Papel de las células plasmacitoides en el control del VHA1

Machmach, Kawthar

Papel de las células plasmacitoides en el control del VHA1

Machmach, Kawthar

Published: 2014-01-01
Repository: idus.us.es
Language: es

Abstract

During the last five years I was focused on my PhD projects about HIV infection and innate immunity under the supervision of Dr Ezequiel Ruiz-Mateos and Dr Manuel Leal. My main research was focused on the role of plasmacytoid dendritic cells (pDCs) in the spontaneous control of HIV infection in ¿HIV elite Controllers¿ (EC). EC are a rare group of HIV-infected patients who are able to maintain undetectable viral loads during a long period of time in the absence of antiretroviral treatment (ART)[1]. Mechanisms responsible for this spontaneous control have been studied with the hope of developing a therapeutic vaccine against HIV. Preserved T cell functionality has been described for EC [2-4]; however, the innate immunity response needs to be characterized. The pDCs are innate immune cells that respond to viral infections, producing up to 1000-fold more alpha interferon (IFN-¿) than other cell types [5] through the stimulation of Toll-like receptor 7 (TLR-7) and TLR-9. The IFN-¿ produced by pDCs not only participates in the inhibition of viral replication but also has an adjuvant effect on different immune cells types[6-8], acting as a critical link between innate and adaptive immunity. The first aim of my thesis was to quantify pDCs levels and to analyze pDCs functionality in relation to the spontaneous control of HIV viremia observed for EC. When I joined the group, we first started characterizing our cohort of EC. We observed how the ability to control spontaneously HIV infection in EC is not infinite and that they eventually progress to AIDS [9]. Once the EC from our cohort were characterized, we started working on pDCs and began collaboration with Dr Jean-Phillipe Herbeuval (CNRS UMR 8147, Necker Hospital, Paris, France), who is an expert on pDCs, and Dr Olivier Lambotte (Bicêtre Hospital, Paris, France) who is an expert on HIV infection in EC. During my 9 months training at Dr Herbeuval¿s laboratory, we obtained very interesting discovery after learning several tools; and demonstrated for the first time the preserved frequency and functionality of pDCs in relation with the capacity to control HIV infection by EC when compared to other group of HIV infected individuals [10, 11]. Moreover, we showed a preserved capacity of pDCs from EC to suppress viral production in HIV-infected T cells and also showed the mechanisms by which pDCs induced this viral suppression. These findings highlight the important role of innate immunity in HIV immunopathogenesis and could have important immunotherapeutic applications. During my training, we also were wondering whether the EC are able to control other persistent infections as Hepatitis C virus (HCV), whose natural history is accelerated in HIV-co-infected patients. In this study [12], we interestingly found a lower HCV viral load in HIV controllers, alongside a different distribution of HCV genotypes in relation to the HIV/HCV co-infected comparison group. Thus, some of these subjects are able to control both infections, suggesting the possibility of shared control mechanisms. In this sense, as we previously observed a preserved capacity of pDCs from EC to suppress HIV replication: we wondered whether pDCs are also implicated in the control of both infections HCV/HIV. For this purpose, I participated tightly with my mentor Dr Ruiz-Mateos in the development of this project and the design of the experiments. In parallel, analyzing shared control mechanisms of HIV and HCV infections, we were also interested in a single nucleotide polymorphism (SNP) rs12979860, near the IL28B gene, which codes for interferon ¿3 (IFN-¿3) and has been associated with the spontaneous clearance of HCV [13] and with sustained virological response (SVR) after HCV-specific treatment [14]. Although it is still unknown how this SNP affects the antiviral activity of IFN-¿, several studies have demonstrated the antiviral activity of IFN-¿ against different viruses, including HIV [15]. However, no association was found between these protective alleles and spontaneous HIV control in African American individuals [16, 17]. We wondered whether this IL28B SNP would be overrepresented in white HIV controllers, thus our aim was to analyze the association of the IL28B SNP with the spontaneous control of HIV infection. Our study demonstrates that the IL28B-CC genotype is independently associated with spontaneous HIV control in white individuals [18]. Our finding adds to the suggestion of common shared mechanisms involved in the control of persistent infections, such as HIV and HCV infection. We are now focusing our efforts to explain these results. As the pDCs also produce IFN-¿3 [19] and we already showed the preserved functionality of pDCs in EC, it is unknown whether this SNP alter IFN-¿3 production by pDCs. Recently, a new transiently induced region that harbors a dinucleotide variant ss469415590 (TT or ¿G), which is in high linkage disequilibrium with IL28B rs12979860, has been discovered [20]. ss469415590 [¿G] is a frameshift variant between IFNL2 and IFNL3 that creates a novel gene, designated IFNL4, encoding the IFN-¿4, which is moderately similar to IFN-¿3. Compared to rs12979860, ss469415590 is more strongly associated with HCV clearance in individuals of African ancestry, although it provides comparable information in Europeans and Asians [20]. We were interested in analyzing whether ss469415590 SNP is associated with the main parameters associated with HIV progression, i. e. HIV viral load and CD4+ T-cells, which have not been studied in HIV-infected patients. For this aim, we included 389 HIV-infected subjects, in chronic phase of HIV infection and ART-naïve. Briefly, we observed a higher prevalence of presenting AIDS-defining illness in subjects¿ carriers of ss469415590 ¿G variant. We showed that ss469415590 TT variant is independently associated with a lower proportion of CDC events.

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Co-director de Tesis,  !"!#!$              ,-...                                      & . 5 ?  . .  6 ? 9&=#? ! . !   !  !  .   (& ! ?  ! . 9-   @ABC?9&=#?D-89&=>5 !!?.5? . EF . !  5   - !5!   . ?9&=#89&=>@%C.!565  !5   @G1 9&= "$( HC  . 89&=  > @"%C . -.    5- 1# 9&= "$( H:.?.##@$;C!56EF. ??89&=>-..?$;! 5   %I . ! J1H   ?   -.! .K..5.-.89&=> 5..%I!? -. . ! ? 9&= ? !56 EF 9-  ? . ! L%I(&EIF..5?. !  . 5 ! -. . . ?   .! 9&=...! ?9&=#!-.?!E1#MF$. ..L..   &   .  ? # .!    3#N ( ! @3N(C . 5 ?     ?..!&!   ? . 6 95 %L @9%C  &  . 9(# K12    . ! 9&=   E1 OF &   ! ,6 !.L. E0F 5  ??5.9(!..! .  ?    -. -! L  .. !  . !.!-!5.6L5%O..5 !   !! 9&= ? %I   EMF   ?? ?.5!.!E2F.5?. %I?9&=!56EF   . .  ?  .  !    !??  L . !  ? 9&= ? 9- .  ! 9&=5..; @%C!.!?#.@&'$# PCQ!!??&..?5EF . % ! &'$#P    .  "$( 5 . ! ? . #D "#2 @"#2C  !. %3 $( ? .!. "#M EF . &'$#P ! 5 %     . .5?5!.6!????! DEF!DEIFE1FD 5-!.?%9&=?- !   .?!..!5?!%- 9&=?E0F.5#-...- %I!55!..?!E2FD ?!D-&'$!?%I.?! EO MF (. ! ! ? .  . 5 .  ? 9&=#  % ? 5  .  EF -.  %I   !EF(%!5 5.!....@9(("C!.? -!?EF.?%!! 9(("  -.  - !   ? !! . ! EIF'!.9&=#%L."(&@!  ?#  ! C  .  D @?#!D%CE1F."(&#LD% @,%C -   5   L   %I    ?9&=#?EF    "(&!?9&=#L%IE0F "(& !  ? .# 1 @"1C#L  E2F . ? %I  L"(&"1    .5? EOMF!5!?!9(("EF;!! "(& !  .  & ? ! 5 % ? 9&= !"(&%IEF   & 9&=  !56 . ?! ? %Q &'$#P !  "(&L5%.5.!!?56-   %     . ?!  9&=  . 56 -   . -  8&' $( (($$ & ( ) ($ $$  &>@R=C8&' $((($$ & *)&+ ,     & ) ( $, %- ( ($,,$ $  ,$ .$)&($!$ >@R&C   :...?.!?  . !  ? 9&=  5     . !?.%!@9%=CEF.? .9(#K12.9&=!.5 .!?9%=?!56EF-9%=# ? ?! %O    E#IF 3 . .  !?5.9&=.%!@9%=C.?#? 9&=?.9%=!.9&=#? E1F " . 9&=H9%= ?  9&=     . !  ? 9%=   . ? (?#( ? -. 9%= .5E0F!..5?9&= !?!?.@9&=#9%=C.   ? D  . .    !   5?..!   . 9&=H9%= #?  %! 9&=  .   5 .!!56--.L%!9&= 59%=.?. 56 -   .  8)$$&  !$& )$$  $ &$   &>@R=9C   &   ! . @$;C M2MO0  . &OK-..??S@D-&OKC.5 -.?9%=E2F!?9%=# ?EOF!..?&$'# S??!!9&=EMF.!.!D-.-.$; ?? .   ? &$'#S 9-  ..  ?  &OK#%%(?#(9&=# !56-5-?!5-.&OKM2MO0  9&= !   (?#( ! EI IF$. -.D!.!5?9&=! . !  . . ? . .Q   5 (?#( !-..-?&OK#%%.%!!56E2F .&OK#%%.5$;!56. EIF555!.?.