A rare (less than 1%) subset of HIV-infected patients, referred to as ‘elite’ controllers, maintain undetectable plasma HIV RNA levels in the absence of antiretroviral therapy (ART) [1,2]. Although controllers often also meet standard definitions of ‘long-term nonprogressors’  (i.e., they do not lose CD4+ T cells and do not develop AIDS), many nonprogressors have detectable viral loads and a small subset of controllers exhibit immunologic and clinical progression .
HIV infection of the cerebrospinal fluid (CSF) compartment (leptomeninges and ventricles) is pervasive in typical antiretroviral untreated patients [5–8]. Although untreated infection of the CSF may be asymptomatic, in some patients, it eventually evolves into HIV encephalitis and AIDS dementia complex (ADC) [9,10]. Central nervous system (CNS) immune activation accompanies this infection and may be an important mediator of neurological injury; this immunoactivation may also persist in the setting of systemically suppressive ART [11,12].
The extent to which controllers harbor CNS HIV infection with its associated immunoactivation has not been examined. Controllers may serve as a model population for understanding the relationship between levels of systemic HIV, CNS viral burden, and neuroimmune activation. We, therefore, examined the virological and immunological features of CSF in eight well characterized controllers, comparing them to three groups of individuals: untreated HIV-infected (HIV-positive), treated HIV-positive with plasma HIV RNA levels less than 50 copies/ml, and HIV-uninfected (HIV-negative).
Study participants and evaluations
Individuals were enrolled in the Sentinel Neurological Cohort (SNC) at San Francisco General Hospital between 2001 and 2008 . Controllers were infected for at least 5 years, maintaining plasma HIV RNA levels below less than 50 RNA copies/ml. Comparison individuals included HIV-negative, HIV-positive treated with plasma HIV RNA levels less than 50 copies/ml, and untreated HIV-positive with blood CD4+ T-cell counts more than 200 cells/μl to exclude those with advanced immunosuppression. Individuals exhibited no active HIV-related neurological disease. Protocols were approved by the UCSF Committee on Human Research, and informed consent was obtained from all participants. CSF was obtained solely for study purposes with concurrent phlebotomy along with clinical assessments as described elsewhere [13–15].
HIV RNA levels were measured in cell-free CSF and plasma using the ultrasensitive (lower limit of detection = 50 copies/ml) Amplicor HIV Monitor assay (versions 1.0 and 1.5; Roche Molecular Diagnostic Systems, Branchburg, New Jersey, USA). Samples with results below the lower limit of detection on the ultrasensitive assay were re-tested using the ultra-ultrasensitive modification with a lower detection limit of 2.5 copies/ml . CSF cell counts and albumin concentrations, blood CD4+ and CD8+ T-lymphocyte counts, and albumin concentrations were measured in the San Francisco General Hospital Clinical Laboratory using standard techniques. Concentrations of CSF and plasma neopterin (American Laboratory Products Company, Windham, New Hampshire, USA) and CSF IP-10 and MCP-1 (R&D Systems, Minneapolis, Minnesota, USA) were measured by commercial immunoassays.
Nonparametric statistics were performed using Stata/SE 9.0 and Prism (version 5; GraphPad, La Jolla, California, USA) software. Group differences were analyzed using Kruskal–Wallis and posthoc testing with α adjusted to 0.025 for multiple comparisons.
Study participant characteristics
Eight controllers were compared with 26 HIV-negative participants, 25 untreated, viremic HIV-positive participants, and 23 treated HIV-positive participants with plasma viral suppression (Table 1). Half of the controllers in this sample were women compared with a majority of male participants in the other groups. Controllers tended to be older than the participants in the other groups (P = 0.051 for comparison of medians) and had higher median blood CD4+ T-cell counts than the other HIV-positive groups (P < 0.001 for each comparison). The controllers also had higher CD4+ T-cell counts than HIV-negative individuals, but this difference was not significant. Controllers had a median known duration of infection of 17 years [intraquartile range (IQR) 12.8–18.3] and were treatment-naive, except for two participants who received ART for less than 1 year, more than 10 years prior to study entry.
