In general, the activated CD4 cells showing intracellular cytokines were higher than CD8 positive population of cells (p < 0.05). These findings indicate that the vaccine induced both a humoral and cellular immunity. Cytokine profile also showed high levels of TNFα, IFN γ and IL2 and CD69 expression in the group of animals immunized at a dose of 10 μg when compared to control group (p < 0.05). Conclusion: A 10 μg dose intramuscular injection of the modified DNA-based vaccine encoding HBsAg in mice induces both high humoral and cellular immune responses.

Characterization of the immune response induced by a commercially available inactivated bluetongue virus serotype 1 vaccine in sheep

The protective immune response generated by a commercial monovalent inactivated vaccine against bluetongue virus serotype 1 (BTV1) was studied. Five sheep were vaccinated, boost-vaccinated, and then challenged against BTV1 ALG/2006. RT-PCR did not detect viremia at any time during the experiment. Except a temperature increase observed after the initial and boost vaccinations, no clinical signs or lesions were observed. A specific and protective antibody response checked by ELISA was induced after vaccination and boost vaccination. This specific antibody response was associated with a significant increase in B lymphocytes confirmed by flow cytometry, while significant increases were not observed in T lymphocyte subpopulations (CD4(+), CD8(+), and WC1(+)), CD25(+) regulatory cells, or CD14(+) monocytes. After challenge with BTV1, the antibody response was much higher than during the boost vaccination period, and it was associated with a significant increase in B lymphocytes, CD14(+) monocytes, CD25(+) regulatory cells, and CD8(+) cytotoxic T lymphocytes.

Caractérisation de DKK1 comme antigène tumoral et manipulation des lymphocytes T CD8 : utilisation de la voie de Wnt en immunothérapie du cancer