-5! @%IC@!5C   .?.&OK#%%.! ? 9&= ?  %! ! ! . . 56 ? .  .  -   . ! 8/01 &$2% $( ) ' )$& &3/454067 $& && $( $ &)  &     $ $ &89&>@R&C   "-!..5!! I0MI11M @  T3C -..   .. D  !5! -. &OK M2MO0 . 5  EI IIF I0MI11M ET3F   ?.?  5- ? 5  @&'$C  &'$  .     &'$I.&'$#SI-..!!&'$# SEIF(.!...?!?.!D-&'$I I0MI11M#T35.?!-..! ? &OK EIIF N.   M2MO0 I0MI11M    -..!?9%=?.9%=  EI#I0F N.. I0MI11M   -.   !9&=#?!D-  &  -.  .      .D . I0MI11M .-.#.! ?9&=.?!.?..-.  ? . &'$I  -.   !   9&=#? . 56 -   . ! 8: &&:64:3;;47 ) ' )$&$&&& $($,! 8$$($  2$&&$ %$,($($!$(&>@!5!C       of H9 T cell apoptosis. It should be noted that there was a tendency for pDCs from EC to induce greater levels of annexin V-positive T cells than those from VIR subjects (Fig. 3B). We also analyzed the ability of pDCs to induce HIV-infected primary autologous CD4 1 T cell apoptosis in 8 healthy donors (Fig. 4). Both unstimulated and CpG-stimulated pDCs induced HIV-infected autologous CD4 1 T cell apoptosis. Reduced response of pDCs from VIR subjects is associated with CD4 expression. According to the results of suppression and apoptosis assays, unstimulated pDCs from VIR subjects showed low degrees of responses to HIV. We also showed by the addition of CQ that the endocytosis pathway is needed for pDC activation. HIV binding to the CD4 expressed by pDCs is essential to activate the endocytosis pathway (7). Thus, we studied CD4 expression on pDCs from HD, EC, and VIR subjects using 3D microscopy (Fig. 5). CD4 was homogeneously expressed on the cell surface of pDCs from EC and HD subjects. In contrast, pDC from VIR subjects showed low levels of membrane CD4. We also observed intracellular CD4 expression in pDCs from VIR subjects, contrasting with pDCs from HD or EC subjects. When pDCs from HD subjects were exposed to HIV MN overnight, we observed the intracellular expression of CD4, as we observed for pDCs from VIR subjects. We also wanted to corroborate these results by flow cytometry; a decrease in CD4 cell surface expression levels in pDCs from VIR compared to HD subjects was observed (see Fig. S2 in the supplemental material). This finding suggests that continuous exposure FIG 1 Quantification of peripheral pDCs and IFN- a production. (A and B) pDC quantification. Percentages (A) and absolute numbers (B) of pDCs (BDCA2 1 CD123 1 ) from 22 healthy donors (HD), 14 elite controllers (EC), 13 relative controllers (RC), 19 HIV-treated patients (HAART), and 25 viremic subjects (VIR) were determined. (C) IFN- a production measured in the supernatants of PBMC cultures, in the presence of a TLR-9 ligand (CpG ODN 2216), from 18 HD, 11 EC, 11 RC, 15 HAART, and 22 VIR subjects. The level of IFN- a production by PBMCs cultured in medium alone was in all the cases below the detection limits (data not shown). (D) Correlation between numbers of peripheral pDCs and HIV RNA copies/ml (log 10 ) in 11 EC, 11 RC, 15 HAART, and 22 VIR subjects. (E) Correlation between numbers of peripheral pDCs and IFN- a produced by CpG-stimulated PBMCs from 18 HD, 11 EC, 11 RC, 15 HAART, and 22 VIR subjects. Pvalues were determined by using a Mann-Whitney U test. ns, not significant (a Pvalue of ,0.05 was considered statistically significant); p,P,0.05; pp,P, 0.005. Machmach et al. 4248 jvi.asm.org Journal of Virology on April 12, 2012 by USE/BTCA.GENERAL UNIVERSITARIAhttp://jvi.asm.org/Downloaded from to HIV in VIR subjects is associated with the internalization of CD4 in pDCs. DISCUSSION In the present study, we show the preserved frequency and functionality of pDCs in relation with the capacity to control HIV infection by EC. The decreased peripheral pDC count observed for VIR subjects was described previously (29) and is possibly due to the migration of these cells to lymph nodes (11, 13). We observed that after successful HAART, the loss of pDC numbers in the chronic phase of HIV infection seems not to be restored entirely, similar to data from a previous study (3). We also observed preserved IFN- a production in PBMCs from EC at even higher levels than in PBMCs from HD subjects. Indirectly, we can assume that this IFN- a production is due mainly to pDC stimulation. Indeed, only pDCs and B lymphocytes express TLR-9 (8), and only pDCs express 1,000 times more IFN- a than other cell types, including B lymphocytes (31). This finding was supported by the correlation observed between IFN- a production by PBMCs after TLR-9 stimulation and pDC counts (r50.464; P,0,001). The higher level of IFN- a production by PBMCs through TLR-9 stimulation observed for EC may be due to the preserved pDC levels and not to a better functionality of pDCs in these patients. In fact, when isolated pDCs were stimulated with TLR-9, we observed similar levels of IFN- a production in HD, EC, and VIR subjects (Fig. 2B). However, when we analyzed IFN- a production by pu- FIG 2 pDC-mediated suppression of HIV production in H9 T cells. Purified pDCs from 7 HD, 7 EC, and 8 VIR subjects were plated overnight at 5 310 4 pDCs/well with or without CpG and cocultured in the presence of HIV-infected T cells. After 5 days of coculture, amounts of p24 and IFN- a in the supernatants were measured by an ELISA. (A and B) Effects of unstimulated (A) and CpG-stimulated (B) pDCs on HIV-infected T cell line virus production. The index of suppression by pDCs is expressed as the difference of log HIV p24 production between the culture containing T cells alone and the coculture of T cells and pDCs [index of suppression 5log HIV p24 (T cells) 2log HIV p24 (T cells 1pDCs)]. (C and D) IFN- a production measured in the supernatants of the coculture. (E) Implication of IFN- a in pDC-mediated viral suppression. IFN- a produced by pDCs was partially neutralized by the addition of an anti-IFN- a antibody. Data are representative of 5 different experiments (pDCs from 3 HD and 2 EC subjects). (F) Blocking of endocytosis by the addition of CQ at 1 m M to wells of CpG-stimulated pDCs. Data are representative of 7 different experiments (pDCs from 2 HD, 1 EC, and 4 VIR subjects). Pvalues were determined by using a Mann-Whitney U test. ns, not significant (a Pvalue of ,0.05 was considered statistically significant); p,P,0.05; pp,P,0.005. Plasmacytoid Dendritic Cells and HIV Controllers April 2012 Volume 86 Number 8 jvi.asm.org 4249 on April 12, 2012 by USE/BTCA.GENERAL UNIVERSITARIAhttp://jvi.asm.org/Downloaded from rified pDCs after HIV stimulation, we observed comparable amounts of IFN- a between HD and EC subjects and considerably reduced amounts for VIR subjects. These results demonstrate a preserved pDC response to HIV in EC. On the other hand, when both HIV controller groups were compared, despite the lower difference in viral loads, we showed differences in pDC percentages and IFN- a production (P50.076 and P50.030, respectively) between them; these data demonstrated how the presence of detectable viral loads, even at lower levels, could affect peripheral pDC levels. Interestingly, when we compared RC and HD subjects, despite the lower pDC levels, PBMCs from RC subjects produced levels of IFN- a similar to those produced by PBMCs from HD subjects. Thus, we can conclude that the preserved pDC numbers are associated with the spontaneous control of HIV viremia. We demonstrated a preserved capacity of pDCs from EC to suppress viral production in H9 T cells. This result is in agreement with data from a previous study (15), where a higher capacity to suppress HIV replication in autologous CD4 1 T cells was found for antiretroviral-naive low-viremic subjects (,12,500 HIV RNA FIG 3 pDC-mediated H9 T cell apoptosis. Unstimulated (A) and CpG-stimulated (B) pDC-induced apoptosis was assessed on H9 T cells from the cocultures of the suppression assays. The data represent data from 6 HD, 5 EC, and 6 VIR subjects. Left panels represent annexin V (A-V)-positive T cells, middle panels represent Topro-III-positive T cells, and right panels represent annexin V-positive/Topro-III-positive T cells. Pvalues were determined by using a Mann- Whitney U test. A Pvalue of ,0.05 was considered statistically significant. FIG 4 pDC-mediated HIV-infected primary autologous CD4 1 T cell apoptosis. Isolated primary autologous CD4 1 T cells from 8 healthy