Pierre De Truchis, Laurence Morand-Joubert, Isabelle Pellegrin Primoinfection Pierre De Truchis, Laurence Morand-Joubert, Isabelle Pellegrin

Mme N, 28 ans Antécédents médicaux: Syndrome dépressif, suivie à Saint-Anne. AVC en décembre 2015, exploré à l'Hôpital Foch. Hémiplégie brachio-faciale gauche et aphasie avec récupération sans séquelle après thrombolyse. Bilan biologique négatif à part une antithrombine III légèrement diminuée. Antécédents chirurgicaux: Appendicectomie. Chirurgie esthétique du nez dans l'enfance. Gynécologiques : Frottis positifs en 2016. Cônisation prévue mais décalée en raison de la persistance de la fièvre. Dr L Morand-joubert

Mme N, 28 ans Mode de vie. Travaille comme hôtesse Vit en couple, pas d'enfant. Tabagisme occasionnel. Alcool occasionnel. Pas de consommation de drogue. Histoire de la maladie: Rapport sexuel non protégé il y a un mois. Voyage au Trinidad et Tobago, il y a un mois et demi. La patiente consulte à l’hôpital pour fièvre, céphalées, vomissements et altération de l'état général avec diarrhées. Pas d'anomalie à l'examen clinique et refus de la ponction lombaire. Que faites-vous? Dr L Morand-joubert

Mme N, 28 ans Retour à domicile avec une prescription de Paracétamol. Elle consulte deux jours plus tard aux urgences. A l’examen clinique, présence d’une pharyngite. Ponction lombaire normale. Prescription Amoxicilline/Acide clavulanique 1 g x 3/jour et retour à domicile. Au domicile, la patient n'arrive pas à prendre les antibiotiques en raison de vomissements importants. Elle consulte 2 jours plus tard dans une clinique où un scanner est réalisé et retrouve une collection rétropharyngée de 29 x 9 mm sans médiastinite. Reprise du traitement par Amoxicilline/Acide clavulanique en intra-veineux. Un bilan complémentaire est réalisé et retrouve une sérologie VIH ELISA positive avec une charge virale VIH à > 10.000.000 cp/ml Prise en charge à Saint-Antoine: Dépistage positif. Western blot VIH-1 négatif CV: 6 850 000 copies/ml CD4: 214/mm3 Que faites-vous? Dr L Morand-joubert

Mme N, 28 ans Le traitement est instauré rapidement par TDF/FTC/RAL en deux fois par jour Un génotypage de résistance est demandé.

Mme N, 28 ans Un Génotype de résistance est réalisé : Sous type CRF02_AG   M41 E44 A62 K65 D67 T69 69SS K70 L74 V75 F77 Y115 F116 Q151 M184 L210 T215 K219 Autres Codons mutés   V90 A98 L100 K101 K103 V106 E138 V179 Y181 Y188 G190 H221 P225 M230 Autres Codons mutés I   L10 V11 I13 I15 G16 K20 L24 D30 V32 L33 E35 M36 M46 I47 G48 I50 F53 I54 Q58 D60 I62 L63 H69 A71 G73 T74 L76 V77 V82 I84 I85 N88 L89 L90 Codons mutés V L E I K M   A49 T66 L74 E92 T97 G118 F121 E138 G140 Y143 P145 S147 Q148 V151 S153 N155 E157 S230 R263 Codons mutés I Q Absence de mutation conférant une résistance aux inhibiteurs nucléosidiques de la RT. Absence de résistance aux inhibiteurs non nucléosidiques de la RT. Pas de résistance aux IP (présence de plusieurs mutations mineures). (pour le TPV/r, données limitées concernant les sous-types non B) Résistance au RAL et EVG. Résistance possible au DTG 50 mg BID et 50 mg QD Dr L Morand-joubert

Que faites-vous devant le résultat de ce génotype ? Dr L Morand-joubert

  Mutations associated with resistance Mutations associated with « possible resistance » Mutations associated with resistance RAL T66K [10] E92Q [1, 2] G118R [10, 17] F121Y [10,17] G140A/S [7] Y143A/C/G/H/R/S [1, 3, 4, 5, 8, 14] Q148E/G/H/K/R [1, 2] V151L [9] N155H/S/T [1, 2, 9] E157Q [2] A49G + S230G/R + R263K [18] EVG T66I/A/K [6] E92Q [6] G118R [17] F121Y [9,17] E138K G140C/S Y143A/C/G/H/R/S [14] P145S [9] S147G Q148H/R/K [6] N155H/S/T [6,9] E157Q [11] R263K [18] T97A [21,22] S147G [21] DTG 50 mg BID G118R [12,13] F121Y [17] S153Y R263K [16] T66K + L74M E92Q + N155H Q148H/K/R + at least 2 mutations among: L74I or E138A/K/T or G140A/C/S [15] Q148R + N155H T66K [9] S153F E157Q [19, 20] Q148H/K/R + 1 mutation among: L74I or E138A/K/T or G140A/C/S [15]    E92Q + N155H [9, 23, 24] Q148R + N155H[9]  DTG 50 mg QD E138A/K/T G140A/C/S Q148H/K/R N155H www.hivfrenchresistance.org

Epidémiologie de la résistance transmise en France entre 2010-2012 (Cohorte PRIMO) Tous les patients (n = 799) Subtype B (n = 536) Subtype non B (n = 263) P value SEXE Hommes, n (%) Femmes, n (%) Données manquantes, n (%) 723 (90,5) 74 (9,3) 2 (0,3) 517 (96,5) 17 (3,2) 2 (0,4) 206 (78,3) 57 (21,7) 0 (0,0) < 0,001 Groupe de transmission Hommes homosexuels, n (%) Hommes ou femmes heterosexuels, n (%) Autre, inconnu, n (%) 605 (75,7) 149 (18,6) 45 (5,6) 460 (85,8) 46 (8,6) 30 (5,6) 145 (55,1) 103 (39,2) 15 (5,7) Patients de l’Afrique sub-Saharienne , n (%) 41 (5,1) 10 (1,9) 31 (11,8) CD4 médian (cells/mm3) [range] Mediane ARN VIH-1 (log copies/ml) [range] 490 [6-4960] 5,5 [2,1-8,1] 495 [6-4960] 5,4 [2,1-8,0] 480 [34-2872] 5,6 [2,1-8,1] 0,538 0,017 Prevalence résistance transmise Au moins une , % (95% CI) PI , % (95% CI) NRTI , % (95% CI) NNRTI , % (95% CI) RPV & ETV , % (95% CI) INT157Q, % (95% CI), n = 331 10,6 (8,6-13,0) 2,0 (1,1-3,2) 5,1 (3,7-6,9) 4,0 (2,8-5,6) 3,3 (2,1-4,7) 1,5 (0,5-3,5) 12,9 (10,2-16,0) 2,8 (1,6-4,6) 6,7 (4,7-9,2) 5,0 (3,3-7,2) 3,5 (2,1-5,5) 1,3 (0,3-3,9) 6,1 (3,5-9,7) 0,4 (0,0-2,1) 1,9 (0,6-4,4) 1,9 (0,6-4,4) 2,7 (3,5-9,7) 1,9 (0,2-6,5) 0,003 0,028 0,003 0,035 0,672 0,526  En France entre 2010-2012, la prévalence de la résistance transmise chez les patients en primoinfection est de 10,6% et reste stable depuis 1996 CROI 2014 – D'après Chaix ML et al., abstract 582 10

Contexte épidémiologique L’épidémie VIH reste active en France, avec: Une estimation de 6900 nouvelles infections (2012) qui ne diminue pas depuis 2007 Une insuffisance de diagnostics au moment de la primo-infection: 11% seulement des découvertes de séropositivité (N= 6584 nouveaux cas en 2014) Une insuffisance de diagnostics à un stade précoce (1) test d’infection récente (infection < 6 mois) ou (2) primo-infection ou > 500 CD4/mm3: 25% et 39% des nouveaux cas Des diagnostics plus précoces chez les HSH que dans les autres groupes de transmission Pour moi le mot Défaut évoque un défaut des techniques! Congrès de la SFLS - 7 octobre 2016 Primoinfection Cécile Goujard et groupe d’experts

Seulement 50% des primoinfections sont asymptomatiques (Robb et al Seulement 50% des primoinfections sont asymptomatiques (Robb et al. NEJM. 2016)

Histoire naturelle de l’infection, menée en Afrique de l’est et en Thaïlande Robb ML et al. NEJM. 2016

Histoire naturelle de l’infection, menée en Afrique de l’est et en Thaïlande Robb ML et al. NEJM. 2016