L’immunothérapie tumorale à médiation cellulaire est un traitement qui utilise le système immunitaire des patients afin d’induire une réponse des lymphocytes T CD8+ (T CD8+) contre la tumeur. Cette réponse est produite suite à la reconnaissance des antigènes par les T CD8+. Ces cibles sont appelées antigènes tumoraux (TAA) et définies comme des protéines exprimées par les cellules cancéreuses mais absentes des tissus normaux. Par une approche bio-informatique, notre laboratoire a identifié Dickkopf-1 (DKK1), une protéine inhibitrice de la voie de Wnt, comme un TAA potentiel. Une immunothérapie à médiation cellulaire efficace requiert l’identification de TAA candidats pertinents. Le traitement de patients par immunothérapie pourrait également être améliorées par l’augmentation de la puissance d’action anti-tumorale ainsi que la persistante des T CD8+ spécifiques aux TAA. Ce projet de doctorat se divise en deux parties : 1- La caractérisation de l’expression de DKK1 dans les cancers communs et la détermination de son immunogénicité afin de valider sa candidature comme TAA. 2- La reprogrammation des T CD8+, de patients atteints d’un cancer commun, vers un phénotype moins différentié afin d’augmenter leur potentiel anti-tumoral et leur persistance. Dans le premier objectif, nous avons caractérisé l’expression de DKK1 dans le cancer du sein et dans d’autres cancers communs. Le profil d’expression de DKK1 a été étudié par RT-PCR et par ELISA dans plusieurs lignées cellulaires de cancer et dans les tissus normaux. L’expression de DKK1 a aussi été étudiée dans des échantillons cliniques provenant de cancers du sein, du poumon et du rein. Trente pourcents (30%) des tumeurs provenant d’un cancer du sein exprimaient DKK1. La moitié des tumeurs DKK1(+) était triple négative, donc pas de récepteurs d’œstrogène et de progestérone et était Her-2/neu(-) (ces patientes ont des possibilités de traitements très restreintes). De plus, 50% des échantillons cliniques de tumeurs du poumon et 30% des tumeurs de rein exprimaient DKK1. Les observations effectuées dans le cancer du poumon ont été, par la suite, corroborées par d'autres groupes qui ont montré une corrélation entre l'expression de DKK1 et un mauvais pronostic. Après avoir confirmée l’expression de DKK1 dans les cancers communs, justifiant ainsi sa candidature comme TAA, nous avons évalué l’immunogénicité de DKK1. Pour ce faire, nous avons effectué des stimulations in vitro de cellules mononucléées du sang périphérique (PBMC) de patient(e)s atteint(e)s d’un cancer du sein ou du poumon avec des peptides dérivés de DKK1 pouvant être présentés par les complexes majeurs d’histocompatibilité (CMH) HLA-A*0201. Des clones de T CD8+ reconnaissant un peptide de DKK1 ont été identifiés et isolés. Par essai multiplex et cytométrie de flux intracellulaire, la polyfonctionnalité d’un ces clones T CD8+ spécifiques à DKK1 a été étudiée et a révélée un profil effecteur, renforçant ainsi la candidature de DKK1 comme TAA. Dans l’ensemble, les résultats obtenus dans cette première partie de thèse suggèrent une possible utilisation de DKK1 en immunothérapie contre les cancers communs, attribuable à son expression dans ces cancers et la possibilité de faire proliférer des T CD8+ effecteurs spécifiques à DKK1 à partir de sang de patients. Dans la seconde partie de cette thèse, je décrirai la manipulation in vitro des T CD8+ de patients atteints d’un cancer commun, afin d’augmenter la force et la durée de leurs fonctions anti-tumorales. Il a été démontré que des lymphocytes moins différentiés sont capables d’une réponse immunologique plus efficace et durable. Nous avons basé ce projet sur l’utilisation d’un inhibiteur pharmacologique de la GSK-3, pour activer de la voie de Wnt chez les T CD8+ et ainsi leur conférer un phénotype moins différentié, partageant des caractéristiques de la cellule naïve et de la cellule mémoire. Des cultures de T CD8+, spécifiques à des antigènes viraux, en présence de l’inhibiteur ont permis d’augmenter la sécrétion d’interféron (IFN)- et leur activité cytotoxique. Ces résultats indiquent un effet de l’activation de la voie de Wnt sur la fonction des T CD8+. Ces observations sont rapportées pour la première fois chez les T CD8+ humains et suggèrent une nouvelle stratégie, applicables à l’immunothérapie du cancer, afin de prolonger la persistance des cellules ainsi que leur activité anti-tumorale. En conclusion, ces travaux de recherche ont mené à la réalisation d’une étape très importante dans la validation de la candidature de DKK1 comme TAA pour les cancers communs, soit la démonstration de son expression dans ces cancers et son absence dans les tissus normaux dérivés d’organes importants. Ces travaux ont également mené à la démonstration de l’immunogénicité de DKK1, par l’identification d’un peptide de DKK1 reconnu par les T CD8+. De plus, l’étude de la polyfonctionnalité des T CD8+ spécifiques à DKK1 a révélée un profil effecteur favorable pour l’obtention d’une réponse anti-tumorale efficace. Ces découvertes pourraient servir à l’élaboration d’une stratégie d’immunothérapie à médiation cellulaire pour les cancers communs. Pour sa part, l’étude phénotypique et fonctionnelle de la modulation de la voie de Wnt dans les T CD8+ a donné lieu à l’observation d’un phénotype encore jamais rapporté chez l’humain, conférant aux T CD8+ un aspect moins différentié avec des caractéristiques propre à un phénotype mémoire. Ces résultats sont pertinents dans l’amélioration de l’immunothérapie du cancer, passant par l’augmentation de la persistance des lymphocytes. En résumé, les résultats présentés dans cette thèse de doctorat fournissent des évidences indéniables quant à la validation de DKK1 comme TAA pour une immunothérapie à médiation cellulaire des cancers communs. Ces résultats fournissent également des preuves quant à la pertinence de la reprogrammation des T CD8+ par l’activation de la voie de la voie de Wnt, afin de générer des lymphocytes médiateurs plus efficaces pour ce type de thérapie.

Caractérisation des lymphocytes T CD4+CD8+ dans le contexte de la sclérose en plaques

Une petite population de lymphocytes T exprimant les deux corécepteurs CD4 et CD8 et appelée double positive (DP), a été détectée dans le sang périphérique de donneurs sains et de patients atteints de diverses pathologies dont la sclérose en plaques (SEP). Nous avons émis l’hypothèse qu’il s’agissait de lymphocytes T hautement activés pouvant contribuer à l’inflammation chronique présente dans la SEP. Nous avons comparé les cellules T DP obtenues du sang de donneurs sains et de patients atteints de la SEP et non traités. La fréquence des cellules DP était similaire chez les patients et les donneurs sains. La proportion de lymphocytes T DP qui exprimaient les chaines du récepteur de l’interleukine-15 (IL-15) était plus élevée que pour les autres populations lymphocytaires. Des mesures d’induction de la phosphorylation du STAT5 (signal transducer and activator of transcription) ont démontré que les cellules DP ont répondu à des doses plus faibles et pour de plus longues périodes à l’IL-15 comparativement aux autres lymphocytes T. Le pourcentage de lymphocytes T DP ayant la capacité de produire l’interféron-gamma et des enzymes lytiques était élevé chez les témoins sains mais ces niveaux étaient significativement réduits chez les patients atteints de la SEP. La caractérisation phénotypique de cellules DP a suggéré que ces cellules ont des propriétés similaires aux lymphocytes T activés. Bien qu’il ne s’agisse que d’une caractérisation partielle, il semble que les lymphocytes T DP perdent une partie de leurs propriétés chez les patients atteints de la SEP.