donors were infected for 6 days with HIV BaL. HIV-infected primary autologous CD4 1 T cells (aCD4 1 HIV 1 ) were cultured in the presence of purified pDCs at a ratio of 1:2. After 24 h, annexin V-positive CD4 1 T cells (A) and Topro-III-positive CD4 1 T cells (B) were assayed by flow cytometry. Differences were analyzed by using a Wilcoxon test. A Pvalue of ,0.05 was considered statistically significant. ns, not statistically significant; p,P,0.05. Machmach et al. 4250 jvi.asm.org Journal of Virology on April 12, 2012 by USE/BTCA.GENERAL UNIVERSITARIAhttp://jvi.asm.org/Downloaded from copies/ml) than for treated subjects and high-viremic subjects ($12,500 HIV RNA copies/ml). However, in our study, we used the same target cells (H9 T cell line) to compare the suppressive faculty of pDCs among the study groups. We demonstrated that pDCs from EC subjects had the same capacity to reduce HIV production as pDCs from HD subjects, whereas pDCs from VIR subjects could scarcely respond. In contrast, when pDCs from all groups were prestimulated with TLR-9, we observed the same behavior for the three groups. These results agree with previous data showing that CpG A- and HIV-stimulated pDCs are not refractory to IFN- a production after restimulation (18). In this work, we show that the magnitude of the response after continuous HIV stimulation is higher in EC than in VIR subjects. This result demonstrates that pDCs from VIR subjects are functional but are not able to be efficiently stimulated by HIV and need to be previously activated by an HIV-independent pathway to display antiviral activity against HIV-infected T cells. This activity was associated with the IFN- a produced by isolated pDCs. These results show that pDCs from EC have preserved functionality to suppress HIV. The great majority of in vitro studies on pDCs suggested that IFN- a is the principal mechanism of viral suppression (4, 17). A previous report demonstrated that the HIV stimulation of pDCs induces a high level of production of IFN- a and a rapid expression of TRAIL, transforming them into IFN-producing killer pDCs (iKpDCs) (5). It was also shown previously that despite TRAIL expression, pDCs could not induce the lysis of autologous CD4 1 T cells (1). However, in the present work, when primary HIV-in- fected autologous CD4 1 T cells were used as target cells, we also observed apoptosis induced by both unstimulated and TLR-9- stimulated pDCs. This discrepancy can be explained because we investigated not lysis but apoptosis using early (annexin V) and late (Topro-III) apoptosis markers, and autologous CD4 1 T cells were productively infected and not only exposed to HIV; in addition, cocultures were maintained not for 6 h as in previous work but overnight in the case of autologous CD4 1 T cells or for 5 days in the case of H9 T cells. Thus, we demonstrated that besides IFN- a , pDCs exert their antiviral effect by inducing T cell apoptosis. Indeed, when p24 1 T cell levels in the coculture were analyzed, we observed a reduction of p24 1 T cell percentages. This effect was also observed when recombinant IFN- a was added to H9 T cells. These observations suggest that the pDC-induced viral reduction is due mostly to H9 T cell-induced apoptosis, which is preserved in EC. This finding is in accordance with our results showing that purified pDCs from EC and HD subjects produced large amounts of IFN- a in response to HIV-1 particles produced by the T cell line. In contrast, the level of IFN- a production by pDCs from VIR subjects was very low and barely suppressed or induced T cell apoptosis, confirming the fact that VIR subjects had an impaired HIV-mediated activation of pDCs. Thus, in an at- FIG 5 Microscopy analysis of CD4 distribution in pDCs. Shown is three-dimensional microscopy of the CD4 distribution in pDCs. pDCs from HD, EC, and VIR subjects and HIV-activated pDCs from HD (HD1HIV) subjects were stained with anti-CD4 antibody (pink), anti-TLR-7 antibody (green), and DAPI (blue). pDC stainings (green, pink, and blue) were merged and analyzed by 3D microscopy. Plasmacytoid Dendritic Cells and HIV Controllers April 2012 Volume 86 Number 8 jvi.asm.org 4251 on April 12, 2012 by USE/BTCA.GENERAL UNIVERSITARIAhttp://jvi.asm.org/Downloaded from tempt to understand why pDCs from VIR subjects were not able to respond to the HIV stimulus, we analyzed CD4 receptor expression on pDCs, which is necessary for HIV binding to pDCs and the subsequent activation of the endocytosis pathway by these cells (6, 7). The very low membrane CD4 expression level on pDCs from VIR subjects could explain the lack of responses when they were cultured in the presence of HIV. However, when pDCs from VIR subjects where stimulated by a CD4-independent activator (CpG), they responded similarly to those from HD or EC subjects. These data also explain the similar behaviors of pDCs among the different groups when a previous stimulation via a CD4-inde- pendent pathway, such as the TLR-9 ligand CpG, was performed. In conclusion, this study shows the qualitative and functional involvement of pDCs in the spontaneous control of HIV viremia. These findings highlight the important role of innate immunity in HIV immunopathogenesis and could have important immunotherapeutic applications. ACKNOWLEDGMENTS This work was supported by Redes Telemáticas de Investigación Cooperativa en Salud (RETICS) (2006, Red de SIDA RD06/0006/0021, 2007 to 2010), Consejería de Salud de la Junta de Andalucía (2008, PI-0270), Consejería de Salud de la Junta de Andalucía (2009, PI-0066), Fondo de Investigacion Sanitaria (PS09-00120), and the French National Agency for AIDS Research (ANRS) EP36 V study. E.R.-M. has a grant from Fondo de Investigaciones Sanitarias (CP08/00172). K.M. has a grant from Redes Telemáticas de Investigación Cooperativa en Salud (RETICS) (2006, Red de SIDA RD06/0006/0021, 2007 to 2010). The Biobank of the Spanish AIDS Research Network is supported by Instituto de Salud Carlos III and the Spanish Health Ministry (grant no. RD06/006/0035). We are grateful to all of the patients who participated in this study; to Manuel Moyano, Maria Teresa Martínez, Angel Ayala, Jesus García, and Juan Manuel Aznar from the Centro Regional de Transfusiones Sanguíneas y Banco de Tejidos Sevilla/Huelva for their support; to Marien Gutierrez, Magdalena Rodriguez, and Paca Cano from the Infectious Diseases Service at HUVR for their clinical support; and to the Biochemistry Department at HUVR for their support. We thank Laurence Meyer, Daniel Séréni, Caroline Lascoux, Olivier Taulera, Jeannine Delgado, David Zucman, Nadège Velazquez, Isabelle Poizot-Martin, and all the other physicians and nurses. The H9 T cell line was obtained through the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH (H9/HTLV-IIIB NIH 1983), from Robert Gallo. REFERENCES 1. 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Machmach et al. 4252 jvi.asm.org Journal of Virology on April 12, 2012 by USE/BTCA.GENERAL UNIVERSITARIAhttp://jvi.asm.org/Downloaded from            Hepatitis C virus replication in Caucasian HIV controllers E. Ruiz-Mateos, 1 K. Machmach, 1 M. C. Romero-Sanchez, 1 S. Ferrando-Martinez, 1 P. Viciana, 1 M. Del Val, 2,3 M. A. Mun˜oz-Fernandez, 4 M. Genebat 1 and M. Leal 1 on behalf of the Cohort of the Spanish AIDS Research Network (CoRIS) 1 Immunovirology Laboratory and Biochemistry Department, Infectious Diseases Service, Virgen del Rocio University Hospital/Biomedicine Institute of Seville (IBIS), Avd. Manuel Siurot s/n, Seville; 2 Centro de Biologı ´a Molecular Severo Ochoa (CSIC-UAM), Madrid; 3 Centro de Microbiologı ´a, Instituto de Salud Carlos III, Madrid; and 4 Laboratory Inmuno-Biologı ´a Molecular, Hospital General Universitario Gregorio Maran ˜o ´n, Madrid, Spain Received September 2010; accepted for publication November 2010 SUMMARY. Whether HIV controllers, patients who spontaneously control HIV viraemia, are able to control hepatitis C virus (HCV) infection, in terms of spontaneous clearance or lower HCV replication, is not well understood. To assess to what extent Caucasian HIV controllers are able to control HCV replication and potential associated factors, plasma HIV-1 and HCV RNA