GENETIC FACTORS LEADING TO LOSS OF VIRAL CONTROL IN HIV ELITE CONTROLLERS PATIENTS José M. Benito, Marcial García, Victoria de Santisteban, Ricardo Ramos, Clara Restrepo, María A. Navarrete-Muñoz, Agathe León, Ezequiel Ruiz-Mateos, Alfonso Cabello, José Alcamí, Miguel Górgolas, Norma Rallón; On behalf of ECRIS integrated in the Spanish AIDS Research Network Good afternoon. I wanna thank the organization for giving me the opportunity to present data form our group. The tittle of my presentation is: “Genetic factors leading to loos of viral control in HIV elite controller patients” Watson JD, Crick FH. Nature. 1953; 171(4356):737-8

CONFLICT OF INTEREST No conflicts of interest to declare I have no conflicts of interest to declare

ELITE CONTROLLERS (EC) HIV patients capable of controlling viral replication to undetectable levels without the need of antiretroviral therapy. They represent less 1% of all HIV infected population Elite Controllers Non Controllers CD4+ As probably most of the audience knows, elite controller are a extraordinary population of HIV patients that have the capacity to control HIV replication to undetectable levels without antiretroviral therapy. They are very rare and their frequency is estimated in less than 1% of the global HIV population. In contrast to the great majority of HIV patients (the non controllers), EC maintain control of viral replication (usually soon after acute infection) and high and stable levels of CD4 counts VL

DIFFERENT FACTORS INVOLVED IN ELITE CONTROL OF HIV Factors associated to virological control Host factors Viral factors HLA-B*57 HLA-B*27 HLA-B*14 HLA-B*52 CCR5∆32 Defective viral strains The factors involved in this expceprional ability of EC patients are not well understood and they are likely multifactorial. Among the host factors, certain HLA alleles have benn strongly associated with the EC phenotype suggesting an important role of virus-specific immune response in this ability. Among the viral factors, the existence of defective viral strains with very low fitness has been reported in some patients

LOSS OF VIROLOGICAL CONTROL Up to 30% of EC experience loss of virological control Leon et al. AIDS. 2016;30(8):1209-20 Weeks Years HIV-RNA plasma levels Acute infection Chronic infection Virological control Loss of However, not all EC patients maintain this ability for prolonged periods of time and in fact a variable proportion of them loose the capacity to control viral replication over time. A previous study from obtained with data from the Spanish EC cohort has shown that as much as 30% of EC patients loose virological control

FACTORS INVOLVED IN LOSS OF ELITE CONTROL Factors associated to loss of virological control Viral factors Host factors Clinical Nadir CD4 Coinfections (HCV, HBV) Viral blips León et al. AIDS. 2016; Madec et al AIDS 2013 Yang et al. AIDS 2017 Chereau et. al Plos One 2017 Epidemiological Some previous studies have analyzed factors associated to the loss of elite control of HIV replication. Among the viral factors, a high frequency of viral blips and the existence of superinfections have been reported in EC patients loosing control. Among the host factors, different clinical, epidemiological and demographic characteristis of the patients hasve been associated to the loss of viral control Risk factor for HIV acquisition Year of seroconversion Superinfections León et al. AIDS. 2016; Madec et al AIDS 2013 Clerc et al. J Clin Virol. 2010 Demographic Gender Age León et al. AIDS. 2016; Madec et al AIDS 2013

FACTORS INVOLVED IN LOSS OF ELITE CONTROL Host-dependent biological mechanisms underlying this phenomenon Clinical HIV-specific T cells response Pro-inflammatory milieu Other host genetic factors? Pernas et al. J Virol. 2017 Pernas et al. J Virol. 2017 Epidemiological El-Far et al. Sci Rep. 2016 However, very few studies have deepened into the biological mechanisms underlying this phenomenon. A recent study performed in the Spanish AIDS research network EC cohort suggest that the level and/or funtionality of virus-specific T cell response as well as a proinflammatory millieu are potentail factors involved in the loss of virological control.IL32 has also been recentluy associated to loss of control. However, other potential host genetic factors underlying this phenomenon have not been explored sofar. Pernas et al. J Virol. 2017 El-Far et al. Sci Rep. 2016 Pernas et al. J Virol. 2017

HYPOTHESIS Expression levels of different genes involved in HIV pathogenesis could influence the ability of EC patients to maintain viral replication control . OBJECTIVE To investigate the association of several genes, previously related to pathogenesis of HIV infection, with the loss of spontaneous virological control in EC patients. Based on the hypothesis that the expression levels of different genes involved in HIV infection pathogenesis may influence the ability of EC patients to maintain control of viral replication, the objective of our study was to investigate the association of several genes previously related to pathogenesis of HIV infection with the loss of spontaneous virological control in EC patients.