Undetectable cerebrospinal fluid HIV RNA in controllers
HIV RNA levels in both plasma (by definition) and CSF of the controllers and treated HIV-positive participants were undetectable by the ultrasensitive assay, and, therefore, all samples from these groups were re-run using the ultra-ultrasensitive method. CSF HIV RNA levels were below the 2.5 copies/ml detection limit of this assay in all eight elite controller participants. This differed from the plasma HIV RNA that was detected in three controllers (range 13–121 copies/ml). In controllers, median CSF HIV RNA levels were equal to those of treated HIV-positive individuals and substantially lower than those of untreated HIV-positive individuals (Fig. 1a). The relationship between CSF HIV RNA levels and group thus paralleled the a priori relationship between plasma HIV RNA level and analysis group.
Lack of cerebrospinal fluid inflammation and blood–brain barrier compromise in HIV controllers
Median CSF white blood cell (WBC) counts in controllers were similar to those in the HIV-negative and treated HIV-positive groups (Fig. 1b) and lower than those measured in the untreated HIV-positive individuals. Similarly, soluble biomarkers of immune activation that are commonly elevated in HIV infection were also not different from uninfected or treated, aviremic participants. This included neopterin, a biopterin precursor derived from GTP that serves as a biomarker of macrophage immunoactivation , which is typically elevated in the plasma and CSF throughout the course of untreated HIV infection and may remain elevated in CSF in the face of seemingly effective ART [12,18]. Controllers had levels of plasma neopterin comparable to that observed in HIV-positive and treated HIV-positive individuals but significantly lower (P = 0.0006) than those in untreated viremic participants (data not shown). Likewise, CSF neopterin in controllers was not distinct from HIV-negative or treated HIV-positive individuals but lower than that in the untreated HIV-positive individuals (Fig. 1c).
CSF MCP-1 (CCL2) is elevated in HIV encephalitis and correlates with CSF HIV RNA levels, independent of the degree of plasma viremia . CSF concentrations of IP-10 (CXCL10), a key chemokine in lymphocyte recruitment which correlates with CSF pleocytosis [20,21], was also comparable in controllers and the HIV-negative and treated HIV-positive groups but lower than that observed in untreated viremic HIV-positive patients (Fig. 1d). The median CSF MCP-1 level in the controllers was comparable to that in the HIV-negative patients and HIV-positive patients on ART (Fig. 1e). The CSF: albumin ratio, a marker of blood–brain barrier integrity [22,23], also did not vary significantly across groups (Fig. 1f).
Discussion and conclusion
HIV-infected persons who are able to control HIV replication are the focus of intense investigation [1,3,4,24–28]. Although a strong adaptive immune response targeting HIV is involved with viral control, at least in some individuals , it may also have detrimental consequences, and high-level T-cell activation in controllers may be associated with both loss of CD4+ T cells and premature atherosclerosis [4,28]. A similar persistent high level of immune activation within the CNS might have neuropathological consequences. However, in the current study, we found that individuals controlling systemic HIV replication also controlled CSF HIV replication, and importantly, this viral control was not associated with evidence of intrathecal immune activation or inflammation associated with untreated HIV infection. These findings suggest that controllers are not at high risk for developing HIV-related CNS damage, either related to compartmentalized viral escape or immune activation.