levels, anti-HCV antibodies, HCV genotype and human leucocyte antigens (HLA) typing were determined in samples from 75 HIV controllers (33 viraemic controllers, <1000 HIV-1 RNA copies/mL, and 42 elite controllers, <40 HIV-1 RNA copies/mL) and compared with 261 HIV-infected noncontrollers. We did not find differences in the HCV spontaneous clearance rates between groups. However, we interestingly found a lower HCV viral load in HIV controllers, alongside a different distribution of HCV genotypes in relation to the comparison group. In addition, HLA-B57 was associated with a lower HCV viral load in the control group and HIV controllers, and conversely, HLA-B35 with higher HCV viral load in HIV controllers. The subrepresentation of HCV genotype 1 and the overrepresentation of HLA-B57 only partly explained the lower HCV viral load found in HIV controllers. In fact, HIV controller status was independently associated with lower HCV viral load, together with HCV genotype non-1, the presence of HLA- B57 and absence of HLA-B35. Caucasian HIV controllers are able to better control HCV replication, in terms of lower HCV viral load levels. These findings support the idea that some common host mechanisms are involved in the defence against these two persistent infections. Keywords: elite controllers, hepatitis C virus, HIV, HIV controllers. INTRODUCTION In HIV infection, there is a rare group of patients (1–5%) who are able to maintain very low levels of plasma viraemia over long periods of time in the absence of antiretroviral treatment. These patients are known as HIV controllers (normally <1000 HIV-1 RNA copies/mL) or elite controllers for those who maintain plasma viraemia below the detection limits [1]. The mechanisms involved in this outstanding phenotype are not well understood at the moment. The overrepresentation of different human leucocyte antigens (HLAs) such as HLA-B57 and the associated cytotoxic T-lymphocyte (CTL) response seems to be a relevant mechanism associated with the natural control of HIV replication [2]. However, important questions remain open concerning the extraordinary immunity of these patients. One important aspect is to know whether HIV controllers are able to control other persistent infections in the same way that they do HIV. Hepatitis C virus (HCV) is of particular interest, whose natural history is accelerated in HIV-co-infected patients [3]. Recently, an increase in the spontaneous clearance of HCV in a cohort of African-Ameri- can HIV controllers infected with HCV genotype 1 has been reported [4]. The aim of the current work was to analyse whether Caucasian HIV controllers were able to control HCV infection in terms of spontaneous clearance and/or HCV viral load levels and whether this fact was associated with different HLAs and the distribution of HCV genotypes. PATIENTS AND METHODS Patients HIV controllers included in this study came from different cohorts of Spanish HIV-infected patients: the HIV-infected Abbreviations: CTL, cytotoxic T-lymphocyte; FIPSE, fundacion para la Investigacion y Prevencion del SIDA en Espan˜a; HCV, hepatitis C virus; HLA, human leucocyte antigens; LTNP, long-term nonprogressors. Correspondence: Manuel Leal, MD, PhD, Infectious Diseases Service, Virgen del Rocio University Hospital/Biomedicine Institute of Seville (IBIS), Avd. Manuel Siurot s/n, 41013 Seville, Spain. E-mail: [email protected] Journal of Viral Hepatitis, 2011, 18, e350–e357 doi:10.1111/j.1365-2893.2010.01431.x Ó2011 Blackwell Publishing Ltd patients cohort (CoRIS) and HIV Biobank of the Spanish AIDS Research Network (RIS), as well as the HIV-infected patient cohorts of the Infectious Diseases Service at the Hospitals of Universitario Virgen del Rocio (Seville), Comarcal de Don Benito (Badajoz) and Navarra (Pamplona). Samples from patients were kindly provided by the HIV BioBank integrated within the Spanish AIDS Research Network (RIS). Blood samples were processed following standard procedures and frozen immediately after their reception. All patients participating in the study gave their informed consent, and protocols were approved by institutional ethical committees. HIV controllers were defined as established by the International HIV Controller Consortium but with the following modifications [2]; these were subjects with positive antibodies for HIV-1, plasma viral load measurement <1000 HIV-1 RNA copies/mL in the absence of antiretroviral therapy (ART) for at least 12 months and with at least three plasma viral load determinations during this period of time. Seventyfive subjects that fulfilled these criteria were included in the present study. HIV-2-infected patients were excluded. HIV controllers were divided in two subsets: (i) ÔHIV–elite controllersÕwere patients were able to maintain plasma viral load below the limit of detection, <40 HIV-1 RNA copies/mL (isolated viral rebounds ÔblipsÕ<1000 HIV-RNA copies/mL were allowed) (n= 42) and (ii) ÔHIV–viraemic controllersÕ, patients who maintained plasma viral load between 40 and 1000 HIV RNA copies/mL (n= 33). ÔLoss of HIV controlÕwas considered when two consecutive viral load measurements were >40 HIV-1 RNA copies/mL for HIV–elite controllers and >1000 HIV-RNA copies/mL for HIV–viraemic controllers. Clinical and demographic characteristics of HIV controllers were compared with a group of 261 HIV-1-infected patients from our cohort (Hospital Universitario Virgen del Rocio) who consecutively visited our Unit until the study was censored, from now on refer to as the Ôcomparison groupÕ. HIV controllers and HIV-2-infected patients were excluded from this comparison group. Patients were asymptomatic and in the chronic phase of HIV or HIV/HCV infection, at the time of the study. METHODS Quantification of plasma viraemia Plasma HIV-1 RNA was measured in fresh samples by a quantitative RT-PCR (COBAS Ampliprep/COBAS Taqman HIV-1 test; Roche molecular systems, Basel, Switzerland) according to the manufacturerÕs instructions. The detection limit was 40 HIV-1 RNA copies/mL. Plasma samples were tested for anti-HCV antibodies using HCV-ELISA (Siemens Healthcare Diagnosis, Deerfield, IL, USA). HCV genotype was determined using a reverse-hybridization assay (InnoLiPA HCV II; Innogenetics, Barcelona, Spain). A quantitative RTPCR amplification was performed for plasma HCV RNA amplification (COBAS Amplicor; Roche Diagnosis, Barcelona, Spain) with a detection limit of 15 IU/mL. Patients with positive antibodies for HCV and negative PCR for plasma HCV RNA levels in the absence of previous HCV-specific treatment were considered to have spontaneous clearance of HCV infection. In the case of several HCV-RNA determinations available during follow-up, the last determination was considered. In the case of HCV-specific treatment, the HCV-RNA determination just before the start of treatment with either pegylated interferon and ribavirin or alpha interferons alone was taken. Besides, for HIV controllers who lost control of HIV viraemia during follow-up, HCV-RNA determinations were considered only during the HIV control period. HLA typing HLA-B* alleles were genotyped using a reverse stripdot-blot kit with sequence-specific oligonucleotide probes (Dynal RELIäSSO HLA-A and HLA-B typing kits; Dynal Biotech, Bromborough, UK) following the manufacturerÕs instructions. Statistical analysis Statistical analyses were performed using the Statistical Package for the Social Sciences software (SPSS 17.0, Chicago, IL, USA) using nonparametric statistical tests in all cases. Median and interquartile ranges were used to describe continuous variables and percentage for categorical ones. Spearman test was used to analyse correlation between continuous variables. Differences between groups were analysed with the Kruskal–Wallis, Mann–Whitney U-test, chi-square test or FisherÕs exact test when appropriate. All the differences between groups with a P< 0.05 were considered statistically significant. To analyse the independent factors associated with HCV viral load, a multivariate linear regression analysis was assessed. Variables showing a Pvalue <0.05 were considered statistically significant. RESULTS The characteristics of the patients are summarized in Table 1. A total