Diagnostic virologique Le diagnostic d’une primo-infection par le VIH est une urgence virologique, évoqué devant Test ELISA négatif avec une forte suspicion clinique ou d’exposition; Test ELISA avec < 5 bandes sur le Western blot; La charge virale ARN-VIH devient l’examen clef du diagnostic de primo-infection et les résultats doivent pouvoir être obtenus en moins de 48 heures La recherche de l’antigène p24 ne doit plus être prescrite pour la confirmation du diagnostic de primo-infection Les autotests et tests rapides (TROD) peuvent être pris en défaut pour le diagnostic de primo-infection: ils peuvent être négatifs en infection aiguë (Western blot négatif) et se positivent inconstamment en infection récente (Western blot indéterminé, < 5 bandes) Le dépistage d’une personne recevant une PrEP repose sur le test ELISA et la PCR VIH Attention à ne pas aller trop loin, les TROD rendent de grands services y compris pour le diagnostic de primo-infection. Au checkpoint, 25% des sujets qui étaient en primo-infection ont été dépistés par TROD, y compris chez des sujets dont le WB était blanc. Ce n’est pas parce qu’ils n’ont pas 100% de sensibilité qu’il ne faut pas les utiliser. La phrase était vraiment trop catégorique!!!! Congrès de la SFLS - 7 octobre 2016 Primoinfection Cécile Goujard et groupe d’experts

Indications et modalités du traitement antirétroviral Tout patient diagnostiqué en primo-infection VIH relève d’un traitement antirétroviral rapide (au mieux 24-48h) associant 2 INTI TDF/FTC 3ème agent IP/r (darunavir/ritonavir, 800/100 mg) INI (dolutégravir) Ce choix est fait en l’absence des résultats du typage HLA-B*5701 et du test génotypique de résistance aux ARV Le traitement sera adapté selon ces résultats Congrès de la SFLS - 7 octobre 2016 Primoinfection Cécile Goujard et groupe d’experts

Indications et modalités du traitement antirétroviral Situations particulières Grossesse : TDF/FTC associé à darunavir/ritonavir (800/100 mg) ou raltégravir (dont la tolérance pendant la grossesse est mieux connue qu’avec les autres INI); le traitement sera intensifié en cas de primo-infection pendant le 3ème trimestre de la grossesse (TDF/FTC + IP/r + raltégravir ou enfuvirtide) Primo-infection au cours d’une PrEP: TDF/FTC associé à IP/r ou INI en attendant le résultat du test génotypique de resistance Primo-infections VIH et VHC concomitantes: traiter le VIH en priorité, en tenant compte d’éventuelles anomalies hépatiques Congrès de la SFLS - 7 octobre 2016 Primoinfection Cécile Goujard et groupe d’experts

Cinétique des marqueurs virologiques sans et sous traitement Ananworanich J et al. EBioMedicine 2016

Cinétique des marqueurs virologiques sans et sous traitement Population totale Population Fiebig 1/2 Ananworanich J et al. EBioMedicine 2016

Bénéfice du traitement sur le réservoir lymphoïde digestif La charge virale plus élevée dans le sang périphérique et dans le LCR dans le groupe ARN+ par rapport au groupe ARN- (p<0,01). Après 24 semaines traitement, les patients du groupe ARN+ dans le colon ont montré une élévation persistante de l’ADN total du VIH dans les cellules mononucléées de la muqueuse colique et une tendance à un ADN total plus élevé dans les cellules mononuclées du sang périphérique (PBMC) Crowell TA et al. Journal of the International AIDS Society 2016, 19:21163

Bénéfice du traitement sur le réservoir lymphoïde digestif Niveau significativement plus élevé de cytokines, de marqueurs inflammatoires et d’activation des cellules CD8+ dans le sang et dans le colon dans le groupe ARN+ par rapport au groupe ARN-. A S24 de traitement, plus de différence dans l’activation immunitaire et l’inflammation entre les 2 groupes. Crowell TA et al. Journal of the International AIDS Society 2016, 19:21163

Rationnel immunologique pour un traitement précoce au cours de la PRIMOINFECTION Réservoir et timing du traitement Immunité muqueuse / CD4+ Th17 Aide à la réponse LB / CD4+ Tfh Epuisement lymphocytaire / Immune Check Point (ICP)

Définition des stades de Fiebig ARN Antigène P24 Specific EIA Présence possible de l’antigène P24 Present on Western Blot Y Stade 1 Stade 4 Plasma Viral RNA (copies per ml) This is another representation of the Fiebig stages. This time, we show the tests that are able to detect the infection. Stage 1: Only HIV RNA Stage 2: HIV RNA and p24 antigen Stage 3: HVI RNA, p24 antigen and specific enzyme immuno-assay (EIA) Stage 4: The HIV infection starts to appear on the Western Blot Y Y Limit of detection of assay for Plasma viral RNA Days following HIV-1 Transmission

ART 2 years (11 patients OPTIPRIM, Fiebig III/IV) HIV reservoirs are believed to be established at the early Fiebig stages I/II as early as 10 days after symptoms onset. ANRS 147 OPTIPRIM, ART (5-drugs vs 3-drugs) Treatment interruption M24 (VL<50, CD4>500) Treatment resomption (VL>5000, CD4<500) Reservoir substudy Effect of ART initiated at the time of the PHI (early ART) on the HIV reservoir [M0 and M24] in peripheral resting CD4+ T cells (naive (TN), central memory (TCM), transitional memory (TTM) and effector memory (TEM) cells) ART 2 years (11 patients OPTIPRIM, Fiebig III/IV) VISCONTI group 1: ART 6 years (10 patients) VISCONTI group 2: 11 post-treatment controllers (PTCs). Background: Therapeutic control of HIV replication reduces the size of the viral reservoir, particularly among central memory CD4+ T cells, and this effect might be accentuated by early treatment. Methods: We examined the effect of ART initiated at the time of the primary HIV infection (early ART), lasting 2 and 6 years in 11 and 10 patients, respectively, on the HIV reservoir in peripheral resting CD4+ T cells, sorted into naive (TN), central memory (TCM), transitional memory (TTM) and effector memory (TEM) cells, by comparison with 11 post-treatment controllers (PTCs). Results: Between baseline and 2 years, CD4+ T cell subset numbers increased markedly (P,0.004) and HIV DNA levels decreased in all subsets (P,0.009). TTM cells represented the majority of reservoir cells at both timepoints, T cell activation status normalized and viral diversity remained stable over time. The HIV reservoir was smaller after 6 years of early ART than after 2 years (P,0.019), and did not differ between PTCs and patients treated for 6 years. One patient, who had low reservoir levels in all T cell subsets after 2 years of treatment similar to the levels in PTCs, spontaneously controlled viral replication during 18 months off treatment. Conclusions: Early prolonged ART thus limits the size of the HIV reservoir, protects long-lived cells from persistent infection and may enhance post-treatment control. HIV-1-infected subjects with PHI were included in the randomized ANRS-147 OPTIPRIM trial conducted in 33 hospitals throughout France and involving adult participants presenting with PHI. Recruitment began in April 2010 for a planned period of 2 years and was completed in July 2011, with follow-up until December 2013 for the last patient. The primary endpoint was the difference between the two arms in the HIV DNA level per million PBMCs at month 24 (M24). Using data from the ANRS C06 PRIMO cohort of patients with PHI,27 we calculated that we would need to enrol 90 patients in order to achieve 80% power to detect a difference in HIV DNA levels of at least 0.42 log10 copies per 106 PBMCs between the two arms at M24.28 The first 12 randomized patients from the OPTIPRIM study who agreed to participate in this HIV reservoir substudy were enrolled immediately (2 year group), receiving either an intensified fivedrug regimen (tenofovir/emtricitabine, darunavir/ritonavir 800/100 added to raltegravir 400 mg twice daily and maraviroc 150 mg twice daily; arm 1) or a three-drug regimen (tenofovir/emtricitabine and darunavir/ ritonavir 800/100 once daily; arm 2). To be enrolled in the trial, patients had to present with biological criteria of acute infection and/or symptomatic PHI, as previously described.25 To determine whether post-treatment control could be observed within a randomized clinical trial, treatment interruption was scheduled at month 24, when patients had reached a viral load of ,50 copies/mL and .500 CD4+ T cells/mm3. Treatment resumption was recommended when the HIV RNA level rose above 50000 copies/mL or the CD4+ cell count fell below 500 cells/mm3 or 30%. Co-enrolment in the ANRS PRIMO cohort was proposed to all patients in order to organize post-trial follow-up. Blood samples, socio-demographic and clinical data were collected before and after 2 years of treatment, and during treatment interruption (month 24 to month 30). The objective of the substudy was to describe the reservoir at the time of the PHI and the impact of two different treatment arms on the CD4+ T cell subset reservoir. The second objective was to compare in a crosssectional study the HIV DNA levels measured after 2 years of treatment with the levels in the VISCONTI groups. The VISCONTI study included two patient groups, both of whom were treated within 10 weeks of infection with a classical three-drug regimen. The first group consisted of 10 subjects treated continuously for .6 years (6 year group) [median 79 months (IQR 38–142)]. The second group consisted of 11 PTCs who controlled the infection for a median of 101 months (IQR 82–107) after interrupting treatment maintained for a median of 31 months (IQR 17–56), as previously reported Initiation of suppressive ARTduring PHI (early ART) has numerous benefits and is now recommended in several guidelines worldwide. Early ART preserves the immune system both quantitatively and qualitatively, notably enhancing the recovery of CD4+ T cell numbers and functions. Moreover, early ART also prevents infection of resting CD4+ T cells and limits cell-associated HIV DNA levels, resulting in a more rapid and profound decline in the HIV reservoir size than when ART is started during the chronic phase of infection, the effectiveness of ART is therefore strongly dependent on the timing of its initiation. In addition, early ART impacts rebound of the HIV RNA level during treatment interruption, allowing greater time off therapy. Among long-term-treated patients, central memory CD4+ T (TCM) cells are the major cellular reservoirs, while in HIV controllers TCM cells are protected and contribute minimally to the reservoir, particularly among those carrying protective alleles such as HLA-B*27 and/or -B*57. Our group has previously shown that early therapy may be associated with sustained HIV control after treatment discontinuation in so-called post-treatment controllers (PTCs) from the VISCONTI cohort, who presented remission with an extremely low level of HIV reservoir, particularly in long-lived naive (TN) and TCM cell subpopulations. We also recently reported that, in acute infection, HIV is widely distributed among all CD4 T cell subsets, including TN and TCM cells. However, the precise impact of ART initiated at the time of the primary HIV infection (early ART) on HIV reservoir size and composition remains unclear. Therefore, we studied how 2 years of early initiated ART alters immunovirological outcomes and HIV reservoir characteristics compared with patients who received 6 years of ART and patients who were PTCs. Cheret, JAC 2015

