Elite controllers are a rare subset (less than 1%) of HIV-1-infected individuals who maintain HIV-1 RNA concentrations in plasma below the lower limit of quantification of assays approved for clinical use (20–50 copies/ml) in the absence of antiretroviral therapy (ART) [1,2]. In some cases, individuals classified as elite controllers are infected with a defective virus [3,4], but most elite controllers are infected with strains of HIV-1, which do not contain any obvious genetic defects . This is also reflected in the fact that replication-competent virus can be isolated and cultured from elite controllers [6–8]. Certain human leukocyte antigen (HLA) class I alleles as well as CD4+ and CD8+ T-cell immune responses are associated with virus control, whereas humoral immunity does not appear to be a major mechanism behind virus control in elite controllers. No single factor, however, seems to be completely protective nor is there one that is strictly required . Despite the low HIV-1 RNA concentrations, evolution occurs over time suggesting that ongoing viral replication occurs albeit at a low level in these individuals [10,11]. It has been suggested that studies of elite controllers could guide the design of HIV-1 vaccines and HIV-1 eradication strategies [12,13]. HIV-1 eradication strategies need to take into account infected cells in other compartments of the body than the blood where HIV-1 persists.
We have previously demonstrated that HIV-1 RNA can be detected in the cerebrospinal fluid (CSF) in individuals on suppressive ART and that these levels are not affected by treatment intensification . Therefore, the central nervous system (CNS) could be a compartment that needs to be taken into account when designing HIV-1 eradication strategies. If HIV-1 vaccines and/or eradication strategies are to be based on the same mechanisms that elite controllers use for viral control, it is of interest how and to which extent elite controllers control HIV-1 infection of the CNS. We have reported that elite controllers control infection of the CNS as reflected in having CSF HIV-1 RNA concentrations below 2.5 copies/ml . Elite controllers also maintain CSF white blood cell counts, CSF:serum albumin ratios and CSF neopterin, monocyte chemotactic protein-1 and interferon gamma-induced protein-10 concentrations similar to HIV-1-uninfected individuals and HIV-1-infected individuals on suppressive therapy . To determine whether HIV-1 RNA can be found in the CSF of elite controllers at levels lower than 2.5 copies/ml, we analysed paired CSF and plasma samples from several time points in 14 elite controllers using the single-copy assay (SCA). SCA is a very sensitive assay that allows HIV-1 quantification down to 0.3 copies of HIV-1 RNA per millilitre in both CSF and plasma .
Materials and methods
We analysed CSF and plasma samples from 14 individuals (of whom eight were the same as in our previous report ) classified as elite controllers, as they had, over a period of more than 12 months, three or more longitudinal plasma HIV-1 RNA determinations below the lower level of quantification of the assay used in the clinical setting in the absence of ART (<40 copies/ml). Informed consent was obtained from all study participants, and CSF was obtained solely for study purposes with concurrent phlebotomy in the context of protocols approved by the University of California San Francisco (UCSF) Committee on Human Research. Permission for analysing the samples was also obtained by the regional ethical review board in Stockholm, Sweden.
SCA has been described elsewhere , but in brief, up to 8 ml of CSF or plasma, with a known amount of replication-competent avian sarcoma-leukosis virus long terminal repeat with a splice acceptor (RCAS) (an avian retrovirus) added as an internal standard, was ultracentrifuged and viral RNA was extracted from the pelleted virions and subjected to complementary DNA synthesis followed by real-time PCR amplification of a 79-base pair region of HIV-1 Gag or a portion of the RCAS genome. HIV-1 RNA levels were determined using a standard curve constructed with HIV-1 of known RNA copy number. To ensure that the extraction process was successful, the level of RCAS was measured using a separate standard curve constructed with RCAS of known RNA copy number. HIV-1 RNA results by SCA each represent the median of triplicate determinations.
As sample volume affects the lower limit of quantification in SCA and our sample volumes varied, the lower limit of quantification also varied. We therefore, as previously described , assigned a value of 0.1 copy below the limit of detection for negative samples to be able to compare HIV-1 RNA concentrations in CSF and plasma.
Statistical analysis was performed using Prism (Version 5.04; GraphPad, La Jolla, California, USA). Proportions were compared using Fisher's exact test. Distributions were compared using Mann–Whitney test.
In total, we analysed 55 samples (28 CSF samples and 27 plasma samples) from 14 individuals. Baseline characteristics for the study population from their first study visit are summarized in Table 1; six individuals were studied at more than one time point.