of 75 HIV controllers, of whom 42 were elite controllers, were recruited. It is important to mention that there was a twofold higher proportion of women in the HIV controller groups compared to the comparison group. In this group, naı ¨ve patients (14.6%) had a median viral load of 4.8 [4.1–5.3] log HIV-1 RNA copies/mL. Sixty per cent of the patients on treatment had undetectable viral load, and the rest of the treated patients had a median viral load of 3.41 [2.8–4.3] log HIV-1 RNA copies/mL. Low plasma HCV load in HIV controllers Owing to the high proportion of HCV infection in HIV controllers, we analysed whether HIV controllers were able to Ó2011 Blackwell Publishing Ltd HCV replication and HIV controllers e351 control HCV infection in terms of spontaneous clearance. Conversely, the proportion of HCV spontaneous clearance was not statistically different to the comparison group (Table 1). However, when HCV load was also quantified, a lower HCV load was found in HIV controllers in relation to the comparison group, with a decrease of 0.7 log HCV IU/ mL in elite controllers (Fig. 1 and Table 1). This result was similar when viraemic and elite controllers were grouped together, although the difference decreased to 0.5 log HCV IU/mL (Fig. 1 and Table 1). We also observed that some elite controllers lost the capability to control the virus during follow-up. We wondered whether this fact could be associated with plasma HCV RNA levels. A tendency to have higher HCV load in elite controllers who thereafter lost the capacity to control HIV was found (Fig. S1). Additionally, it was found that HCV PCR+ HIV controllers had lower CD4+ T-cell levels than HCV PCR– HIV controllers (P= 0.022, Mann–Whitney U-test). Although this tendency was repeated in the elite controllers, statistical significance was not reached (P= 0.131, Mann–Whitney U-test). However, these differences were not observed in the comparison group (data not shown). Unusual distribution of HCV genotypes in HIV controllers HIV controllers showed lower prevalence of HCV genotype 1, and reciprocally, the proportion of HCV genotype 3 increased when compared to the general HIV population. No differences were found in the distribution of genotype 4 (Table 1). HLA-B57 and HLA-B35 are associated with HCV load levels in an opposite way All the HLA-B alleles present in the study subjects were analysed and only HLA-B57+/)and HLA-B35+/)subjects correlated with HCV load levels. HLA-B57 was associated with lower HCV load levels in all the study subjects (Fig. 2a). This was true for the comparison group Table 1. Characteristics of the patients Comparison group (n= 261) Elite controllers* (n= 42) HIV controllers (n= 75) P* P Female sex (%) 52/261 (20.0) 17/42 (40.5) 32/75 (42.7) 0.005 <0.0001 Caucasian race (%) 258/261 (99.0) 41/42 (97.6) 73/75 (97.3) 0.451 0.310 Age (years) 43.0 [38.3–47.1] 43.0 [39.0–46.3] 43.0 [38.0–46.0] 0.926 0.211 Time since diagnosis (years) 12.1 [5.1–16.8] 15.1 [6.0–20.8] 13.9 [5.0–20.1] 0.004 0.033 CD4+ T-cells/mm 3 à428.5 [247.8–594.3] 649.3 [442.8–1028.0] 664.0 [444.0–915.0] <0.0001 <0.0001 Naı ¨ve for ART§ 38/261 (14.6) 31/42 (73.8) 52/75 (69.3) <0.0001 <0.0001 Clinical stage C (%) 63/213 (29.6) 2/40 (5.0) 3/60 (5.0) 0.007 <0.0001 Risk group (%) IDU–120/261 (46.0) 27/42 (64.3) 41/75 (54.7) 0.031 0.192 Sexual 131/261 (50.2) 13/42 (31.0) 32/75 (42.7) 0.030 0.295 Anti-HCV+ (%) 134/261 (51.3) 32/42 (76.2) 50/75 (66.7) 0.003 0.019 HCV RNA+ (%)** 95/134 (71.0) 22/29 (75.9) 35/47 (74.5) 0.656 0.709 HCV clearance with HCV treatment 17/134 (12.7) 1/29 (3.4) 3/47 (6.4) 0.485 0.584 Spontaneous HCV clearance 22/134 (16.4) 6/29 (20.7) 9/47 (19.1) 0.581 0.670 Log HCV RNA (IU/mL) 6.4 [5.7–6.9] 5.7 [4.8–6.5] 5.9 [5.0–6.6] 0.005 0.007 HCV genotypes Genotype 1 56/96 (58.3) 8/23 (34.8) 15/36 (41.7) 0.061 0.117 Genotype 3 17/96 (17.7) 10/23 (43.5) 15/36 (41.7) 0.013 0.006 Genotype 4 22/96 (22.9) 4/23 (17.4) 5/36 (13.9) 0.780 0.335 HLA-B57+ (%) 6/83 (7.2) 6/31 (19.4) 8/40 (23.3) 0.085 0.065 HLA-B35+ (%)àà 12/83 (14.5) 5/31 (16.1) 8/40 (20.0) 1.000 0.448 *Elite controllers vs comparison group; HIV controllers (elite plus viraemic) vs comparison group. Mann–Whitney U-test was used; àLast determination; §Antiretroviral treatment; –Injecting drug user; **Not available in three patients in the elite and HIV controller groups; HCV genotype determination available for those HCV RNA +, including patients with HCV clearance on HCV treatment, where HCV genotype was assayed before the start of HCV-specific treatment; ààHLA typing was assayed in patients where DNA samples were available. Pvalues depicted in bold for significant variables. Ó2011 Blackwell Publishing Ltd e352 E. Ruiz-Mateos et al. (a 0.6 log IU/mL decrease) (Fig. 2b). In HIV controllers, where this allele is overrepresented (Table 1), these results were reproduced and HCV load levels were even lower, with a 1.34 log IU/mL decrease (Fig. 2b). Besides, we found that HLA-B57+ subjects had a 3.6-fold increase in the proportion of spontaneous clearance of HCV in HIV controllers (43% in HLA-B57+ vs 12% in HLA-B57)subjects, P= 0.101, chi-square test). On the other hand, HLA-B35 has been traditionally associated with disease progression in HIV infection [5]. However, the association of HLA-B35 with HCV is not clear. In the light of the results with HLA-B57, we also wondered whether HLA-B35 was reciprocally associated with higher levels of HCV. We found that HLA-B35+ HIV controllers had higher HCV load than patients negative for this allele (Fig. 3a). An additional interesting finding with HLA-B35 was based on the association of HLA-B35 with HIV disease progression [5]. This idea prompted us to analyse whether HLA-B35+ was associated with the loss of the capability to control HIV in controllers. The proportion of HLA-B35+ HIV controllers who lost the capacity to control HIV viraemia was higher than the proportion of HLA-B35+ HIV controllers who persistently controlled the virus (Fig. 3b). This loss of control was assumed when both elite controllers became viraemic controllers and viraemic controllers became noncontrollers. When loss of HIV control was only considered for elite controllers (from elite controllers to viraemic controllers), this tendency was also maintained (43% of HLA- B35+ subjects lost control compared to 8.3% of HLA-B35+ elite controllers with persistent control, P= 0.062, FisherÕs exact test). Elite controller status, HCV genotypes, HLA-B57 and HLA-B35 are independently associated with HCV load levels Multiple linear regression analysis was performed to pinpoint which variables were independently associated with HCV Fig. 2 Influence of HLA-B57 on HCV viral load. HLA typing and HCV load were available in 54 patients in the comparison group and in 25 patients in the HIV controllers group. (a) HLA- B57+ patients showed lower levels of HCV load than HLA-B57)patients. (b) This was true for both patients in the comparison and HIV controller groups. Mann–Whitney U-test was used. Values represent median and interquartile ranges. Fig. 1 Influence of HIV controller status on HCV load. HCV viral was available in 81 patients in the comparison group, 34 patients in the HIV controller group and in 21 patients in the elite controllers group. HIV controllers, including the subgroup of elite controllers, showed lower levels of HCV load than the comparison group. Values represent median and interquartile ranges. Ó2011 Blackwell Publishing Ltd HCV replication and HIV controllers e353 load. Variables included in the model were elite controller status, HCV genotype 1, HLA-B57, HLA-B35, CD4+ T-cell counts, time since diagnosis, sex and naı ¨ve for antiretroviral treatment. This analysis showed that when we controlled for the above potential confounders, the variables elite controller status, the absence of genotype 1 and HLA-B35 and the presence of HLA-B57 were independently associated with lower HCV load (n= 63, R 2 = 0.476, Table 2). This analysis showed no colinearity among variables which means that each one exerts an additive effect in lowering HCV load. DISCUSSION Results presented herein show how Caucasian HIV controllers exhibited better control of HCV replication, measured as plasma HCV