Decrease in the HIV reservoir after 2 years of early ART 2 years of early ART normalized the activation status of all CD4+ subsets Decrease in the HIV reservoir after 2 years of early ART Decrease in the HIV reservoir after 2 years of early combined ART. The total HIV DNA content was evaluated in total CD4+ T cells (CD4 TLy), TN, TCM, TTM and TEM CD4 cell subsets. Measurements were made during the acute infection (D0, baseline, black symbols) and after 2 years of early combined ART (M24, early combined ART for 2 years, grey symbols). Only significant P values are shown. Results are expressed as log10 HIV DNA copies/million PBMCs and black lines represent the medians. Open symbols represent values below the detection limit. Cheret, JAC 2015

In contrast to 6 years of treatment, 2 years of early ART is not sufficient to reach the low HIV reservoir status of PTCs Two years of early combined ART is not sufficient to reach the low HIV reservoir status of PTCs. Reservoir size and compositionwere evaluated in TN, TCM, TTM and TEM CD4 cell subsets. Measurements were made in subjects who began treatment early for 2 years (grey circles) or 6 years (black circles), and in the unique control of the infection after interruption of an early treatment that represents the PTC group (black triangles). The grey star represents putative PTC Patient 4 and the grey square represents putative PTC Patient 8, both from the 2 year treatment group. (a) Results are expressed as log10 HIV DNA copies/mL of blood, and values below the detection limit were calculated for each assay according to available cell numbers. (b) The contribution of TN, TCM, TTM and TEM cells to the HIV reservoir was estimated by taking into account the frequency and infection level of each subset. Results are expressed as the percentage of the HIV blood cell reservoir in the different groups of subjects. Values under the detection limit are represented by open symbols. In contrast with 6 years, 2 years of early ART did not reduce the size of the HIV reservoir to the level observed in PTCs, particularly in the long-lived TCM subset.23 This may be due to the relatively long lifespan of the reservoir, and demonstrates that not only the timing but also the duration of ART is an important determinant of HIV reservoir decay. This challenges a study that showed that levels of cellular HIV DNA correlated with the timing of ART initiation but not with its duration,17 although this was based on a comparison of 55 patients treated during the chronic phase with only 6 patients treated during the acute phase of the infection. One limitation of our study is the cross-sectional nature of the comparison of the different patient groups. However, each group was highly homogeneous, all the patients started treatment during primary HIV infection, and all samples were tested with the same methodologies by the same operators in the same laboratories over a 2 year time frame. Another limitation is that only half the patients in the 2 year group received a standard three-drug ART comparable to that of the 6 year group, while the other half received intensified five-drug ART. However, we show that there was no difference between the two regimens in terms of reservoir size and distribution. Not only the timing but also the duration of ART is an important determinant of HIV reservoir decay. Cheret, JAC 2015

TH17 et Infection HIV Main Role in Adaptive Immunity Tfh Activate infected macrophages Provide help for B cell Ab production; switching to IgE Increase neutrophil response Suppressor T cells Long-term non-progressors display a greater number of Th17 cells than HIV-infected typical progressors, Salgado et al, Clin, Immunol 2011 Th17 cells, HIV and the gut mucosal barrier , S. Dandekar, M.D. George, A.J. Baumler Curr Opin HIV AIDS, 5 (2010) Altered balance between Th17 and Th1 cells at mucosal sites predicts AIDS progression in simian immunodeficiency virus-infected macaques. V. Cecchinato, C.J. Trindade, A. Laurence, J.M. Heraud, J.M. Brenchley, M.G. Ferrari, et al. Mucosal Immunol, 1 (2008) The Relative Balance between Th17 and Regulatory T cell subsets is Critical for Progression of HIV Infection. Maina EK, Bukusi EA, Martha S, Lartey M and Ampofo WK,JACR 2014

Infection précoce des TH17 (CXCR5+) dans la muqueuse Rôle +++ dans l’immunité muqueuse, Protection de la barrière intestinale Réponse immunitaire anti-bactérienne, anti-fongique

TH17 et Infection HIV

25 subjects Fiebig III/IV/V 42 subjects with AHI, staged according to Fiebig at time of HIV diagnosis (+ sigmoid biopsy + phlebotomy). 17 subjects Fiebig I/II 25 subjects Fiebig III/IV/V 5 treatment-naıve subjects with CHI 10 age-, gender- and risk groupmatched HIV-uninfected Median time since history of HIV exposure = 16 days Median CD4+T = 465 cells/mm3, Median plasma HIV RNA = 5.5 log10 copies/mL Median sigmoid colon HIV RNA = 2.6 log10 copies/mg tissue Kinetics of Th17 depletion, microbial translocation and subsequent immune activation in early acute HIV infection  Effect of early initiated ART on these events We evaluated the kinetics of Th17 depletion, microbial translocation and subsequent immune activation in early acute HIV infection and the effect of early initiated ART on these events. We discovered that a collapse of Th17 cell number and function, accompanied by local and systemic immune activation, occurs already during acute HIV infection. However, early initiation of ART preserved Th17 number and function and fully reversed any initial HIV-related immune activation. These findings argue for the importance of early events during HIV infection setting the stage for chronic immune activation and for early and aggressive treatment during acute HIV infection. Schuetz, PlosPath 2014

CD4+ staining (% Area lamina propria) decreases with progression of Fiebig stage in the sigmoid colon when compared to FI/II. CD4+ staining (% Area lamina propria) correlated inversely with the colonic (b) and plasma (c) HIV RNA in FI/II, FIII and FIV/V. Percentage area of lamina propria CD4+ staining (% Area LP) decreases with progression of Fiebig stage in the sigmoid colon when compared to FI/II. The percent area of the lamina propria that stained for CD4+T cells correlated inversely with the colonic (b) and plasma (c) HIV RNA in FI/II, FIII and FIV/V. Schuetz, PlosPath 2014

Th17 (function : 3CK= IFNg, IL2, IL22) Frequency and function of Th17 mucosal CD4+T cells decreases by progression of Fiebig stage. Th17 (%) Th17 (function : 3CK= IFNg, IL2, IL22) Frequency of IL-17 and/or IL-22 expressing mucosal CD4+T cells decreases by progression of Fiebig stage. A decrease in the frequency of IL-17 (e), IL-22 (f), IL-17/IL-22 (g)-producing cells was seen as well as in the subpopulation of triple-cytokine producing (IL-2, IL-22, IFNc) Th17 cells (h) from FI (black circle)/FII (red circle) to FIII and FIV (red square)/FV (black square). Frequency of single and double cytokine producing cells is calculated from percentage CD4+T cells, while triple-cytokine producing cells are calculated from percentage of CD4+IL17+T cells. All comparisons To quantify the expression of Il-17 and IL-22 in CD4+T cells, mucosal and peripheral mononuclear cells were stimulated for 5 hours with 40 ng/mL PMA and 1 mM Ionomycin. Gating strategy of sigmoid colon Th17 and Th22 CD4+T cells is shown for a FI subject (a) and representative flow cytometry plots including unstimulated controls (unstim) gated on CD4+T cells showing expression of IL-17 and/or IL-22 in FI (b), FIII (c) and FV (d) in the sigmoid colon. were made to FI/II: *p#0.05, **p#0.01 and ***p#0.001. Schuetz, PlosPath 2014