HIV-1 RNA concentrations in cerebrospinal fluid and plasma
Table 1 summarizes the results of SCA HIV-1 RNA concentration measurements, while complete results are provided in Table 2. As, as noted above, sample volume affects the lower limit of detection for SCA, we first compared the volumes for CSF and plasma samples to ensure that a difference in the volumes did not cause a bias in our measurements. There was no significant difference in the volumes (P = 0.47). The internal standard failed for three samples, and these results were excluded from analysis. This left measurements from 52 samples (26 CSF and 26 plasma) for analysis. Median and range for the measurements are shown in Table 1. The proportion of samples with detectable HIV-1 RNA was significantly lower for CSF than plasma (P = 0.02). HIV-1 RNA concentrations were also significantly lower in the CSF than in the plasma samples (P < 0.0001). Of note, a sample from an individual who had been classified as an elite controller (836) according to the study definition above had an HIV-1 RNA concentration of 189 copies/ml in plasma at the time of sampling for our study, yet interestingly maintained control of infection in the CNS with an undetectable HIV-1 RNA concentration in CSF by SCA (Table 2). For six of the 14 individuals, we had samples from multiple time points. Among the individuals with samples from multiple time points, the CSF concentrations varied between less than 0.3 and 0.6 copies of HIV-1 per millilitre and plasma concentrations between less than 0.2 and 45 copies/ml. HIV-1 RNA could be detected in the CSF in two out of six individuals and in plasma in four out of six individuals (Table 2). Individual 769 (Table 2) was of special interest, as samples were available from primary infection with the first sampling estimated to be at 13 weeks after HIV-1 exposure. HIV-1 RNA concentrations fluctuated in plasma between less than 0.5 and 41 copies/ml, while CSF HIV-1 RNA was maintained at very low levels (<0.3 to 0.6 copies/ml). Plasma measurements using a standard assay (Abbott RealTime HIV-1; Abbot Laboratories, Abbot Park, Illinois, USA) with a limit of detection of 40 copies/ml showed emergence of detectable HIV-1 RNA at the two latest time points, corresponding with the increase in viral load measured by the SCA.
We have previously reported that elite controllers control CNS HIV-1 infection well . In contrast to the previous report, this study demonstrated, using a more sensitive assay for HIV-1 RNA concentration measurement and analysing more samples from more individuals, that elite controllers can occasionally have detectable HIV-1 RNA in their CSF. We found that five out of 26 CSF samples were positive for HIV-1 RNA, as shown in Table 1. The absence of HIV-1 RNA in the CSF of most elite controllers, and the very low levels detected in the CSF of some elite controllers could be due to two different reasons: first, CNS HIV-1 infection occurs in elite controllers but is very well controlled or second, HIV-1 does not establish infection in the CNS in elite controllers, but occasionally HIV-1-infected cells migrate into the CSF from the periphery. A previous report has demonstrated that viral evolution in the plasma may occur in elite controllers with a median HIV-1 RNA concentration of 1 copy/ml , so even exceeding low levels of HIV-1 RNA in the CSF may not definitively indicate complete lack of ongoing replication within the CNS.
The HIV-1 RNA concentrations were significantly lower in the CSF than in the plasma of elite controllers (P < 0.0001). HIV-1 RNA was less frequently detected in the CSF compared with the plasma (P = 0.02). The fact that we could measure HIV-1 RNA in the plasma of elite controllers is consistent with prior reports of measurable HIV-1 RNA in the plasma of elite controllers [17–19]. Our findings that HIV-1 RNA levels in CSF are lower than in plasma are consistent with measurements in these fluids for both untreated and successfully treated individuals [14,20,21].
For the six elite controllers in whom we had samples from multiple time points, we could see fluctuating concentrations of HIV-1 RNA in plasma in four out of six sample series while concentrations were maintained at a very low level in CSF. This could be an example of viral replication generating immune-escape mutations followed by adaptations in the immune response to regain control over the infection in plasma while maintaining immune control in CSF (Table 2). One of the elite controllers (individual 769) was sampled as primary infection. In this case, we also noted fluctuating levels of HIV-1 RNA in plasma with very low levels of HIV-1 RNA in the CSF (Table 2). However, HIV-1 RNA was detectable in CSF and plasma at the two last time points, which could indicate that this is the beginning of a change in the individual's status as an elite controller. Early viral control in plasma that is lost over time has previously been described by Goujard et al..
A weakness of this study is that we did not ensure the primers and probe matched the viral strains in the elite controllers by sequencing as has been done by an earlier study using SCA to quantify the HIV-1 RNA concentration in plasma from elite controllers. In this study, a primer or probe mismatch that was thought to be significant was found in 11% of the 62 elite controllers from whom it was possible to obtain a sequence . Our reported numbers could therefore underestimate the prevalence of HIV-1 RNA in CSF and plasma.
In conclusion, using a more sensitive method in a larger study population, this study confirms our previous finding that elite controllers also control CNS HIV-1 infection well. In contrast to our previous findings, this study shows that HIV-1 can be detected in the CSF of some elite controllers, although at significantly lower levels than in the plasma. It is, however, unclear whether this means that virions are produced within the CNS or whether this is due to migration of infected cells into the CNS. These results may suggest that viral eradication strategies that mimic the natural host mechanisms underlying elite control of infection may also provide effective control of CNS HIV infection, though this will need to be tested directly.
We thank the participants who volunteered for these studies.