loads, when compared with a comparison group. Furthermore, they presented a different distribution of HCV genotypes with an overrepresentation of genotype 3. Genetic determinants, associated with HIV progression, were also associated with HCV loads. These findings support the idea that some common host mechanisms are involved in the defence against these two persistent infections. The absence of differences in the proportion of spontaneous HCV clearance between the comparison group and HIV controllers agrees with previous work that showed how different immune mechanisms are operating against HIV, cytomegalovirus and HCV, at least in relation to CTL responses [6]. However, we found that HIV controllers had lower levels of HCV loads. Hence, both persistent infections should share common control mechanisms although at a different level. On the other hand, the association between loss of HIV control and higher levels of HCV load can be related, although in a different scenario, with previous observations that showed increased disease progression in HIV–HCV-co-infected patients with higher viral load [7]. The clinical implications of this lower HCV load in the response to HCV-specific treatment and the natural history of HCV in HIV controllers are unknown at the moment, although recently no differences in liver histology between HIV controllers and non-HIV controllers with chronic HCV infection were reported [4]. In this same work, significantly lower CD4 cell counts in HIV controllers without chronic HCV infection compared to HIV controllers with chronic HCV infection were found. These data are in line with those presented in the current work. In this study, another peculiarity of the HIV controller cohort was the different distribution of HCV genotypes compared to the general HIV-infected population. The reasons for this different distribution are unknown at the moment. One possible explanation could be related to the different distribution of specific HLAs in elite controllers. In this sense, a variable influence of HLA on various genotypes has been reported [8]. We can also hypothesize that the presence of genotype 1 would favour the loss of HIV control, and hence, this genotype could be underrepresented in the HIV controller group. This idea could be supported, although at a different level, by the higher progression rate in Fig. 3 Influence of HLA-B35 on HCV load and loss of HIV control. HLA typing was available in 40 patients and both, HLA typing and HCV load were available in 25 patients in the HIV controllers groups. (a) HLA-B35+ HIV controllers showed higher levels of HCV load than HLA-B35- patients. Mann–Whitney U-test was used. Values represent median and interquartile ranges. (b) There was an overrepresentation of HLA-B35+ patients who lost HIV control. FisherÕs exact test was used. Table 2 Results of the multivariate analyses PB (95% CI)* Constant (Log HCV load) 6.0 (5.6, 6.2) Elite controller status <0.0001 )0.577 ()0.973, )0.180) HCV Genotype 1 0.005 0.690 (0.336, 1.044) HLA-B57 0.024 )0.625 ()1.164, )0.087) HLA-B35 0.025 0.570 (0.073, 1.066) CD4+ T-cells/mm 3 0.654 <0.001 ()0.001, 0.001) Time since diagnosis 0.494 0.012 ()0.023, 0.047) Female sex 0.680 0.098 ()0.375, 0.570) Naı ¨ve for ART 0.401 0.298 ()0.408, 1.003) Multivariate linear regression analysis, Pvalues depicted in bold for significant variables; *B, Regression coefficient, changes in the estimation of the mean of the Log HCV load (constant) when the rest of the variables do not change. CI, confidence interval; ART, antiretroviral treatment; Dependent variable expressed as estimation of the mean. Ó2011 Blackwell Publishing Ltd e354 E. Ruiz-Mateos et al. HIV-co-infected people with HCV genotype 1 found in some studies [9,10]. The lower HCV loads found in HLA-B57+ subjects point once again to some common immunogenetic host mechanisms involved in the defence against these two viruses. It has been widely documented that the HLA-B57 is overrepresented in HIV controllers [11]. The reasons why this host genetic factor has been associated with HIV control are beginning to be understood [12]. Recent studies point to the presence of a strong CTL response and a viral fitness cost to the virus that arises from HLA-B57 escape mutations [13]. This process could also take place in HCV infection and might be associated with a common immunological cellular mechanism such as efficient elimination of infected cells by HLA-B57-restricted CTLs recognizing distinct epitopes from HIV or HCV. Studies relating HLA-B57 with HCV are scarce, and an association has only been found between the presence of HLA-B57 and the spontaneous clearance of HCV [14–16]. This agrees with our results, although the contrast was not statistically significant because of the difficulty in finding a high number of patients who were HIV controllers, HLA-B57+ and exposed to HCV. Following this reasoning, we found the opposite effect with HLA-B35, i.e. higher HCV loads in HLA-B35+ HIV controllers. In relation to this observation, HLA-Cw04 has been shown to be associated with HCV persistence and this allele was in strong linkage disequilibrium with HLA-B53 and HLA-B35. However, only HLAB-53 and the Cw04-B-53 haplotype were weakly associated with viral persistence [15]. This finding again shows how a genetic factor associated with progression in HIV infection shares features with HCV infection, at least in relation to HCV loads. Apart from this, the additional observation of the association between the presence of HLA-B35 with the loss of HIV control is in agreement, although in a different scenario, with the disease progression in HIV infection [5]. Apart from these HLAs, other genetic factors, such as IL28B polymorphism, which has been shown to be associated with HCV clearance, genotypes and response to treatment, should be explored to define their association with the lower HCV loads seen in Caucasian HIV controllers [17–20]. A limitation of this work is the low number of HLA-typed patients, and another inherent limitation of this work is its cross-sectional nature. In this sense, studies have shown loss of control and/or HCV re-infection when patients were followed longitudinally particularly in HIV-co-infected patients [21,22]. However, we have to assume that the re-infection frequency in these patients is anecdotal because they have abandoned injecting drug use and HCV re-infection by sexual transmission is rare. Different variables as CD4+ T-cell counts [23], sex [24,25] and even the influence of antiretroviral therapy [26] have been associated with HCV load. These variables were statistically different between the comparison group and the HIV controller groups, the same happened with the time since diagnosis. To avoid potential confounders, all these variables were controlled for by their introduction in a multivariate model together with elite controller status, HCV genotype 1, HLA-B57 and HLA-B35. Only elite controller status, the absence of HCV genotype 1 and HLA-B35, and the presence of HLA-B57 were independently associated with the lower HCV load. The reason why elite controller status per se is independently associated with lower HCV load is not yet clear. One limitation of the present study is that we do not provide experimental data to answer this question. However, we can hypothesize, based on observations in long-term nonprogressors (LTNP), how common immunological control mechanisms against these two persistent viruses could explain the lower HCV load found in HIV controllers. In fact, a strong HCV Th1 cell response with higher HCV peptide recognition has been found in LTNP when compared to progressors [27,28]. In a recent study, Sajadi et al. [4] have shown an increased rate of HCV spontaneous clearance in Ônatural viral suppressorsÕ(<400 HIV-RNA copies/mL), when compared to HCV–HIV-co-infected and HCV-mono-infected control groups. In that study, the cohort was entirely composed of African-Americans. This fact could explain the discrepant results found in our cohort composed mainly of Caucasians. Another difference is that variations in HCV load between HIV controllers and reference groups were not found. This could be explained, apart from the