The magnitude of CSF infection is typically variable across patients; however, it correlates strongly with plasma HIV infection and CSF WBC count [15,29]. CSF pleocytosis, like HIV RNA levels, is reduced, usually to normal levels, in antiretroviral-treated patients [15,30]. The reduced CSF inflammatory response in the context of ART has been presumed secondary to reduction of local antigenic stimulus or of more direct HIV-related chemotaxis. In the controllers, the absence of both virus and inflammatory response suggests that virus does not reach the CNS in appreciable amounts and does not establish local replication, or that local immunity is capable of suppressing any replication. The low level of local inflammation may suggest reduced access or replication rather than continued robust antiviral immune surveillance, although these issues require more direct assessment of local virus-specific immune responses to resolve more fully. Our findings are also consistent with the hypothesis that local CSF HIV RNA levels drive intrathecal inflammation rather than simply reflecting a ‘spillover’ of systemic immune activation.
In five of the eight controllers, CSF neopterin was above the published normal reference value of 5.3 nmol/l . However, nine of 26 of the HIV-uninfected controls also had values higher than the reference range, and the levels were similar between the two groups. Our HIV-negative comparison individuals are asymptomatic individuals recruited from the local community in an effort to match lifestyle and exposure confounders with our HIV-positive participants. The fact that intrathecal macrophage activation was similar between controllers and HIV-negative participants argues against significant macrophage activation in the CNS due to HIV infection in controllers.
Overall, the finding of CSF WBC counts, soluble markers of local immune reactions including neopterin, MCP-1, and IP-10, and CSF: plasma albumin ratios similar to those of uninfected individuals in the setting of undetectable CSF HIV RNA levels is consistent with previous reports [15,30], but novel as a characteristic of those with intrinsic, rather than treatment-induced, viral suppression.
One caveat of this study relates to sample selection among controllers. The controllers participating in this study were the result of convenience sampling and volunteering for lumbar puncture. However, it is not fully representative of the controller population, based on their elevated median CD4+ T-lymphocyte counts in comparison to a larger group of controllers followed at our institution (727 cells/μl) . Although we did not examine blood CD8+ T-cell activation at the time of CSF analysis for this study, flow cytometry assays using expression of CD38 and HLADR as an indicator of blood T-cell activation were previously performed through a separate study of these controllers . The eight individuals in this study had lower median CD8+ T-cell activation (9.3%, 8–12) in blood than the overall group of 38 elites studied (14.9%, 9–22). Hence, the question remains whether CSF immunoactivation and inflammation might occur in those controllers who harbor relatively elevated systemic T-lymphocyte activation and/or might appear in patients in this group who progress to measurable immunosuppression and clinical AIDS. We have previously reported that CSF HIV viral burden correlates with blood CD8+ T-lymphocyte activation, although the mechanism underlying this relationship remains uncertain . Further investigation into the scope of HIV-specific CD8+ T-cell responses in the CSF is necessary to understand the mechanisms and effects of HIV RNA control within the CNS in this group of controllers.
In our sample of controllers with preserved CD4+ levels and low systemic immune activation, the profile of CSF HIV infection was similar to that of HIV-negative and HIV-positive individuals with treatment-induced viral suppression and distinct from that of typical, viremic untreated HIV-positive individuals. Longitudinal studies and those focusing on neurological outcomes such as neuropsychological testing and examinations should provide further insight into the neurological prognosis of these patients.
R.W.P. has received research support for an investigator-initiated study from Merck and Co.
We gratefully thank the study participants who generously participated in these studies. We also thank the National Institutes of Health (grants R01MH62701, R01NS37660, K23MH74466, K24AI069994, and UL1RR024131), the Centers for AIDS Research at UCSF (AI27763 and MH59037), the UCSF Clinical and Translational Science Institute (UL1 RR024131-01), and the American Foundation for AIDS Research (106710-40-RGRL) for research support.
Author contributions: J.P. performed the analysis and drafted the manuscript. S.D. and P.H. assisted in interpretation of the findings. T.L. directed the virology assays. E.L. and R.H. recruited and interviewed participants. R.W.P. and S.S. conceived of the study, performed participant study visits, and edited the manuscript.
The data presented here were presented in part at the 15th Conference on Retroviruses and Opportunistic Infections; February 2008; Boston, MA; Oral Abstracts: Insights into Neuropathogenesis.