Impact of early ART initiation on CD4+Th17 cells. ART initiatiation during FI/FII vs FIII/IV for 6 months were able to maintain mucosal CD4+T cells, Th17 cells and Th17 triple cytokine-producing Th17 cells … with no differences when compared to HIV- CD4+ Impact of early ART initiation on CD4+ and CD8+T cells as well as Th17 cells. Subjects that initiated ART during FI (black circle)/FII (red circle) for 6 months were able to maintain mucosal CD4+T cells (a), CD4+CCR5+ (b), Th17 cells (c), triple cytokine-producing Th17 cells (d), Th22 cells (e), and IL-17 and/or IL-22 (f) producing CD4+T cells with no differences when compared to HIV-. In addition their CD8 activation in the mucosa (g) and periphery (h) normalized after 6 months of ART. blue dotted line: median of HIV- individuals; purple dotted line: median of CHI individuals. Schuetz, PlosPath 2014

Impact of early ART initiation on CD8+T. ART initiation during FI/FII vs FIII/IV for 6 months were able to maintain CD8 activation in the mucosa (g) and periphery (h) normalized after 6 months of ART. … with no differences when compared to HIV- CD4+ mucosa PB Impact of early ART initiation on CD4+ and CD8+T cells as well as Th17 cells. Subjects that initiated ART during FI (black circle)/FII (red circle) for 6 months were able to maintain mucosal CD4+T cells (a), CD4+CCR5+ (b), Th17 cells (c), triple cytokine-producing Th17 cells (d), Th22 cells (e), and IL-17 and/or IL-22 (f) producing CD4+T cells with no differences when compared to HIV-. In addition their CD8 activation in the mucosa (g) and periphery (h) normalized after 6 months of ART. blue dotted line: median of HIV- individuals; purple dotted line: median of CHI individuals. Early initiation of ART prevents loss of mucosal Th17 cells preserves their functional status and normalizes systemic and local immune activation  Very Early and agressive treatment during acute HIV infection Schuetz, PlosPath 2014

Tfh-LB TFH cells are professional B helper T cells = CD4+ cells that help to differenciation / maturation LB Interaction between Tfh and LB  efficient Antibody immune response Identification of immune parameters associated with T and B memory response preservation  control of viral rebound if treatment interruption Schematic view of dentritic cell (DC) participation in the naive CD4+ T cells differentiation into follicular helper T cells (Tfh) lineage by means of an increase in Bcl-6 expression and downregulation of its antagonist Blimp-1 under the influence of a combination of elements, including IL-21, inducible co-stimulator (ICOS), T cell receptor (TCR) and likely CD28 (A). Since T cells are primed during interaction with DC in the T cell zone and B cells reside in the B cell follicle, antigen-specific T cells and their cognate B cells must migrate towards a secondary lymphoid organ to meet each other and promote the generation of germinal centers by differentiation of primed B cells (B).  The HIV-specific T cell memory response diminishes rapidly even after the initiation of anti-retroviral treatment (ART), and there is no control of viral rebound if treatment is interrupted. Restoration or preservation of memory T cells or B cells with treatment, to allow for control of virus replication after ART is stopped, is therefore very important. CD4+ T cells, in particular T follicular helper (Tfh) cells, have a major role in mediating antiviral immunity by providing help to B cells, which is key to a strong and efficient anti-HIV antibody response. The progressive nature of immune dysfunction in HIV infected individuals has implied that early treatment could play a critical role in reducing immune defects and in preserving T and B cell memory responses against HIV infection [1–3]. Despite the fact that antiretroviral treatment (ART) has been pivotal in reducing the viral burden in persons infected with HIV, the concurrent decline in the HIV-specific T and B cell memory response poses a great risk, as treatment interruption can lead to a loss in the control of viremia [4–7]. The need to identify immune parameters that are associated with preservation of the memory response during HIV infection is therefore important to providing clues for therapies going forward. Even with ART, low level HIV replication in lymphoid tissues has been shown to maintain a state of chronic immune activation [8]. B cell hyperactivation, a hallmark feature of HIV infection, is characteristically evidenced by elevated serum immunoglobulin [9, 10], and also includes changes within the circulating B cell compartment, some of which cannot be reversed by ART as evident in chronic individuals [11]. These changes include an increase in their activation, proliferation, immature and terminal differentiation markers [12–16], as well as a reduction in CD27+ memory cells [17–19]. CD4 T follicular helper cells (Tfh) are specialized in providing help to B cells and support B cell maturation and differentiation to long-lived plasma cells in the germinal center (GC) [20]. It therefore follows that if an efficient HIV-specific B cell response is to be achieved, Tfh function must be preserved. With access to human lymphoid tissue limited, there has been an increased interest in the study of CD4+CXCR5+ T cells in the blood known as memory circulating Tfh (cTfh), that are also very efficient at inducing B cell differentiation and providing B cell help; and much progress has been made to characterize and understand their biology [6, 21–23]. We have previously shown that germinal center Tfh cells in HIV positive lymph nodes are dysregulated and that the B cell response is severely reduced compared to those from HIV negative lymph nodes [24]. We have also recently shown that HIV-associated microenvironment can affect the differentiation and phenotype of cTfh cells, and that these cells from chronic aviremic individuals treated in very late stages (3–6 months after transmission) are dysfunctional in providing B cell help compared to elite controllers or healthy individuals [6]. It is therefore plausible that individuals who undergo very early ART could preserve their CD4+ Tfh function, and that phenotypic perturbations of T and B cell populations that are characteristic of acute HIV infection are better resolved in very early treated individuals. As the HIVassociated microenvironment likely affects the Tfh program [6], it is also possible that in very early acute HIV-infected individuals, there is less immune activation and therefore fewer inflammatory signals, which would reduce the risk for adverse effects on Tfh phenotype and function. Peripheral T Follicular Helper Cells Are the Major HIV Reservoir Within Central Memory CD4 T Cells in Peripheral Blood from chronic HIV infected individuals on cART. Suresh Pallikkutha, et al, J Virol 2015 HIV and Tfh Cells: Circulating New Ideas to Identify and Protect. Ann Chahroudi, Guido Silvestri. Immunity 2016

function of circulating T follicular helper cells (cTfh) HIV Dysregulation Tfh in lymph node (HIV-associated micro-environnement) B cell response reduced cTfh cells obtained from HIV infected individuals who were treated late (months after transmission) displayed a Tfh1 inflammatory phenotype that is not capable of providing help to B cells. How early this impairment occurs? RV254 cohort (cohorte Thaï) of HIV-infected very early acute (stage 1/2) and late acute (stage 3) individuals Immune restauration Reservoir decrease RV254 was used to study T helper- B cell responses in acute HIV infection and the impact of early ART on T and B cell function. function of circulating T follicular helper cells (cTfh) Phenotype of cTfh and memory B cell populations The unique RV254 cohort provided the best setting to analyzeimmune responses during very early acute HIV, as the study was able to enroll individuals that were infected for less than 2 weeks and initiated treatment approximately 1–2 days after recruitment. We aimed to study the capacity of memory circulating Tfh (cTfh) cells to promote B cell help in acute HIV infection, and found the interaction to be dysfunctional in the later stage compared to the very early stages, accompanied by increased levels of proinflammatory cytokines and a reduction in regulatory cytokines. High levels of plasma viremia correlated with low cTfh-mediated B cell antibody production in later stage acute individuals; and memory B cells were significantly decreased but could be restored with ART, compared to chronically infected individuals, who could not normalize this compartment compared to healthy individuals. Overall, we show that the cTfh- B cell interaction and B cell memory compartment is altered in late stage acute infection, mainly attributed to an increase in inflammation and skewing of the response away from helper to proinflammatory. Identifying individuals for treatment in the earliest stages of acute infection, prior to immune damage, could preserve cTfh function and the anti-HIV B cell response, therefore reducing the chances of viral rebound upon the cessation of ART. The unique RV254/SEARCH010 Thai cohort is strategic in that HIV infected participants are recruited during the earliest stage of acute infection and receive ART almost immediately [25, 26]. This population of individuals offers the best setting to accurately analyze the benefits of early ART on immune preservation and function. Using cTfh cells as surrogate B-cell helper T cells, we examined T-helper mediated function in stage 1/2 (4thG stages 1 and 2 grouped) (early acute; median average 12 and 17 days respectively from history of exposure) and stage 3 (4thG stage 3) (late acute; median average 18 days from history of exposure) [27]; and investigated the phenotype and immune activation status of cTfh and memory B cells before and post ART. We hypothesized that cTfh-mediated B cell help in untreated late acute individuals will be reduced compared to very early acute individuals and lead to an increased proinflammatory environment. Now, we turn to pivotal studies involving early antiretroviral treatment. The RV254/SEARCH010 study in Thailand, implemented by the U.S. Military HIV Research Program (MHRP) showed that early ART limits the persistence of the HIV reservoir in long-lived CD4+ T cells subsets: Most participants were enrolled at Fiebig I and III. Very early ART protected all memory CD4+ T cell subjects from infection, including long-lived central memory T cells. ART in Fiebig I was associated with the preservation of poly-functional gut Th16 cells; however, elevated plasma biomarkers of gut repair and microbial translation persisted. The RV254 cohort of HIV-infected very early acute (4thG stage 1 and 2) (stage 1/2) and late acute (4thG stage 3) (stage 3) individuals was used to study T helper- B cell responses in acute HIV infection and the impact of early antiretroviral treatment (ART) on T and B cell function. To investigate this, the function of circulating T follicular helper cells (cTfh) from this cohort was examined, and cTfh and memory B cell populations were phenotyped. Impaired cTfh cell function was observed in individuals treated in stage 3 when compared to stage 1/2. The cTfh/B cell cocultures showed lower B cell survival and IgG secretion at stage 3 compared to stage 1/2. This coincided with lower IL-10 and increased RANTES and TNF-α suggesting a role for inflammation in altering cTfh and B cell responses. Elevated plasma viral load in stage 3 was found to correlate with decreased cTfh-mediated B cell IgG production indicating a role for increased viremia in cTfh impairment and dysfunctional humoral response. Phenotypic perturbations were also evident in the mature B cell compartment, most notably a decrease in resting memory B cells in stage 3 compared to stage 1/2, coinciding with higher viremia. Our coculture assay also suggested that intrinsic memory B cell defects could contribute to the impaired response despite at a lower level. Overall, cTfh-mediated B cell responses are significantly altered in stage 3 compared to stage 1/2, coinciding with increased inflammation and a reduction in memory B cells. These data suggest that early ART for acutely HIV infected individuals could prevent immune dysregulation while preserving cTfh function and B cell memory. Muir et al. PLOSPath 2016