race differences, by most of the subjects being HCV genotype 1 (approximately 90%) and, as we have shown, HCV genotype is associated with HCV load. In this regard, it is important to mention that HIV controllers from the Sajadi cohort had almost fourfold higher HCV load than elite controllers from our cohort owing to the prevalence of genotype 1. Although in this previous work genetic factors were not analysed, our findings underlined how different race groups of HIV controllers with different genetic and immunological backgrounds can influence HCV load levels. In summary, the results presented herein show how Caucasian HIV controllers exhibit better control of HCV replication when compared with a comparison group. This is associated with a different distribution of HCV genotypes, absence of HLA-B35 and the overrepresentation of HLA- B57. Additional immunological mechanisms should be explored to explain how HIV controller status influences partial HCV control to design future therapeutic strategies against both infections. ACKNOWLEDGEMENTS This work was supported by Redes Telema ´ticas de Investigacio ´n Cooperativa en Salud (RETICS; 2006, Red de SIDA RD06/0006/0021 and RD06/0006/0033, 2007–2010), Consejeria Salud Junta de Andalucia grants CS2008/PI0270 and CS2009/PI0066, Fondo de Investigacion Sanitaria grant PS09/00126 and by a research grant sponsored by Ó2011 Blackwell Publishing Ltd HCV replication and HIV controllers e355 Janssen-Cilag [Correction added on 9 March 2011, after online publication: Ô(RETICS; 2006, Red de SIDA RD06/ 0006/0021, 2007–2010)Õwas corrected to Ô(RETICS; 2006, Red de SIDA RD06/0006/0021 and RD06/0006/0033, 2007–2010)Õ]. E. R-M. was supported by Fondo de Investigacion Sanitaria grant CP08/00172. K.M. was supported by RETICS; 2006, Red de SIDA RD06/0006/0021, 2007– 2010, MC. R-S. was supported by Fundacion para la Investigacion y Prevencion del SIDA en Espan˜a (FIPSE) grant 36624/06 and by Fondo de Investigacion Sanitaria grant PI06/0915 and S. F-M has a grant from the Fondo de Investigaciones Sanitarias (FIS06/00176). We particularly acknowledge the patients in this study for their participation. The Biobank of the Spanish AIDS Research Network is supported by Instituto de Salud Carlos III and Spanish Health Ministry (Grant no. RD06/006/ 0035). We thank Dr Amalia Martin and Dr Francisca Gonzalez-Escribano and staff from the Microbiology Service and Immunology Service at Virgen del Rocio University Hospital, respectively, for their technical assistance. We also thank Marien Gutierrez, Francisca Cano and Magdalena Rodriguez for their support to the patients. We also acknowledge Dr Nieves Nogales and Dr Julio Sola for their clinical support. We also thank to Maria Tavares for critical reading of the manuscript. CONFLICT OF INTERESTS The authors do not have any association that might pose a conflict of interest. REFERENCES 1 Lambotte O, Boufassa F, Madec Y et al. 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Specific human leukocyte antigen class I and II alleles associated with hepatitis C virus viremia. Hepatology 2010; 51: 1514–1522. 17 Montes-Cano MA, Garcia-Lozano JR, Abad-Molina C et al. Interleukin-28B genetic variants and hepatitis virus infection by different viral genotypes. Hepatology 2010; 52: 33–37. 18 Suppiah V, Moldovan M, Ahlenstiel G et al. IL28B is associated with response to chronic hepatitis C interferon-alpha and ribavirin therapy. Nat Genet 2009; 41: 1100– 1104. 19 Tanaka Y, Nishida N, Sugiyama M et al. Genome-wide association of IL28B with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis C. Nat Genet 2009; 41: 1105–1109. 20 Thomas DL, Thio CL, Martin MP et al. Genetic variation in IL28B and spontaneous clearance of hepatitis C virus. Nature 2009; 461: 798–801. 21 Grebely J, Conway B, Raffa JD, Lai C, Krajden M, Tyndall MW. Hepatitis C virus reinfection in injection drug users. Hepatology 2006; 44: 1139– 1145. 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Clin Infect Dis 2002; 35: 873–879. 27 Alatrakchi N, Di Martino V, Thibault V, Autran B. Strong CD4 Th1 responses to HIV and hepatitis C virus in HIV-infected long-term non-progressors co-infected with hepatitis C virus. AIDS 2002; 16: 713–717. 28 Valdez H, Carlson NL, Post AB et al. HIV long-term non-progressors maintain brisk CD8 T cell responses to other viral antigens. AIDS 2002; 16: 1113–1118. SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. HCV load and loss of HIV control. HCV load was available in 21 patients in the elite-controller group. Some elite-controllers lost the capability to control HIV; these patients showed a tendency to have higher HCV load just before the loss of control compared to elite-controllers who did not lose the control of HIV. Mann– Whitney U-test was used. Values represent median and interquartile ranges. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article. Ó2011 Blackwell Publishing Ltd HCV replication and HIV controllers e357 B R I E F R E P O R T IL28B Single-Nucleotide Polymorphism rs12979860 Is Associated With Spontaneous HIV Control in White Subjects K. Machmach,1,3 C. Abad-Molina,2M. C. Romero-Sánchez,1,3 M. A. Abad,1,3 S. Ferrando-Martínez,1,4 M. Genebat,1I. Pulido,1P. Viciana,1 M.F. González-Escribano,2M. Leal,1,a E. Ruiz-Mateos,1,3,a and the HIV Controllers Consortium of the AIDS Spanish Network 1 Laboratory of Immunovirology, Biomedicine Institute of Seville, Clinic Unit of Infectious Diseases, Microbiology and Preventive Medicine, and 2 Immunology Service, Virgen del Rocío University Hospital, IBiS/CSIC/SAS/University of Seville and 3 Biochemistry Department, Virgen del Rocío University Hospital, Seville; and 4 Laboratory of Molecular Immuno-Biology, Gregorio Marañón, General University Hospital, Madrid, Spain The single-nucleotide polymorphism (SNP) rs12979860 near the IL28B gene has been associated with the spontaneous clearance of hepatitis C virus. We sought to determine whether this SNP could be associated with the spontaneous control of human immunodeﬁciency virus (HIV) infection. We studied the prevalence of the IL28B CC genotype among 53 white HIV controllers, compared with the prevalence among 389 HIV-infected noncontrollers. We found that the IL28B CC genotype was independently associated with spontaneous HIV control (odds ratio [OR], 2.669; P= .017), as were female sex (OR, 7.077; P≤.001) and the presence of HLA-B57 and/or B27 (OR, 3.080; P= .017). This result supports the idea that common host mechanisms are involved in the spontaneous control of these 2 chronic infections. Keywords.HIV controllers; IL28B, and SNP. The spontaneous control of human immunodeﬁciency virus (HIV) infection is observed in a rare group of individuals who have become known as HIV controllers. These individuals are able to maintain undetectable or very low viral loads in the absence of antiretroviral treatment over a long period [1]. Several genetics factors have been associated with this control, including the overrepresentation of different HLA antigens, such as HLA-B57 [1]. Curiously, the presence of HLA-B57 has also been related to the spontaneous clearance of hepatitis C virus (HCV) infection [2], suggesting shared mechanisms of viral control for both infections. We have previously reported the ability of white HIV controllers to maintain lower HCV loads than other noncontroller HIV-infected subjects, as well as an association of HLA-B57 with lower HCV levels in HIV controllers [3]. Recently, a single nucleotide polymorphism (SNP) near the IL28B gene, which codes for interferon λ3 (IFN-λ3), has been associated with the spontaneous clearance of HCV [4] and with sustained virological response (SVR) after HCV-speciﬁc treatment [5]. Although it is still unknown how this SNP affects the antiviral activity of IFN-λ, several studies have demonstrated the antiviral activity of IFN-λ against different viruses, including HIV [6]. However, no association was found between these protective alleles and spontaneous HIV control in African American individuals [7,8]. We wondered whether this IL28B SNP would be overrepresented in white HIV controllers, and, thus, our aim was to analyze the association of the IL28B SNP with the spontaneous control of HIV infection. METHODS All patients included in this study came from Spain and were white. The HIV controllers included in this study came from the HIV-infected patient cohort of the Infectious Diseases Service of Virgen Del Rocío University Hospital (Seville) and from a National Multicenter Cohort from the HIV Controllers Consortium of the AIDS Spanish Network (some of these subjects came from HIV-Infected Long-Term Nonprogressors Cohort and the prospective HIV-Infected Patients Cohort, both from the AIDS Spanish Network). The sample included 53 HIV controllers, deﬁned as subjects who tested positive for antibodies against HIV had a plasma HIV-1 load of <1000 HIV-1 RNA copies/mL in the absence of antiretroviral therapy (ART) for at least 12 months, and had at least 3 plasma HIV-1 load determinations during this period. Thirtyfour of these HIV controllers were naive to ART; the remaining 19 patients had received ART, with a median interval of 4.42 years (range, 2.76–7.83 years) between their most-recent use of ART and enrollment in the study. The HIV controllers Received 26 April 2012; accepted 11 September 2012. a M. L. and E. R.