1. Deeks SG, Walker BD. Human immunodeficiency virus controllers: mechanisms of durable virus control in the absence of antiretroviral therapy. Immunity 2007; 27:406–416.
2. Walker BD. HIV controllers: an untapped source of clues to overcoming HIV infection. Res Initiat Treat Action 2007; 12:21–22.
3. Mikhail M, Wang B, Saksena NK. Mechanisms involved in nonprogressive HIV disease. AIDS Rev 2003; 5:230–244.
4. Hunt PW, Brenchley J, Sinclair E, McCune JM, Roland M, Page-Shafer K, et al
. Relationship between T cell activation and CD4+ T cell count in HIV-seropositive individuals with undetectable plasma HIV RNA levels in the absence of therapy. J Infect Dis 2008; 197:126–133.
5. Ellis RJ, Hsia K, Spector SA, Nelson JA, Heaton RK, Wallace MR, et al
. Cerebrospinal fluid human immunodeficiency virus type 1 RNA levels are elevated in neurocognitively impaired individuals with acquired immunodeficiency syndrome. HIV Neurobehavioral Research Center Group. Ann Neurol 1997; 42:679–688.
6. Gisslen M, Fuchs D, Svennerholm B, Hagberg L. Cerebrospinal fluid viral load, intrathecal immunoactivation, and cerebrospinal fluid monocytic cell count in HIV-1 infection. J Acquir Immune Defic Syndr 1999; 21:271–276.
7. Gisslen M, Hagberg L, Fuchs D, Norkrans G, Svennerholm B. Cerebrospinal fluid viral load in HIV-1-infected patients without antiretroviral treatment: a longitudinal study. J Acquir Immune Defic Syndr Hum Retrovirol 1998; 17:291–295.
8. Price RW, Staprans S. Measuring the ‘viral load’ in cerebrospinal fluid in human immunodeficiency virus infection: window into brain infection? Ann Neurol 1997; 42:675–678.
9. Navia BA, Cho ES, Petito CK, Price RW. The AIDS dementia complex. II: Neuropathology. Ann Neurol 1986; 19:525–535.
10. Price RW, Brew BJ. The AIDS dementia complex. J Infect Dis 1988; 158:1079–1083.
11. Eden A, Price RW, Spudich S, Fuchs D, Hagberg L, Gisslen M. Immune activation of the central nervous system is still present after >4 years of effective highly active antiretroviral therapy. J Infect Dis 2007; 196:1779–1783.
12. Yilmaz A, Price RW, Spudich S, Fuchs D, Hagberg L, Gisslen M. Persistent intrathecal immune activation in HIV-1-infected individuals on antiretroviral therapy. J Acquir Immune Defic Syndr 2008; 47:168–173.
13. Spudich S, Lollo N, Liegler T, Deeks SG, Price RW. Treatment benefit on cerebrospinal fluid HIV-1 levels in the setting of systemic virological suppression and failure. J Infect Dis 2006; 194:1686–1696.
14. Price RW, Paxinos EE, Grant RM, Drews B, Nilsson A, Hoh R, et al
. Cerebrospinal fluid response to structured treatment interruption after virological failure. AIDS 2001; 15:1251–1259.
15. Spudich SS, Nilsson AC, Lollo ND, Liegler T, Petropoulos C, Deeks S, et al
. Cerebrospinal fluid HIV infection and pleocytosis: Relation to systemic infection and antiretroviral treatment. BMC Infect Dis 2005; 5:98.
16. Havlir DV, Bassett R, Levitan D, Gilbert P, Tebas P, Collier A, et al
. Prevalence and predictive value of intermittent viremia with combination HIV therapy. JAMA 2001; 286:171–179.
17. Huber C, Batchelor JR, Fuchs D, Hausen A, Lang A, Niederwieser D, et al
. Immune response-associated production of neopterin. Release from macrophages primarily under control of interferon-gamma. J Exp Med 1984; 160:310–316.
