The HIV-1 Nef accessory protein induces B cell abnormalties and autoimmunity

The HIV-1 Nef protein is known to be the major determinant of HIV-1 pathogenesis. It causes multiple abnormalities not only in the virus main target CD4+ T cells, but also other lymphocytes and myeloid cells. Studies of patients living with HIV-1 suggest that it impairs B cell functions. Poudrier J. (2001) examined the impact of the HIV-1 Nef accessory protein on B cells based on the expression of Nef in CD4+ cells of the CD4C/HIV-1Nef Tg mouse model, and established that it induced Ig switch defects and impairment in germinal centres(GC), as well as increased secretion of immunoglobulin M (IgM). This study aimed to determine the effect of Nef on B cell populations and functions in a mouse model. First, we examined changes in B cell subsets beginning in the bone marrow and continuing to the periphery. The presence of B cells in the bone marrow of Tg mice was normal in comparison to WT mice, which can be explained by the low expression of Nef in CD4+ cells. In the periphery, abnormalities of B cell subsets reflected the effect of the Nef protein expression on CD4+ cells, which led to the decline in the percentage of early immature transitional B cells and enrichment of mature cells that seemed to be correlated with down-modulation of surface expression CD21 and CD23. Second, in the presence of the Nef protein we observed the dysregulation in B cell activity, namely an increase in their proliferation and activation and may correlate with the increase in their percentage. Third, we explored a possible involvement of the HIV-1 Nef protein in HIV-related B cell lymphoma by using an Ig gene-translocation mouse model with Burkitt lymphoma. Finally, we investigated the presence of autoimmunity in the serum of the CD4C/HIV-1Nef Tg mouse model, which showed that expression of Nef alone in CD4+ T cells can trigger anti-DNA antibodies with IgM specificity and the deposition of IgM in the kidney glomeruli. These results suggest an active involvement of the HIV-1 Nef protein in B cell abnormalities and autoimmunity.La protéine Nef est un déterminant majeur dans la pathogenèse de l’infection au virus de l’immunodéficience humaine de type 1 (VIH-1). Non seulement Nef induit de multiples anormalités aux cellules T CD4+ (qui sont les cibles majeures du VIH), mais également aux autres lymphocytes et cellules immunitaires. Des études réalisées chez des patients vivant avec une infection au VIH-1 suggèrent que Nef altère les fonctions des cellules B. Les travaux par Poudrier J. (2001) ont examiné l’impact de la protéine accessoire Nef du VIH-1 sur les cellules B. Ils se sont basés sur l’expression de Nef dans les cellules CD4+ du modèle de souris transgénique (Tg) CD4C/HIV-1Nef et ont établi que cela induisait une défectuosité dans le changement de classe des immunoglobulines (Ig) et un dysfonctionnement au niveau des centres germinaux (GC). Ils ont aussi observé un niveau de sécrétion plus élevé des immunoglobulines M (IgM).La présente étude vise à déterminer les effets de Nef sur les populations et les fonctions des cellules B dans un modèle de souris. Premièrement, nous avons examiné les changements au niveau des sous-populations de cellules B qui naissent dans la moelle osseuse et qui migrent en périphérie. La présence des cellules B dans la moelle osseuse des souris transgéniques était normale en comparaison avec les souris sauvages, ce qui pourrait être expliqué par le niveau d’expression faible de Nef dans les cellules CD4+. En périphérie, les anormalités des sous-populations des cellules B reflètent l’effet de l’expression de la protéine Nef sur les cellules CD4+, ce qui engendre un déclin du pourcentage des cellules B transitionnelles immatures et précoces ainsi qu’un enrichissement des cellules matures qui semble corréler avec une régulation à la baisse de l’expression des marqueurs de surface CD21 et CD23. Deuxièmement, en présence de la protéine Nef, nous avons observé une dérégulation de l’activité des cellules B, plus précisément au niveau de leur prolifération et activation et ceci pourrait être en relation avec leur pourcentage augmenté. Troisièmement, nous avons exploré une implication possible de la protéine Nef de VIH-1 dans le lymphome de cellules B associé au VIH-1 en utilisant un modèle de souris atteint du lymphome de Burkitt avec un translocateur de gène Ig. Enfin, nous avons investigué la présence d’auto-immunité dans le sérum du modèle de souris Tg CD4C/HIV-1Nef. Les résultats montrent que l’expression de Nef seule dans les cellules T CD4+ peut déclencher la production d’anticorps anti-ADN avec une spécificité pour les IgM et le dépôt des IgM dans le glomérule rénal. Ces résultats suggèrent une implication active de la protéine Nef de VIH-1 dans les anormalités des cellules B et dans l’auto-immunité