cTfh-mediated memory B cell help is impaired in late stage acute HIV-infected individuals Reduced Tfh help to LB in stage 3 versus stage 1/2 Alterations in Tfh program toward a Th1 profil cTfh-mediated memory B cell help is impaired in late stage acute HIV-infected individuals. PBMCs from W0 stage 1/2 (n = 9) and stage 3 (n = 4–7) individuals were sorted. cTfh (CD3+CD4+CD45RA-CXCR5+CXCR3-) or the less efficient T-helper cells (CD3+CD4+CD45RA-CXCR5+CXCR3+) denoted CXCR5+CXCR3+ were placed in culture with autologous resting memory B cells (CD19+CD10- CD21+CD27+) in the presence of SEB. Quantification of cTfh-mediated B cell help was carried out by measuring (A) total and (B) HIV-specific IgG ELISA in 7 day culture supernatant. Absolute numbers of live (C) B cells were quantified. (E) Correlation analysis was carried out between the absolute number of B cells and HIV-specific IgG. (F) HIV-specific IgG ELISA in 7 day culture supernatant from cultures of CXCR5+CXCR3+ T cells and memory B cells was also quantified. For (A)-(D) and (F) bars represent mean ±SD. Symbols on the graphs represent stage 1/2 individuals (black circles) and stage 3 individuals (black squares). IL-10 and TNF-α levels in cocultures of cTfh and B cells from late stage acute individuals influence HIV-specific IgG production. Cocultures of cTfh and autologous resting memory B cells fromweek 0 stage 1/2 (n = 9) and stage 3 (n = 7) individuals were analyzed for the presence of cytokines including RANTES, (B) TNF-α and (C) IL-10. Correlation analysis was carried out between (D) TNF-α; (E) IL-10 and HIV-specific IgG. Symbols on the graphs represent stage ½ individuals (black circles) and Stage 3 individuals (black squares). Specifically, we showed cTfh cells obtained from HIV infected individuals who were treated relatively late (months after transmission) displayed a Tfh1 inflammatory phenotype that is not capable of providing help to B cells. This is corroborated by findings from our own studies showing that cTfh from chronic individuals can be polarized to a more Th1 profile through the production of IL-2, TNF-α and IFN-γ [6]. cTfh cells can be further phenotyped into Tfh1, Tfh2 and Tfh17 subsets that provide help to B cells at varying levels and are characterized depending on their expression of CXCR3 and CCR6 [21, 36–38]. The impact that proinflammatory cytokines have on cTfh polarization in this setting would be of great interest. The regulatory cytokine IL-10 has been shown to be important in B cell proliferation and maintenance [30–32, 39–41], as well as promoting Tfh-dependent responses in HIV and in other disease models [23, 24, 42]. In fact, lack of IL-10 production by CD4+ T helper cells in Xlinked lymphoproliferative disease (XLP) patients impaired the development of B cell responses [43]. The increase in IL-10 and decreased proinflammatory cytokines, as well as the positive correlation between IL-10 and coculture HIV-specific IgG we observed in stage ½ individuals compared to stage 3, suggests that IL-10 could not only be important in regulating the early acute cytokine environment during HIV infection, but also in promoting cTfh-dependent B cell responses. Muir et al. PLOSPath 2016

Impact of early ART in PHI on blood memory B cell subsets Administration of ART at stage 1/2 and at stage 3 preserved the frequency of resting memory B cells (similar frequencies to HIV negative subjects). Initiating ART at the earliest stages of HIV infection is key to preserving immune memory, and could prove most effective in controlling viral replication after treatment interruption It is worth noting that the frequency of resting memory B cells at week 0 of stage 1/2 is similar to that of healthy individuals (P = 0.406); however the frequencies at week 0 of stage 3 are significantly reduced compared to healthy subjects (P = 0.0004) and is similar to that from chronic (P>0.999). In addition, we did not see significant differences between the stages, prior to ART, in the frequency of total CD19+ B cells, IgD+ resting naïve B cells (CD21+-CD27-), activated memory B cells (CD21-CD27+) or exhausted tissue-like B cells (CD21-CD27-) between stage 1/2 and stage 3 (Fig 5D–5G). In this study, we found that the cTfh-B cell interaction is significantly altered in later stage acute HIV infection. Supernatants from these cocultures showed that total and HIV-specific IgG from stage 3 individuals not yet on ART are significantly reduced compared to stage ½ individuals. We further found that the reduced levels of HIV-specific antibodies coincided with reduced B cell numbers in the coculture supernatants. Our previously published reports have demonstrated that the HIV microenvironment can negatively impact Tfh mediated B cell help [6, 24]. We therefore sought to investigate how early this impairment occurs and examine the underlying mechanisms using the unique RV254 cohort of acute HIV-infected subjects. This work has showed that cTfh cells are impaired in their ability to provide B cell help as early as stage 3 (~18 days) of acute HIV infection. The reduction in absolute numbers of B cells after coculture of cells from stage 3 individuals correlated with increased HV-specific IgG secretion, which provides further evidence that the B cell response, as evidenced by antibody production, is directly linked to cTfh-mediated help. These results indicate that the cTfh-mediated B cells response is better preserved at the earliest stage of HIV infection and that identifying individuals very early on during HIV infection has the potential to preserve cTfh-dependent B cell responses. To further characterize the dysregulation in cTfh-B cell interaction, we performed a coculture assay where we replaced cTfh cells from stage 3 (defective) with cTfh from healthy individuals (effective) in coculture with memory B cells (obtained from stage 3). We found that replacing cTfh showed signs of increased total and HIV-specific antibody levels (S5A and S5B Fig), as well as increased IL-10 and reduced RANTES levels (S5C and S5D Fig). This does suggest that reducing inflammation and hence reversing impairment of the cTfh-mediated B cell response can potentially be achieved by normalization of cTfh function. In order to further discern the contribution of memory B cells and determine the extent of B cell impairment prior to coculture with cTfh, we measured memory B cell proliferation and antibody production following polyclonal stimulation with CpG oligonucleotide. Our results indicated that similar proliferation profiles between the two stages and apparent non-significant increase in total IgG production in stage 1/2 when compared to stage 3 (S6A and S6B Fig). Overall, this suggested some level of intrinsic memory B cell defect that could contribute to impaired antibody response in stage 3. Of note, we observed similar expression of the survival molecule Bcl-2 on memory B cells in both stages (S6C Fig). Overall our results suggest that the impairment of the cTfh-B cell interaction is primarily due to impaired interaction of cTfh and memory B cells, however we cannot overlook the contribution of intrinsic memory B cell defect to this phenomenon. Additionally, we found that cocultures of cTfh cells obtained from patients at stage 3 are polarized towards a proinflammatory phenotype. Our results imply that this skewing of cTfh cells towards a non-cTfh program is associated with elevated levels of viral load observed at stage 3 when compared to stage 1/2. This has been confirmed by the significant correlation between viral load at week 0 and the impaired cTfh function as measured by the production of total and HIV-specific antibodies in culture supernatants. It is not clear from our study, however, whether this impairment is a direct effect of the virus or indirectly through viral-induced microenvironmental changes. Previously published data has suggested elevated proinflammatory cytokine levels including RANTES and TNF-α during chronic HIV infection [28, 29]. Rapid initiation of ART for maintaining low viremia and preserving immunity is a key objective of the RV254 study. We determined that stage 1/2 individuals had significantly lower viral load than stage 3 and consequently less plasma HIV-specific IgG and terminally differentiated B cells compared to stage 3 individuals. We know one of the characteristic effects of increased viremia on the B cell response is hypergammaglobulinemia [9, 10]. In addition, it has not been fully established whether cTfh interactions with memory B cells represent exactly what happens in the follicular microenvironment. However, based on our results, it could be that the hypergammaglobulinemia we observe in the plasma in stage 3 is partly due to antigen overload and hyper activation of the B cells which could occur in the extrafollicular areas of lymphoid tissues as damage to the lymph node has been shown to occur very early in acute infection [33]. This may play a greater role in driving B cell hyperactivation in stage 3. Overall, these results confirm a role for increased viremia in not only increasing plasma antibody responses but also mediating inflammation and skewing immune memory in acute HIV infection, resulting in impaired T cell- B cell interactions. Resting memory B cells play a critical role in eliciting strong humoral immunity and were significantly lower in stage 3 than in stage 1/2. Interestingly, the frequencies of these resting memory B cells in both stage 1/2 and stage 3, but not chronic individuals were restored to levels similar to what is observed in healthy subjects. These results strongly suggest that intervention at the earliest stages of infection can maintain the integrity and survival of resting memory B cells, which are significantly capable of strengthening humoral defenses and very important in preventing HIV disease progression. This is similar to other studies comparing the effects of early ART versus initiation at the chronic phase, where the resting memory B cell subset was better restored with early treatment [44, 45]. As we investigate what occurs in the periphery, we are cognizant that this environment may not necessarily represent what occurs in GCs in secondary lymphoid tissue. Using cTfh and memory B cells as surrogates allows us to interpret what may be going on generally and these results will help is to refine our future investigations. Muir et al. PLOSPath 2016