18. Abdulle S, Hagberg L, Svennerholm B, Fuchs D, Gisslen M. Continuing intrathecal immunoactivation despite two years of effective antiretroviral therapy against HIV-1 infection. AIDS 2002; 16:2145–2149.
19. Cinque P, Vago L, Mengozzi M, Torri V, Ceresa D, Vicenzi E, et al
. Elevated cerebrospinal fluid levels of monocyte chemotactic protein-1 correlate with HIV-1 encephalitis and local viral replication. AIDS 1998; 12:1327–1332.
20. Kolb SA, Sporer B, Lahrtz F, Koedel U, Pfister H, Fontana A. Identification of a T cell chemotactic factor in cerevrospinal fluid of HIV-1-infected individuals as interferon-γ inducible protein 10. J Neuroimmunol 1999; 93:172–181.
21. Cinque P, Bestetti A, Marenzi R, Sala S, Gisslen M, Hagberg L, Price RW. Cerebrospinal fluid interferon-gamma-inducible protein 10 (IP-10, CXCL10) in HIV-1 infection. J Neuroimmunol 2005; 168:154–163.
22. Berger JR, Avison M. The blood brain barrier in HIV infection. Front Biosci 2004; 9:2680–2685.
23. Andersson LM, Hagberg L, Fuchs D, Svennerholm B, Gisslen M. Increased blood-brain barrier permeability in neuro-asymptomatic HIV-1-infected individuals: correlation with cerebrospinal fluid HIV-1 RNA and neopterin levels. J Neurovirol 2001; 7:542–547.
24. Emu B, Sinclair E, Favre D, Moretto WJ, Hsue P, Hoh R, et al
. Phenotypic, functional, and kinetic parameters associated with apparent T-cell control of human immunodeficiency virus replication in individuals with and without antiretroviral treatment. J Virol 2005; 79:14169–14178.
25. Saez-Cirion A, Lacabaratz C, Lambotte O, Versmisse P, Urrutia A, Boufassa F, et al
. HIV controllers exhibit potent CD8 T cell capacity to suppress HIV infection ex vivo and peculiar cytotoxic T lymphocyte activation phenotype. Proc Natl Acad Sci U S A 2007; 104:6776–6781.
26. Hatano H, Delwart EL, Norris PJ, Lee TH, Dunn-Williams J, Hunt PW, et al
. Evidence for persistent low-level viremia in individuals who control HIV in the absence of antiretroviral therapy. J Virol 2009; 83:329–335.
27. Ferre AL, Hunt PW, Critchfield JW, Young D, Morris M, Garcia J, et al
. Mucosal immune responses to HIV-1 in elite controllers: a potential correlate of immune control. Blood 2009; 113:3978–3989.
28. Hsue PY, Hunt PW, Schnell A, Kalapus SC, Hoh R, Ganz P, et al
. Role of viral replication, antiretroviral therapy, and immunodeficiency in HIV-associated atherosclerosis. AIDS 2009; 23:1059–1067.
29. Price RW. The two faces of HIV infection of cerebrospinal fluid. Trends Microbiol 2000; 8:387–391.
30. Marra CM, Maxwell CL, Collier AC, Robertson KR, Imrie A. Interpreting cerebrospinal fluid pleocytosis in HIV in the era of potent antiretroviral therapy. BMC Infect Dis 2007; 7:37.
31. Hagberg L, Dotevall L, Norkrans G, Larsson M, Wachter H, Fuchs D. Cerebrospinal fluid neopterin concentrations in central nervous system infection. J Infect Dis 1993; 168:1285–1288.
32. Sinclair E, Ronquillo R, Lollo N, Deeks S, Hunt P, Yiannoutsos C, et al
. Antiretroviral treatment effect on immune activation reduces cerebrospinal fluid HIV-1 infection. J Acquir Immune Defic Syndr 2008; 47:544–552.