Protecting HIV-1-infected cells from ADCC: role of Nef

ABSTRACT Despite the enormous efforts that are being made to develop new therapeutic strategies to fight HIV-1 infection, a better understanding of the elements contributing to HIV-1 virulence remains necessary to improve the effectiveness of these therapies. Emerging evidences suggest the importance role of Fc-mediated effector functions of anti-gp120 (glycoprotein-120) antibodies in the prevention and limitation of viral spread. This effector response was highlighted in the correlates of protection of the RV144 vaccine trial, the only trial that showed some levels of protection. However, the recognition of such antibodies relies on the necessity of CD4 and Envelope (Env) interaction that results in Env-conformational rearrangement and exposure of CD4-induced (CD4i) epitopes. The implication of the HIV-1 accessory protein Nef was suggested to be a major player in modulating the exposure of CD4i-epitopes and preventing the elimination of infected cells. Indeed, this multifunctional accessory protein is well characterized for its ability to regulate the surface expression of several receptors including CD4, MHC-I, CD28, and NKG2D ligands. Interestingly, the impairment of some Nef activities was described in elite controllers (EC), a rare subset of infected subjects who can control viral replication in the absence of antiretroviral treatment. The work presented in this thesis, focuses on the relation between Nef's defective activities in EC, in modulating the surface levels of CD4 and NKG2D ligands, and antibody-dependent cellular cytotoxicity (ADCC) response. We demonstrated that the inability of Nef isolated from EC to fully remove CD4 from the surface of infected cells leads to Env-CD4 interaction, which ultimately render these cells susceptible to ADCC. We also show that Nef's ability to reduce cell surface levels of NKG2D ligands also protects infected cells from ADCC. Cumulatively, our results suggest that in addition to the exposure of ADCC-mediating epitopes induced by the presence of CD4 at the cell surface, the accumulation of NKG2D activating ligands promotes NK cell cytotoxicity. Finally, during my PhD studies I also uncovered a new HIV-1 Env conformation (State 2A) that is vulnerable to antibody attack, rendering cells susceptible to ADCC. Importantly, this conformation is counteracted by Nef and might explain why its ability to downregulate CD4 from the cell surface is highly conserved and important for HIV-1 pathogenesis. Altogether, the findings presented in this thesis emphasize the potential impact of ADCC in the development of new antiviral approaches while providing a better understanding of HIV-1 mechanisms of immune evasion. Keywords: HIV-1,Env, Nef, CD4, NKG2D ligands, CD4i Abs, ADCCRÉSUMÉ Malgré les énormes efforts pour développer de nouvelles stratégies thérapeutiques contre l'infection par le virus d'immunodéficience humaine (VIH-1), il n'existe à l'heure actuelle aucun vaccin efficace contre ce virus. La prévention de l'infection au VIH-1 exige de comprendre les mécanismes de virulence de ce virus. Il a été montré que les anticorps contre le VIH-1 possèdent une capacité à induire une réponse effectrice dépendante de leur portion Fc. Cette réponse effectrice joue un rôle important dans la prévention de l'infection, et aussi dans la protection observée dans le RV144, le seul essaie vaccinal anti-VIH à avoir démontré un certain degré de protection. Cependant, les anticorps capables d'induire cette réponse contre le VIH-1 sont connus pour reconnaître les glycoprotéines de surface du virus (Env) dans une conformation dite « ouverte ». Cette conformation est adoptée lors de la liaison d'Env avec son récepteur cellulaire CD4 (épitopes CD4i).La protéine accessoire Nef joue un rôle important dans la modulation de l'exposition des épitopes CD4i en empêchant l'élimination des cellules infectées. De plus, Nef intervient dans la diminution de l'expression membranaire des molécules impliquées dans les activités clés de la défense immunitaire, comme CD4, CMH-I, CD28 et des ligands de NKG2D. Il a été décrit que chez des patients particuliers et assez rare dits patients contrôleur d'élite (EC) la fonction de Nef est altérée. Ceci conduit à la suppression de la réplication de VIH-1 chez les patients EC en absence de traitement antiviral.Les travaux de cette thèse se concentrent sur l'étude de la relation entre le rôle affecté de Nef chez les EC et la modulation de l'expression de CD4, les ligands de NKG2D ainsi que la réponse cytotoxique dépendante des anticorps (ADCC). Nous avons démontré que l'incapacité de Nef chez les EC à diminuer l'expression de CD4 sur les cellules infectées conduit à l'interaction de CD4 et Env, ce qui résulte dans l'exposition des épitopes CD4i et donc augmente la susceptibilité des cellules infectées à la réponse ADCC. Nous avons également montré que l'incapacité de Nef de réduire les niveaux des ligands de NKG2D permet l'activation des cellules NK et rende aussi les cellules infectées plus sensibles à l'ADCC. Finalement, mes études doctorales ont permis de découvrir et caractériser une nouvelle conformation d'Env qui est bien reconnue par les anticorps ciblant le domaine interne de la glycoprotéine gp120 et qui interviennent de manière efficace dans la réponse ADCC. La modulation de la réponse ADCC par Nef représente un axe important de la perspective des réponses effectrices dépendante de Fc. Les études présentées dans cette thèse soulignent l'impact potentiel de l'ADCC sur le développement de nouvelles approches thérapeutiques contre l'infection au VIH-1.Mots-clés : VIH-1, Nef, ADCC, CD4, NKG2D, Env, Fc, EC