PD-1 / PD1-L : contrôler la fin de l’activation Immune check points : PD1 PD-1 / PD1-L : contrôler la fin de l’activation Phase effectrice PD-1 = marqueur surtout lié à l’activation et la différenciation en effecteurs Les cellules PD-1+ ne sont pas nécessairement sénescentes/dysfonctionnelles (Utzchneider et al. 2013, Legat et al. 2013, Hong et al. 2013) Permet retour au repos après une activation Down-régulation rapide après exposition aigue à l’antigène (épigénétique) (Sharpe 2007) The rate at which HIV-1 infected individuals progress to AIDS is highly variable and impacted by T cell immunity. CD8 T cell inhibitory molecules are up-regulated in HIV-1 infection and associate with immune dysfunction. When a CD8 T cell encounters its cognate antigen, the up-regulation of T cell inhibitory molecules tightly controls the subsequent T cell activation [6–8], and inhibits autoimmunity [9–11]. However, the persistence of antigen can overcome homeostatic controls and lead to permanent CD8 T cell dysfunction or exhaustion [12–16]. In HIV-1 infection T cell exhaustion is associated with the up-regulation of surface molecules called immune checkpoint receptors (ICRs) such as PD-1, Tim-3 and Lag-3 [12,17–20], which have also been associated with the size of the HIV reservoir and time to viral rebound after therapy cessation[21,22]. Signal inhibiteur Signal inhibiteur Induction de gènes inhibiteurs de la réponse retour au repos après une activation Down-régulation rapide après exposition aigue à l’antigène CD8 inhibitory molecules up-regulated in HIV and associated with immune dysfunction Bardoll, Nat rev cancerol 2012

Substudy Immune Check Receptors (ICR), n=122 SPARTAC Randomised controlled trial of therapy in early HIV infection : impact of short course ART in PHI on clinical outcomes: SOC, ART12, ART48 Trial endpoint : time to CD4 count <350 cells/μl or initiation of antiretroviral therapy Substudy Immune Check Receptors (ICR), n=122 SOC: no immediate ART, n=41 ART12: 12 weeks ART, n=44 ART48: 48 weeks ART, n=37 Expression of 3 exhaustion markers—PD-1, Tim-3, Lag-3 – on CD8 T cells from the closest pre-therapy time-point to seroconversion was correlated with: surrogate markers of HIV-1 disease (pVL and CD4 T) the trial endpoint We evaluated participants (n = 122) recruited to the SPARTAC randomised clinical trial to determine whether CD8 T cell exhaustion markers PD-1, Lag-3 and Tim-3 were associated with immune activation and disease progression. Expression of PD-1, Tim-3, Lag-3 and CD38 on CD8 T cells from the closest pre-therapy time-point to seroconversion was measured by FC, and correlated with surrogate markers of HIV-1 disease pVL and CD4 T cell and the trial endpoint: time to CD4 count <350 cells/μl or initiation of antiretroviral therapy. To explore the functional significance of these markers, co-expression of Eomes, T-bet and CD39 was assessed. Hoffmann et al. PLOSPath 2016

Of the 122, 41 were randomised to receive no immediate ART (SOC), 44 12 weeks ART (ART12) and 37 48 weeks ART (ART48). The median (IQR) time since the estimated date of seroconversion at randomisation was 77 (54, 98) days. The median (IQR) times for participants in ‘early’ (12 weeks after seroconversion) and ‘late’ (>12 weeks after seroconversion) were 60 (42, 72) and 104 (93, 122) days, respectively. MSM were significantly over-represented in the early PHI group. The median (IQR) baseline CD4 T cell count and HIV-1 pVL were 550 (435, 675) cells/μl and 4.70 (4.0, 5.3) log10 RNA copies/ml, respectively, without significant differences in participants enrolled or > 12 weeks into PHI. Hoffmann et al. PLOSPath 2016

Up- régulation des ICR (PD-1 and Lag-3) en PHI significantly higher expression in TEM compared with all other T cell subsets patients sampled earlier (<12 weeks vs >12 W) had significantly higher % of expression of ICR on CD8 CD38 T cells Association between expression of PD-1 on CD8CD38 T with surrogate markers of progression: pVL et CD4+T Similar to previous reports in PHI we found that naïve, central memory (TCM), effector memory (TEM) and TEMRA constituted 14.1, 4.4, 49.6 and 25.9% (median values) of the CD8 T cell population, respectively (Fig 4a). PD-1 and Lag-3 had similar distributions, with significantly higher expression in TEM compared with all other T cell subsets. Although Tim-3 expression was highest amongst TEM, there was relatively less expression on TCM compared with PD-1 or Lag-3. Expression of all three markers on naïve cells was very low (especially for PD-1 and Tim-3). Hoffmann et al. PLOSPath 2016

Association between exhaustion marker (PD-1) and clinical outcome after PHI Trial endpoint= time to CD4 count <350 cells/μl or initiation of antiretroviral therapy PD-1 quartiles: « dose-effet » Association of exhaustion markers with clinical progression Next we evaluated PD-1, Lag-3 and Tim-3 expression and progression to the SPARTAC trial Primary endpoint (either CD4 T cell count <350 cells/μl or (re)initiation of ART). PD-1 expression at baseline on CD8 and CD38 CD8 T cells predicted clinical progression (log rank test, p = 0.049 and p = 0.014, respectively) (Fig 2a and 2d). This effect was most evident in patients recruited within 12 weeks of infection, when compared with those recruited after 12 weeks Hoffman, PlosPath 2016

CONCLUSION Exhausted HIV-specific T cell immunity / Expression of ICR (PD-1, Tim-3, Lag-3) increased in PHI compared with HIV-uninfected controls predominantly found on the CD8 activated TEM subset association between ICR expression and pVL, CD4 count and disease progression Interest for interventions aimed to: reverse T cell exhaustion and restoring T cell functionality : most successful if applied shortly after HIV-1 acquisition, before an exhausted T cell phenotype is established. reactivate the latent HIV reservoir to facilitate immune-directed killing in novel curative HIV strategies. Therapies directed against these markers could impact disease progression or vaccine efficacy. This study measures three exhaustion markers—PD-1, Tim-3, Lag-3 –in individuals with HIV recruited to a randomised controlled trial of therapy in early HIV infection called SPARTAC. We find a complex picture in which these markers alone, together and in combination with other markers that reflect T cell activation (CD38) help predict the speed of clinical progression and immune decline, with differing effects dependent on the duration of infection. We propose that therapies directed against these markers could impact disease progression, vaccine efficacy or even newer curative strategies. Expression of PD-1 on CD8 and CD38 CD8 T cells correlated with pVL and CD4 count at baseline, and predicted time to the trial endpoint. Lag-3 expression was associated with pVL but not CD4 count. For all exhaustion markers, expression of CD38 on CD8 T cells increased the strength of associations. In Cox models, progression to the trial endpoint was most marked for PD-1/CD38 co-expressing cells, with evidence for a stronger effect within 12 weeks from confirmed diagnosis of PHI. The effect of PD-1 and Lag-3 expression on CD8 T cells retained statistical significance in Cox proportional hazards models including antiretroviral therapy and CD4 count, but not pVL as co-variants. Expression of ‘exhaustion’ or ‘immune checkpoint’ markers in early HIV-1 infection is associated with clinical progression and is impacted by immune activation and the duration of infection. New markers to identify exhausted T cells and novel interventions to reverse exhaustion may inform the development of novel immunotherapeutic approaches. Hoffman, PlosPath 2016