(Undetectable plasma HIV-RNA during follow-up) PATIENTS 7 Study population Persistent Controllers (PC) (Undetectable plasma HIV-RNA during follow-up) 13 EC patients (From the HIV controllers cohort of the Spanish AIDS Research Network (ECRIS) 6 Transient Controllers (TC) For this task we retrospectively recruited 13 EC patients from the Spanish EC cohort, 7 of whom maintained virological control during follow-up (named persitent controllers, PC) and six who loose virological control during follow-up (named transient controllers, TC). (Plasma HIV-RNA > 50 copies/mL in two consecutive measurements during 1 year of follow-up)

STUDY DESIGN 6 7 We analyzed two cellular samples from each patient. In the group of transient controllers one sample 1-2 years before (named T1 sample) and one sample 1-2 years after (named T2 sample) the loss of virological control. In the group of persistent controllers two samples separated by a time period of 1-2 years (T1 and T2 samples)

STUDY DESIGN 6 7 We compared the genes expression profile between the two groups of patients at each timepoint (so this was an intergroup comparison)

STUDY DESIGN 6 7 We also compared the genes expression profile at the two different timepoints in each group of patients (so this was an intragroup comparison)

METHODS PCA analysis Differential expression (FDR<0.05) Criopreserved PBMCs Component 1 Component 2 PCA Analysis software RNA extraction Differential expression (FDR<0.05) Gene expression Group A Group B We started from criopreserved PBMCs. RNA was extracted using a commercial extraction method and RT-PCR for 43 different genes previously related to HIV pathogenesis was performed with an Applied Biosystems thermocicler. Raw data was analyzed using StatMiner software and two different statistical approaches were undertaken: a principal component analysis to test the capacity of the genes expressionprofile to discriminate between groups of patients or between timepoints; and a differential genes epxression analysis using a false discovery rate treshold of 0.05. RT-PCR

RESULTS This table shows some relevant characteristics of TC and PC groups of patients. Boths groups were comparable in terms of age, gender, CD4 count at T1 and at T2, and proportion of patients coinfected with HCV. HIV viral load was undetectable at both timepoints in PC patients and detectable at T2 in transient controllers patients with a median of 2,8 log copies The only difference (almost significant) was the length of EC status that, as expected, was lower in TC patients compared to PC patients

RESULTS Genes expression profile was able to discriminate between TC and PC patients at T1 (PCA analysis) TC PC Principal component analysis was able to partially discriminate between TC and PC patients at timepoint 1, this is when both groups of patients presented undetectable HIV viral load and before TC patients lost viral control. So, 5 out of seven PC and 4 out of six TC clustered together. This suggests that genetic expression profile is associated with the phenotype of EC patients and precedes the lost of viral control in TC patients.

Several genes were down-regulated at T1 in TC compared to PC RESULTS Several genes were down-regulated at T1 in TC compared to PC FC = 0,78 FC = 0,68 p= 0.046 25 2.5 p= 0.006 PC TC FC = 0,59 p= 0.024 20 2.0 1.5 Relative mRNA gene expression 15 Relative mRNA gene expression 10 1.0 In fact the differential genes expression analysis revealed the existence of significant differences in some interesting genes between TC and PC at timepoint 1. There was a significant downregulation of CDKN1a, IFI16 and CTR9 genes in TC compared to in PC as can be seen in this graph, with fold changes values (in TC compared to PC) ranging from around 0.6 for CDKN1A gene to 0.8 for IFI16 gene. Interestingly an anti-HIV activity (by different molecular pathways) has been demonstrated for all these three genes. 5 0.5 0.0 CDKN1A IFI16 CTR9

Several genes were up-regulated at T2 compared to T1 in TC RESULTS Several genes were up-regulated at T2 compared to T1 in TC T2 T1 Relative mRNA gene expression p= 0.03 2 4 6 8 10 ABCA1 [Fold change= 3,5] p= 0.04 0.00 0.05 0.10 0.15 0.20 IL10 [Fold change= 2,2] IL21 [Fold change= 4,6] 1 3 5 p= 0.047 PAF1 [Fold change= 1,5] 0.0 0.5 1.0 1.5 2.0 p= 0.012 TRIM26 [Fold change= 1,32] Another interesting (though more expected) finding of our study was the significant increase in the expression level of some genes in TC patients after they lost virological control. This did not happen in PC that maintained virological control. Interestingly these genes have either an ant-viral activity such as PAF1 and TRIM26 or an important role in regulating anti-viral immunity such as IL21 and IL10. Lastly ABCA1 gene regulates cholesterol metabolism and has anti-HIV activity by restricting HIV trans-infection. These results suggest that these genes upregulate their expression in response to the increase in HIV replication

CONCLUSIONS Our results demonstrate a down-modulation of different genes with anti-HIV activity in EC patients that precede the loss of spontaneous viral replication control in these patients. These genes could be considered potential biomarkers of loss of spontaneous control and could provide new insights for the clinical management of these exceptional group of patients. The main conclusions of our study are:

ACKNOWLEDGEMENTS We would like to thank all patients and healthy donors who participated in the study. This study has been funded by: Project CP14/00198 integrated in the State Plan for Scientific and Technical Research and Innovation. ISCIII-Sub-Directorate General for Research Assessment and Promotion. European Regional Development Fund (ERDF). N Rallón is a Miguel Servet investigator from the ISCIII (CP14/00198). M García is co-funded by CP14/00198 project and the Intramural Research Scholarship from IIS-FJD. C Restrepo was funded by project RD12/0017/0031 and is currently funded by project RD16/0025/0013. To finish I wanna thank all the organizations that have collaborated in this study

RESEARCH TEAM Research laboratory in HIV and Viral Hepatitis, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz UAM; Hospital Universitario Rey Juan Carlos And of course to the most important part of any scientific study: the people who made it possible. Thanks for your attention.