Effects of the I559P gp41 Change on the Conformation and Function of the Human Immunodeficiency Virus (HIV-1) Membrane Envelope Glycoprotein Trimer

The mature human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer is produced by proteolytic cleavage of a precursor and consists of three gp120 exterior and three gp41 transmembrane subunits. The metastable Env complex is induced to undergo conformational changes required for virus entry by the binding of gp120 to the receptors, CD4 and CCR5/CXCR4. An isoleucine-to-proline change (I559P) in the gp41 ectodomain has been used to stabilize soluble forms of HIV-1 Env trimers for structural characterization and for use as immunogens. In the native membrane-anchored HIV-1BG505 Env, the I559P change modestly decreased proteolytic maturation, increased the non-covalent association of gp120 with the Env trimer, and resulted in an Env conformation distinctly different from that of the wild-type HIV-1BG505 Env. Compared with the wild-type Env, the I559P Env was recognized inefficiently by polyclonal sera from HIV-1-infected individuals, by several gp41-directed antibodies, by some antibodies against the CD4-binding site of gp120, and by antibodies that preferentially recognize the CD4-bound Env. Some of the gp120-associated antigenic differences between the wild-type HIV-1BG505 Env and the I559P mutant were compensated by the SOS disulfide bond between gp120 and gp41, which has been used to stabilize cleaved soluble Env trimers. Nonetheless, regardless of the presence of the SOS changes, Envs with proline 559 were recognized less efficiently than Envs with isoleucine 559 by the VRC01 neutralizing antibody, which binds the CD4-binding site of gp120, and the PGT151 neutralizing antibody, which binds a hybrid gp120-gp41 epitope. The I559P change completely eliminated the ability of the HIV-1BG505 Env to mediate cell-cell fusion and virus entry, and abolished the capacity of the SOS Env to support virus infection in the presence of a reducing agent. These results suggest that differences exist between the quaternary structures of functional Env spikes and I559P Envs

Characterization of ligand binding to selected HIV-1_BG505 variants by cell-based ELISA.^a

<p>Characterization of ligand binding to selected HIV-1_BG505 variants by cell-based ELISA.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122111#t002fn001" target="_blank">^a</a></p

Effect of a reducing agent on the ability of Env variants to mediate infection and cell-cell fusion.

<p>(<b>A</b>). Normalized amounts of recombinant luciferase-expressing HIV-1 pseudotyped with the wt and mutant HIV-1_BG505 ΔCT Envs were spin-inoculated onto Cf2Th-CD4/CCR5 cells before adding increasing concentrations of DTT (0–5 mM). (<b>B</b>). The ability of the indicated Env variants to mediate cell-cell fusion in the presence of increasing concentrations of DTT was evaluated. The data shown are from a single experiment and are representative of results from at least three independent experiments, performed in quadruplicate.</p

Insights into the I559P phenotypes from the structure of the soluble gp140 SOSIP.664 Env trimer.