Primo-infection VIH Laurence Morand-Joubert, St Antoine, Paris VIROTEAM 2016 Primo-infection VIH Laurence Morand-Joubert, St Antoine, Paris Isabelle Pellegrin, Bordeaux Pierre de Truchis, Garches

Mr JY, 42 ans 1 Né 1969, célibataire Antécédents: HDM: Ulcère gastrique 1985 Traumatisme cervical avec hernie discale et radiculalgie C6 2005 Lombosciatique discale + canal lombaire étroit opérée 2010 intervention sur kyste pilonidal HDM: Séjour Dakar Aout 2011, prophylaxie mefloquine° 30/08: gingivite aigue, traitement amoxicilline 5 jours 19/09: fièvre 40° , frissons, douleurs dorsolombaires 20/09: douleurs abdominales, diarrhée hydrique, vomissements Consultation Urgences: Fièvre 38°5, diarrhée NFS: Hb: 10,5 g/dl; VGM 95; GB 13850, PNN 85%, lymphocytes 8%, monocytes 5%; plaquettes: 162000/mm3 CRP: 270 mg/l PdT

Mr JY, 42 ans 2 Examen clinique: Examens complémentaires: Fièvre persistante >38° Diarrhée hydriques, douleurs abdominales, épreintes, faux besoins « Ralentissement » idéo-moteur Examens complémentaires: Hyperleucocytose PN, lymphopénie, anémie Créatininémie 124μM/l, K+: 3,1 mM/l Hémocultures nég ECBU stérile Ex. des selles, coproculture: nég PdT

Mr JY, 42 ans (A) Quels autres examens complémentaires demandez vous ? Répéter hémocultures et coprocultures Frottis sanguin – GE Sérologie leptospirose Sérologie CMV Sérodiagnostic de Widal Sérologie HIV Antigénémie p24 PCR CMV et EBV PCR VIH PdT

Mr JY, 42 ans 3 Résultats bilan initial: Traitement initial: Hyperleucocytose, anémie inflammatoire Insuffisance rénale fonctionnelle Albuminémie 27 g/l Bactériologie nég Rectite aigue (rectosigmoidoscopie) PCR gonocoque nég, PCR chlamydiae (2re) nég TPHA-VDRL nég Sérologie VIH+ Elisa Traitement initial: ceftriaxone+ metronidazole + doxycycline PdT

Signes cliniques de la PI (B. Hoen, 2010) symptomes fréquence fièvre 90% Céphalées myalgies 40% Signes digestifs nausées, vomts Dysphagie diarrhée candidose 25% 50% 33% 20% Éruption cutanée Ulcérations muqueuses Signes neurologiques méningite lymph. Méningo-encéphalite PRN Neuropathie périph. 3% 5% 10% Hématologie Thrombopénie Leucopénie Lymphopénie Sd mononucléosique 80% Cytolyse hépatique

« Neurologic signs and symptoms frequently manifest in acute HIV infection »  139 patients évalués en primo-infection, 19 jours (3-56) après infection estimée Signes neurologiques dans les 12 semaines: =73 patients (53%): Guillain Barré: 1 Neuropathie périphérique sensitive: 31% Troubles moteurs: 34% Troubles cognitifs: 33% Patients avec signes neurologiques ont une CV initiale plus haute: 5.9 log vs 5.4 log (p=0,006) J. Hellmuth et al., Neurology, 2016, 87: 149

Mr JY, 42 ans (B) Comment confirmer l’hypothèse de la Primo-infection VIH 2è sérologie VIH (Elisa) Antigénémie p24 Western Blot VIH PCR ADN-VIH qualitative Test sérologique d’avidité Ac Charge virale VIH (ARN) PL avec CV dans le LCR PdT

Mr JY, 42 ans 4 Résultats virologiques Western-Blot VIH: 4 bandes + Sérologie CMV: IgG+6,3Ut/ml; IgM+ 83 UA/ml PCR CMV nég Sérologie EBV: IgG VCA+ 210 UA/ml, IgM VCA+ 44 UA/ml, IgG EBNA+ 76UA/ml PCR EBV + 255 copies/ml PdT

Représentation schématique des marqueurs virologiques au cours de la primo-infection par le VIH en l'absence de traitement Taux des marqueurs anti-gp 160 anti-gp 120 anti-gp 41 anti-p24 ARN-VIH Seuil de détection des marqueurs Ag p24 Contage J 0 11-12 14-15 20-21 28-29 Temps (jours) Fenêtre virologique ARN VIH plasmatique Antigénémie P24 ADN proviral Anticorps anti-VIH positifs par ELISA Fenêtre sérologique Ac anti-VIH positifs Western - Blot

Mr JY, 42 ans (C) Quels examens complémentaires demandez vous dans le cadre du bilan initial de la PI ? Contrôle du Western Blot sur un 2è prélèvement Charge virale VIH (PCR-ARN) Prélèvement HLA B57-01 Quantiféron-TB PCR CMV Typage lymphocytaire CD4/CD8 Mesure des l’activation immunitaire cellulaire (CD38+, DR+) Mesure des paramètres d’activation inflammatoire (CRPus, IL6, D-dimères, IP-1, CD14s,..) Génotypage de résistance sur la RT et la Protéase Génotypage de résistance sur l’Intégrase Génotropisme boucle V3 PCR ADN sur les PBMC circulants PdT

Mr JY, 42 ans (D) Quelle conduite thérapeutique préconisez vous? Démarrage immédiat des ARV le jour de la réception du test sérologique VIH Traitement ARV différé de 3 jours pour permettre annonce et ETP Traitement ARV reporté pour attendre la guérison des troubles digestifs et du syndrome infectieux Traitement ARV reporté jusqu’à confirmation du niveau d’immunodépression (CD4<500) en dehors de l’épisode infectieux aigu PdT

Cohorte PRIMO, patients recevant des ARV CD4<350 CD4 350-500 CD4>500 E Krastinova, PLoS One 2013; C. Goujard, 2015

Traitement précoce PI: SPARTAC 366 patients traités en PHI (<6 mois) Randomisation 3 bras: Bras contrôle (abstention thérapeutique) Traitement 12 semaines Traitement 48 semaines

SPARTAC

Mr JY, 42 ans 5 Données biologiques initiales CV= 149000 c/ml CD4: 322/mm3 (35%), CD8: 285/mm3, T4/T8= 1,13 Contrôle J7: CD4: 662/mm3 (32%), CD8:852/mm3 Génotypage: en attente PdT

Mr JY, 42 ans (E) Quelle schéma thérapeutique ARV choisissez vous en première intention ? Tenofovir +FTC+Rilpivirine Tenofovir+FTC+Raltégravir Tenofovir +FTC+cobicistat/elvitegravir Tenofovir +FTC+ritonavir/Atazanavir300 Tenofovir+FTC+ritonavir/Darunavir600x2/j Tenofovir+FTC+ritonavir/Darunavir800x1/j Tenofovir+FTC+dolutégravir50 Abacavir+3TC+dolutégravir PdT

Mr JY, 42 ans 5b Données biologiques initiales CV= 149000 c/ml CD4: 322/mm3 (35%), CD8: 285/mm3, T4/T8= 1,13 Contrôle J7: CD4: 662/mm3 (32%), CD852/mm3 Génotypage: sous-type B RT: sauvage PRO: 36I 62V 63P 71T 77I Int: 157Q V3: FPR: 6,2% PCR-ADN: 3,8 log PdT

(log10 copies/million PBMC 17 Essai OPTIPRIM-ANRS147 : penta- vs tri- thérapie dans la primo-infection à VIH (3) Diminution de l’ADN proviral A M24 pas de différence entre les 2 bras (≈ -1,4 log c/106 PBMC) 1 3 6 12 18 24 (log10 copies/million PBMC ADN proviral 2 4 5 Bras 2 Bras 1 Dans cet essai, un traitement antirétroviral renforcé (5 médicaments) n'a pas montré de bénéfice supplémentaire par rapport à une trithérapie conventionnelle en termes de réduction de l'ADN proviral au terme de 24 mois de traitement. Bras Penta (n = 45) Bras Tri (n = 45) Médiane ADN-VIH log10 c/106 PBMC (IQR) 2,35 (2,05 ; 2,50) 2,25 (1,71 ; 2,55) Médiane Δ ADN-VIH log10 c/106 PBMC (IQR) -1,41 (-1,61 ; -1,22) -1,44 (-1,75 ; -1,10) Médiane ADN-VIH log10 c/106 CD4 (IQR) 2,82 (2,56 ; 3,06) 2,77 (2,27 ; 3,00) Médiane Δ ADN-VIH log10 c/106 CD4 (IQR) -1,71 (-1,92 ; -1,46) -1,66 (-1,97 ; -1,28) Cheret A, CROI 2014, Abs. 549LB