This study was supported by grant #108021-49-RFRL from The Foundation for AIDS Research (amfAR) and by grant R21MH096619, R21NS069219 and R01MH081772 from the National Institutes of Health (NIH).
R.W.P. and S.P. designed the study. B.S., R.W.P. and S.S. oversaw and characterized the study individual cohort. E.L., J.P., R.W.P. and S.S. collected samples and interviewed individuals. V.D. analysed samples. V.D. and S.P. analysed the data. B.S., R.W.P., S.P., S.S. and V.D. wrote the manuscript.
Conflicts of interest
R.W.P. has received funding from Merck and Co. to support an investigator-initiated research study and an honorarium from Abbott for a conference presentation.
1. Hubert JB, Burgard M, Dussaix E, Tamalet C, Deveau C, Le Chenadec J, et al. Natural history of serum HIV-1 RNA levels in 330 patients with a known date of infection. The SEROCO Study Group
2. Grabar S, Selinger-Leneman H, Abgrall S, Pialoux G, Weiss L, Costagliola D. Prevalence and comparative characteristics of long-term nonprogressors and HIV controller patients in the French Hospital Database on HIV
3. Learmont J, Tindall B, Evans L, Cunningham A, Cunningham P, Wells J, et al. Long-term symptomless HIV-1 infection in recipients of blood products from a single donor
4. Learmont JC, Geczy AF, Mills J, Ashton LJ, Raynes-Greenow CH, Garsia RJ, et al. Immunologic and virologic status after 14 to 18 years of infection with an attenuated strain of HIV-1. A report from the Sydney Blood Bank Cohort
. N Engl J Med
5. Miura T, Brockman MA, Brumme CJ, Brumme ZL, Carlson JM, Pereyra F, et al. Genetic characterization of human immunodeficiency virus type 1 in elite controllers: lack of gross genetic defects or common amino acid changes
. J Virol
6. Blankson JN, Bailey JR, Thayil S, Yang HC, Lassen K, Lai J, et al. Isolation and characterization of replication-competent human immunodeficiency virus type 1 from a subset of elite suppressors
. J Virol
7. Julg B, Pereyra F, Buzon MJ, Piechocka-Trocha A, Clark MJ, Baker BM, et al. Infrequent recovery of HIV from but robust exogenous infection of activated CD4(+) T cells in HIV elite controllers
. Clin Infect Dis
8. Lamine A, Caumont-Sarcos A, Chaix ML, Saez-Cirion A, Rouzioux C, Delfraissy JF, et al. Replication-competent HIV strains infect HIV controllers despite undetectable viremia (ANRS EP36 study)
9. Deeks SG, Walker BD. Human immunodeficiency virus controllers: mechanisms of durable virus control in the absence of antiretroviral therapy
10. O’Connell KA, Brennan TP, Bailey JR, Ray SC, Siliciano RF, Blankson JN. Control of HIV-1 in elite suppressors despite ongoing replication and evolution in plasma virus
. J Virol
11. Mens H, Kearney M, Wiegand A, Shao W, Schonning K, Gerstoft J, et al. HIV-1 continues to replicate and evolve in patients with natural control of HIV infection
. J Virol
12. Walker BD, Burton DR. Toward an AIDS vaccine
13. Deeks SG, Autran B, Berkhout B, Benkirane M, Cairns S, Chomont N, et al. Towards an HIV cure: a global scientific strategy
. Nat Rev Immunol
14. Dahl V, Lee E, Peterson J, Spudich SS, Leppla I, Sinclair E, et al. Raltegravir treatment intensification does not alter cerebrospinal fluid HIV-1 infection or immunoactivation in subjects on suppressive therapy
. J Infect Dis
15. Probasco JC, Deeks SG, Lee E, Hoh R, Hunt PW, Liegler T, et al. Cerebrospinal fluid in HIV-1 systemic viral controllers: absence of HIV-1 RNA and intrathecal inflammation
16. Palmer S, Wiegand AP, Maldarelli F, Bazmi H, Mican JM, Polis M, et al. New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma
. J Clin Microbiol
17. 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 human immunodeficiency virus in the absence of antiretroviral therapy
. J Virol
18. Dinoso JB, Kim SY, Siliciano RF, Blankson JN. A comparison of viral loads between HIV-1-infected elite suppressors and individuals who receive suppressive highly active antiretroviral therapy
. Clin Infect Dis
19. Pereyra F, Palmer S, Miura T, Block BL, Wiegand A, Rothchild AC, et al. Persistent low-level viremia in HIV-1 elite controllers and relationship to immunologic parameters
. J Infect Dis
20. 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
21. Price RW, Spudich S. Antiretroviral therapy and central nervous system HIV type 1 infection
. J Infect Dis
2008; 197 (Suppl 3):S294–S306.
22. Goujard C, Chaix ML, Lambotte O, Deveau C, Sinet M, Guergnon J, et al. Spontaneous control of viral replication during primary HIV infection: when is ‘HIV controller’ status established?
. Clin Infect Dis