-M. contributed equally to the study. Correspondence: Ezequiel Ruiz-Mateos, PhD, Immunovirology Laboratory, Infectious Diseases Service, Biomedicine Institute of Seville (IBIS)/Virgen del Rocio University Hospital, Laboratory 211, Avd Manuel Siurot s/n 41013, Seville, Spain ([email protected]). The Journal of Infectious Diseases © The Author 2012. 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DOI: 10.1093/infdis/jis717 BRIEF REPORT •JID •1 Journal of Infectious Diseases Advance Access published December 13, 2012    (& 5)E2!''!+F!""FG8 8''''009*NA A0''!+F!""FGA'''0A0680' 95* 40 N 4  ''0 4 5)E2!''!+F!""FGO/  A0 A !L<6'86454''                        '  )*+C 0   A 60 ''!+F!""FG I<<  O/J   B'@A0 52D'F.FD+G 0 8 A0'  6  5)E2!  0 5)EP! A' 'A 0   '0  ''0 A0 9* 052D0 8NAA05)E2!''!+F!""FG'''0 A0680'95*40'%0'  ,&  00 4 DD ' 800 95*40 800' 6 4 006 000A06E'8'A6 0  ! < 6' 54 ''  95* 6  A   -C<80454''A'60'4O/60 I<<;<<C !FQ: <<;O/C F+Q  O/;O/C F"QJ 5)E2!<< 60 A' 0   6 ''0 A0 9* '80'  R8SGG" TS".+ F"Q5S +.F"FU 5)E2!O/;O/ R8SGGG TS!G++ F"Q5SFF"++F+.U  R8SGGG. TSG+ F"Q5SG.U  ! < R8SGGG TSGFF" F"Q5SGFFGFFDU0095*6A80 ''008'0 54''''5)E2!<<60R8SGG!!S""D!F"Q5S"! G!U  95* 6 R8VGGG S"".. F"Q5SD+G "U A 80  ''0 0 ! <  0'  6' A0 ! < W"G ;X     %C < ''0 4 5)E2! 0 8' A0    80' 0 0 95* '' 8'' 80 0  '0 '' '0440640'<045)EPP!''40 A06804080060'0 9*0'95* 40     /A ''0 '0' I/NJ 04  ' 0 8 8'IE1J'F.FD+G0'F8'00A '404PI5)EPJ'BA'52DRU<'F.FD+GA' ''0 0 0 '80'  000  4 800'  6' I9*J409*4095*40800'R!U00 A '0 0 06 880' 4 52D;5)EP 6  '00   '0 440 6'  95* R".U N 'A 00 0 46 0 8 52D A' ''0 A0 0 '80' 0 4 6  95* 40RDU 0 A0'0 00'060 ''!+F!""FG I<<  O/J A '   B '@ A0 52D 'F.FD+G '  '6 RF GU ''!+F!""FG RO/U '  4'40 60 0A 04   I5)E2J  5)E2 ' 00 0'  6  '05)E2!05)EP!A'00'0 '05)EP RFU 0 0 6  0 40 4 0' 80  BA 5)E2! ''!+F!""FGO/''004060''''0A0080 452DRGUN80'F.FD+G''!+F!""FG''0 ''0 A00'80'49*400'8'09*000RF UN0''!+F!""FG'''0A0680' 95*40800''BA 5  A0  0' 0 0 ' 8606 0 0B 00 ''!+F!""FG 8 8' ' ''0 A0 ''8''080'  0 6' 40'95*<408'0'0 A'0 0''040    5)E2! 60' A0   6 80'  95*40 00608 Y6800'    .,  800'0''0 AA00095*40 800' 0 4 0 540' ''' 6 0 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G.  !<;  VGGG  GFF!  GFFGFF+  GGG  GFF"  GFFGFFD  295*E 8';  GGG  DG!  !+!".!  G!F  "!+  G..! 5)E2!''!+F!""FG <;< O/;< O/;O/ GG+3  GGF GGG!   G. !.G   GDFGK!D! +++"  GGG3  GGG GGG    "!D !G++   "GKFFF F""++F+.    18$#11 %41$   60060'' ''A84'!<' 80 6' " '%0' A0 ! W"G ;X A C !D A <<;<<: !" <<;O/   O/;O/ 34C 4: ''0 A0 0 '84 5)E2!''!+F!""FG0 8'86VGG"A''00'0 '40 60'C''440:5406E088          !<  60 ''060 ''   0'0'   F"Q5   F"Q5  /IJ GFG" !!G. +.FG.+.+     I 'J  GGG  !DF.  .F!DD  GG.  "+!  ".GG!  54'' GGG +! F.""+"+F G+DG F"+! .G+DD""F  295*E8';  VGGG  ".DD DD!F.!F  VGGG  ""FD.  D++""! 5)E2!''!+F!""FG <;< O/;< O/;O/  4 3   GG GG.    ."+D" DDFF    ".F"."." D"DDDG!    GG!. G+    ".+D !.D    !FDGD"D !GG.!"!!         IFNL4-TT/TT IFNL4-TT/∆ G IFNL4-∆ G/∆G 0 5 10 15 20 25 0.085 0.090 0.002 0.021 AIDS-defining illness (%) A) B) IFNL4-TT/TT IFNL4-TT/∆G IFNL4-∆G/∆G 0 250 500 750 1000 1250 1500 1750 2000 0.070 0.7750.011 0.007 CD4 T cell counts (Cell/ul)             )C J < 80 4 54 '' A' 60  ' 4 O/ 60C <<;<<C F;D": <<;O/C +;+.  O/;O/C .;+ J  ! < 6' A '65)E2!<<;<<80'4O/60C<<;<<C!+G;X5[C "+.:<<;O/C!";X5[CD""D"O/;O/CCD;X5[CF!"GF 00'0'@0'0<'00A'0 06' 0'6''806 86VGG"A''00'0 '40                                  ! "      #       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  ! "!  %#   "    '(.     "#"%"  '(.  %         # !" " "    '(.    '"    '"#"%4"" '(.!%  3      %  5%  "  "0""" )16,#   "   ""%*   ".."!7869#122(:    "!7  . "!7 ;69#122  ( : #""#"0 ""%""3% 5% "!7      "  "  %   "!7#  %   "!7 "   #  %""<.0#!!" """%4. ."%'(.="")19,  #""""    5%    "!7  "   %   "!7"!"!!"! ! "  !  .      .'(.="! %"%5%" "    7    "  % "  '(.=   ")1>#1*," "%""'(.=   <#    '(."  % ?% )91,     "   < # % "  """%*@)11,# #  . "" %*@    "   #   !  " ! ! ""  <.0. " %    !  !"     !"  " . #""%*@ "A#""    B    "   !"      "" %*@     1      0   "#    ! '(.=# %   ! "   #  9*@      ""  C#  !  "  9*@!!'(.= 0!"%."" "0."#5% """%5%"!7 ""'(.=.6"! #'(.="!%"!7   ! "  "   #      "!7    .   % -" " C.%692!  " %* )1B# 1+, "#     "  %   "!7!""#D"%* %"%"% "%*% '"#%* %  % !%*% "!7" "" #%"!7"!%*. %4#5%"!7 !%"" "   %*. # "   <.0  %4#    "#""D"" %"    "   "  "   ""  #  D"      "&   !              %"%"#" 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.6F922+A>6+O0**.+ B?7#5<#RP#5. <.("A*B1O>12.* +N"F#&!%N#%#<"%/@ D" .  "  " 922/A90O6220.96 /-'#QS#N<-Q#7.6 <F9262 >>2O6116.+ 0FC.%#<!C%#<!&#!%/ "" -(F$F922+A62*OB++B./6 62 "C.N 5# '.NC F# N ?#      %1+@%9/..#. %*@..%"A/O*+6./6 66 F  N#%N $  "   "#""#"A9>*O6*9.B9 69   # NC  .       " ("922*A1O0/+.01 6>&Q#N#!!#"!N#- F!E 5   " "       (? Q%60/>A>6O9/1.0 6*NQ# F#!!%#P-(4". " !""%4(" ..."."". "922BA66+O>/.*B 61 < E # " ' <  !    "    %"&"9229A6*O*>9.B 6B F" # F # 4 '#     " "  6 .".FE9226A0/O02B.69 6+FE#F#34#<.  O         -% " 922*A>90O9/2./ 6/F%#T<" %"NN9229A9O+>0.1B 60'F#F#"F#.".     "  "   " 9"%"9226A626O926.62 92 N E# N E# ? #   -    " "O       #   #""E922/A669O*10/.B2/ 96EQN#F 3#"<5#%*@"    "      Q 5 N 922*A922O+*0.10 99F4#?#?F#-< "%*@..6E9220A66*O>/1*.B> 9> -  Q# ? F# E N#   " ! %69>@   %66@    "!   .6  E 9226A0/O>26B.96 9* -  Q# " <# ? #   5         !" "   Q   922*A602O6//0.09 913#4#F4N!"Q-"   %  <.  %  ."   ! .   +." '(. - (  F $ F  922+A62*O6+*1>./ 9B < N# N %#  -&#     6.   ""%*F ""922*A92O6+1./9 9+FQ-#NF#-N#%<."! F600+A9++O/6/.96 9/ !" Q-# E # 4Q%#   ('. ."  <  .6.      " ! . E9221A621O9*1/.B* 90!"Q-#4Q%#E#%*@."!"  " .6O    6 .# <:1. E9221A62BO>19*.>6 >2 !" Q-# ( Q# E #    "   !"      "  .    "".D"9220A9>O>1.*2 >6 !" Q-# 3# E # " ('.. "    %*@   ! .6O     '(." -(F$F9221A629O6>0+*.0 >9?P#?"C#Q#F"%   <.E 1+      4A6*2OB/B.B0B6 >>?-#%O#! "4"AB6O699B.>* >*E4#E(#!. NF#5% "  " "# . %/@    Q  922/A/9O6226+.>6 >1 &   5# ?  :% .O #  ##"%":FA/O69. 99 >BF7NN#F (#%.6 ""A9*O6B/0.01 >+  <#  %<# N N-#   4   <9/E  "%"("9220A*B6O+0/./26 >/4#'Q#Q#4<9/E% ."("9220A*B6O>00.*26 >0"3#3S#P<#<!!"" "6Q9220A/>O>/>*.*9 *2F7NN#F (##<9/E %FA*O9/9.* *6 F N# ?  4# % #   - " .9/E    % " # !"   !"  .6      .:"A91O>/1.+ *9(#%#(F#" .9/E ""!.%*A91O9B0.+6 *> - ".& <# N" E# 3#     "  '(<> <9/E'(<* %"(4A*1O6B*.+6 **E!F##%#<9/E:.4 %Q5NA962O6620.6B *1 ? E# (  # E  %#     '(<*   .!%." % *B -#?"N#-N#'(<*.4 F"%%"Q *+FE# EN#'#'"7"F<Q. 7""#"# 9221A>*>O91B.BB */ F '-# ?  (# F N#    "     6 .""!F6000A9/*O6/>1.+ *0 4" 4#  N%# '" <# " # E" Q  <" PQ  " <.> %*2.Q5N600+A6/1O6626.66 12E3#%N%N-.." "F"9221A6+O91>.B6 16NQ#Q"QF#%3#" -<F&922+A9O*1/ 19 &UE N# N &# F! <#   F      "       '(.."  "Q%A696O62//.626 1>N"."N#. ""%E9221A/>O1+/./> 1*4"?E#%#EE#F$!%5" '(. " !         .6Q"922*A6+>O+9B0.+B 11%Q#-5#"%#!    . "" %*@    "  " .."QA/*O9+B9.+> 1B !" Q-# F 4N  !   692:%*   !.6"VF922BA/O>./ 1+ E F# N? ?# F ! N#   5  .6  . .(Q% 9221A661O>9B1.+1 1/ F E# FC.R"7 #  <#   " " " ".D"% """Q600+A9BO6.1 10Q-#&!%N#%#%%/@  " " "#  % "#  "    D"# "# #  " .9 Q9220A/>O9+9/.*9 B2 " # Q # - ?#       % " 6>"" ."" 9220A12O626+.90 B6F!%#-#-(#EF<%! %"""" Q600+A6+1O6B*./ B9 P 3#  F# < # < F   5F 5   % " %%.6Q9221A606O*.62