<p>(<b>A</b>). The ribbon structure (PDB 4TVP) of one protomer of the HIV-1_BG505 soluble gp140 SOSIP.664 Env trimer [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122111#pone.0122111.ref038" target="_blank">38</a>] is shown, oriented with gp41 at the top of the figure. The gp41 subunit is colored green, the gp120 outer domain is colored blue, and the gp120 inner domain and N- and C-termini are colored orange. The approximate location of isoleucine 559 within the disordered gp41 segment (residues 548–568) (dotted line) is indicated by an asterisk. The gp41 α7 helix forms a coiled coil at the trimer axis. The SOS disulfide bond linking gp120 residue 501 and gp41 residue 605 is labeled. The F240 Cluster I gp41 epitope is highlighted in magenta, and is buried near the trimer axis in the soluble gp140 SOSIP.664 structure. The gp120 core is in the CD4-bound conformation, except for the V1/V2 stem, which is not shown. The β-sandwich of the gp120 inner domain, which is involved in the non-covalent association of gp120 and gp41 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122111#pone.0122111.ref090" target="_blank">90</a>], loosely interacts with the gp41 fusion peptide in this structure. (<b>B</b>). The soluble SOSIP.664 Env trimer structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122111#pone.0122111.ref038" target="_blank">38</a>] was fitted as a rigid body into the tomogram of the HIV-1 virion Env trimer (grey) (EMD 5019) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122111#pone.0122111.ref040" target="_blank">40</a>], using UCSF Chimera software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122111#pone.0122111.ref091" target="_blank">91</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122111#pone.0122111.ref093" target="_blank">93</a>]. The soluble gp140 SOSIP.664 ribbon structure is colored as in <b>A</b> and, in addition, the structure of the gp120 V1/V2 and V3 variable regions is shown in red. In the upper panel, the Env spike is oriented such that the viral membrane is at the top of the image. The gp41 disordered region (residues 548–568), including isoleucine 559, and the α7 helix of the soluble gp140 SOSIP.664 structure are out of the virion Env density, as is the α0 helix of the gp120 inner domain. The lower panel shows a view from the perspective of the target cell. Note the density in the gp120 arms of the virion Env tomograms that is not explained by the soluble gp140 SOSIP.664 structure. The approximate angle of approach used by the CD4-binding site (CD4BS) antibodies to engage the HIV-1 Env trimer is indicated by the red arrow. Because these antibodies must access their epitopes by bypassing the adjacent gp120 protomer, their binding is potentially sensitive to changes in the orientation of the gp120 subunits in the quaternary Env trimer structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122111#pone.0122111.ref042" target="_blank">42</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122111#pone.0122111.ref081" target="_blank">81</a>].</p

Binding of MAbs and sera from HIV-1-infected individuals to Env variants.

<p>The binding of VRC01 (<b>A</b>), b12 (<b>B</b>), CD4-Ig (<b>C</b>), 4e10 (<b>D</b>), F240 (<b>E</b>), 7b2 (<b>F</b>), 2.2B (<b>G</b>) or sera from HIV-1-infected individuals (patient sera, PS#1) (<b>H</b>) and PS#2 (<b>I</b>) to HIV-1_BG505 ΔCT Env variants expressed on the cell surface is shown. Means and SEM derived from at least five independent experiments performed in quadruplicate are shown. Statistical significance is indicated, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122111#pone.0122111.g001" target="_blank">Fig 1</a> legend.</p

Ligand binding to HIV-1_BG505 full-length wild-type and I559P Env variants.

<p>The binding of wild-type (wt) and I559P HIV-1_BG505 Env full-length (gp160) variants expressed on the cell surface was measured by cell-based ELISA, and is plotted relative to the binding of the 2G12 antibody. The ligands are arranged according to the relative binding to wt Env. The means and SEM derived from four independent experiments performed in quadruplicate are shown. Statistical significance was assessed using a paired t-test, and the degree of significance is indicated: ***—p < 0.001, **—p < 0.01, *—p < 0.05 and ns—not significant.</p

Phenotypes of HIV-1_BG505 ΔCT mutants.

<p>Phenotypes of HIV-1_BG505 ΔCT mutants.</p

MAb binding to HIV-1_BG505 Env variants on the cell surface.

<p>The binding of PGT121 (<b>A</b>) and PG9 (<b>B</b> and <b>C</b>) to HIV-1_BG505 Env ΔCT variants expressed on the cell surface was measured by cell-based ELISA. The data in (<b>C</b>) were generated in the presence of 80 nM sCD4. The means and SEM derived from at least three independent experiments performed in quadruplicate are shown. Statistical significance was assessed using a paired t-test, and is indicated as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122111#pone.0122111.g001" target="_blank">Fig 1</a> of the main text.</p