Indications et modalités du traitement antirétroviral Tout patient diagnostiqué en primo-infection VIH relève d’un traitement antirétroviral rapide (au mieux 24-48h) associant 2 INTI TDF/FTC 3ème agent IP/r (darunavir/ritonavir, 800/100 mg) INI (dolutégravir) Ce choix est fait en l’absence des résultats du typage HLA-B*5701 et du test génotypique de résistance aux ARV Le traitement sera adapté selon ces résultats Congrès de la SFLS - 7 octobre 2016 Primoinfection Cécile Goujard et groupe d’experts

Résistance aux ARV du VIH au moment de la primo-infection La fréquence de mutations de résistance aux ARV observée sur les virus des patients en primo-infection est stable ces dernières années, avec en 2014, une fréquence de virus ayant au moins une mutation de résistance de 9,3%, avec : Résistance INTI: 4,3% Résistance INNTI: 8,4% (dont 6% à rilpivirine ou étravirine) Résistance IP: 2,4% Résistance INI: 2,7% (mutations E157Q et R263K) Congrès de la SFLS - 7 octobre 2016 Primoinfection Cécile Goujard et groupe d’experts

Mr JY, 42 ans: Evolution 6 Combinaison ARV choisie: Traitement ARV démarré 6 jours après l’admission Combinaison ARV choisie: TenofovirDF+FTC+RTV100/Darunavir 800 x1/j dates ARV CV CD4 CD8 22/09 - 149000 322 (35%) 285 28/09 TDF+FTC+rDRV 662 (32%) 852 15/10 :* 4610 554 (42%) 448 4/11 338 549 (41%) 509 13/01 <20 550 (42%) 484 28/02 *(Syphilis 2re) 528 (29%) 1035 23/06 798 (38%) 809 PdT

Mr EM, 29 ans 1 Antécédents: HDM: Né 1987, hétérosexuel, origine mahgrebine; en concubinage, pas de rapports avec sa partenaire actuelle ? séjour de 15 mois en prison Antécédents: Ulcère gastrique 2009 Lésions congénitale genou (genu valgum), Tt orthopédique et chirurgical Cannabis: 3-4 joints/j HDM: Depuis 9/04/16: fièvre d’apparition progressive, 39°C, algies diffuses/ courbatures, odynophagie Persistance de la fièvre, amenant à consulter aux urgences le 15/04: Sd grippal, nausées et vomissements, angine erythémateuse, rash cutané Hospitalisation SMIT 15/04/16: fièvre, dysphagie, pharyngite et gingivite, vomissements, éruption maculeuse diffuse tronc PdT

Primo infection VIH Rash: 12 à 32% PdT

Mr EM, 29 ans 2 Examens complémentaires initiaux: NFS: Hémoglobine 15g/dl, plaquettes: 105000/mm3; leucocytes 3500/mm3, PNN 72%, lymphocytes 14,6% (510/mm3), monocytes 12,8% BH: Bilirubine 16, ASAT: 71, ALAT: 168; gGT: 76 Albuminémie: 36 g/l; Cholestérol T: 2,5 mM/l EPP: hyperα2Gb 14%, hypoγGb 10% CRP: 65 mg/l PCR grippe et virus respiratoires négatifs Test rapide VIH (urgences) négatif Hémocultures négatives PdT

Mr EM, 29 ans (A) Quels Examens Complémentaires demandez vous d’emblée: IgM anti HAV, Ag HBs et IgM antiHBc, sérologie HCV et PCR HCV IgM HEV et PCR HEV Sérologie parvovirus B19 biopsies cutanées Prélèvement bactériologique de gorge Fibroscopie oeso-gastrique Sérologie VIH Elisa Antigénémie p24 PCR ARN- VIH Sérologie CMV et PCR CMV Sérologie EBV et PCR EBV Sérologie syphilis PdT

Mr EM, 29 ans 3 Résultats initiaux: Sérologie EBV+ IgG et IgM Sérologie CMV +IgG, limite IgM Sérologie VIH EIA + Western Blot VIH: négatif, 0 bande Ag HBs nég, sérologie HCV nég, PCR VHC nég PCR CMV nég PCR EBV nég Quelle interprétation faites vous concernant ces résultats ? (B) PdT

Mr EM, 29 ans 4 Résultats complémentaires: Sérologie VIH EIA + PCR ARN-VIH: 3.376.000 c/ml CD4: 191/mm3 (39%), CD8: 190/mm3 Antigène p24+ index 30,7; test neutralisation + 99,5% PdT

Primo infection / classification (Fiebig) The stages of acute human immunodeficiency virus infection as characterized by detection of viral particles and evolving antibody responses [13]. ELISA, enzyme-linked immunosorbent assay. Myron S. Cohen et al. J Infect Dis. 2010;202:S270-S277 © 2010 by the Infectious Diseases Society of America

Mr EM, 29 ans 5 Contrôle CV J2: PCR ARN = 5.750.700 c/ml Génotypage de résistance: sous-type B RT: - 138A PRO: 63P 71T Int: sauvage Traitement ARV débuté: (18/04) TenofovirDF+FTC+cobicistat/elvitégravir: 1 cp/j dates ARV CV CD4 CD8 16/04 - 3376700 191 (39%) 190 18/04 TDF+FTC+c/EVG 5750700 20/05 :* 99 660 (47%) 432 <20 (4%) 50

(prévalence de résistance, %) La transmission de variants résistants aux INNTI augmente chez les HSH en Europe (2) Prévalence de résistance (%) Evolution chez HSH (prévalence de résistance, %) 12 * p < 0,001 Comparaison avec HSH 15 HSH 10 Hétérosexuel Toutes classes p = 0,19 8 UDIV 10 * 6 * INTI p = 0,22 p = 0,008 4 * 5 * * IP 2 INNTI p = 0,006 Toutes classes INTI INNTI IP 1/2003 1/2004 1/2005 1/2006 1/2007 Chez les HSH diminution globale de la transmission des virus résistants aux IP des virus résistants aux INTI (TAM ou T215 « révertants ») continuent à circuler augmentation des virus résistants aux INNTI Frentz D, EACS 2011, Abs. PS1/5

Chez les patients en primo-infection : Etude PRIMO 2007-2012 (1) Caractéristiques des patients (n = 1 205) Prévalence des sous-types B et « non-B » 1996 à 2012 Homme 88,4 % Pays de naissance France 73,2 % Afrique sub-saharienne 5,6 % Autre/inconnu 21,2 % Groupe à risque Homosexuel masculin 69,7 % Hétérosexuel 20,6 % 9,7 % CD4/mm3 502 (3 -1 580) ARN VIH-1 (log10 c/ml) 5,4 (< 1,8 - 8,1) % 33% 1996- 1998 1999- 2000 2001- 2002 2003- 2004 2005- 2006 2007- 2012 Estimation : les patients de l’étude représentent 50 % des primo-infections en France Chaix ML, IHDRW 2013

Mr EM, 29 ans (C) Quelle stratégie thérapeutique envisagez vous à long terme ? Traitement ARV continu, sans aucune interruption prévue pour les 20 prochaines années Arrêt programmé du traitement ARV après 1 an, et surveillance de la remontée de CV Poursuite du traitement ARV pendant 5 ans avant d’envisager une interruption car le patient pourrait être contrôleur post-Tt Intensification du traitement ARV pour permettre une réduction du réservoir viral Contrôle de la quantification du réservoir par mesure de l’ADN dans les PBMC, avec interruption si PCR-DNA <2,4 log

Corrélation entre délai de mise sous ARV en primo-infection et baisse du réservoir viral 11 Objectif : modélisation de la  à court-terme et à long-terme de l’ADN VIH total chez les patients ayant initié les ARV pendant la primo-infection 327 patients de la cohorte ANRS PRIMO 3 mois après infection 2 mois après infection 1,5 mois après infection 1 mois après infection 0,5 mois après infection (mois) Pente de décroissance de l’ADN VIH total après initiation des ARV (log10 c/106 PBMC) L’impact sur le réservoir viral est en faveur d’un traitement très précoce (15 premiers jours) après la primo infection. Passé ce délai la décroissance du réservoir est peu différente quelque soit le moment d’initiation du traitement. Impact de la précocité des ARV uniquement sur la 1ère pente de décroissance Plus tôt les ARV sont initiés, plus rapidement le réservoir baisse au cours des 8 premiers mois = -0,171 ; -0,131 et -0,068 log10 c/106 PBMC/mois quand les ARV sont commencés 15 jours, 1 mois et 3 mois après l’infection, respectivement Laanani M, CROI 2015, Abs. 373

PLoS Pathog. 2013 Mar; 9(3): e1003211.

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