The Berlin Patient: New Research Presented at the HIV Resistance Workshop in Sitges, Spain Two Days Ago
Reported by Jules Levin. See below discussion with study author, the Author's Summaries & Conclusions and Background & Methods
Challenges inherent in detecting HIV persistence during potentially curative interventions.
SA Yukl1, T Chun2, MC Strain3, J Siliciano4, E Eisele4, R Buckheit4, YC Ho4, JK Wong1, MP Busch5, G Hutter6, DD Richman3, RF Siliciano4, SG Deeks1
1University of California, San Francisco, San Francisco, CA 2National Institute of Health, Bethesda, MD 3Unversity of California, San Diego, San Diego, CA 4Johns Hopkins University, Baltimore, MD 5Blood Systems Research Institute, San Francisco, CA 6Institute of Transfusion Medicine and Immunology, University Heidelberg, Mannheim, Germany
Background. The size of the HIV reservoir during long-term effective antiretroviral therapy and in "elite" controllers is close to the limit of detecting using standard assays. This imposes challenges for the design and assessment of potentially curative interventions. We applied a series of measurements of HIV persistence to the study of the "Berlin Patient", who underwent a hematopoietic stem cell transplant from a donor who was homozygous for the CCR5 delta-32 deletion and who had exhibited clinical evidence of being cured. Our objectives were to (1) determine if HIV had been fully eradicated as a consequence of the transplant and (2) define the potential role of various reservoir measurements in cure research.
Methods. The subject underwent a series of intensive virologic and immunologic studies beginning approximately five years after this transplantation. Replication-competent virus was measured in two laboratories, and HIV DNA and RNA levels (from blood and rectal mucosa) were measured in several laboratories using different approaches.
Results. A large volume apheresis was performed and 9 billion PBMCs evaluated for the presence of replication-competent virus. All wells were negative for HIV p24, indicating that the frequency of replication-competent HIV was therefore estimated to less than one infected cell per 1.4 billion CD4+ T cells. A repeat experiment in a second laboratory confirmed these findings. Using a variety of assays and approaches, very low levels of HIV RNA were intermittently detected in plasma, although sequence analysis of these variants were different from each other and different from those present before the transplant. Digital PCR for HIV DNA was negative for 1 copy per 2 million cells with a 95% confidence limit of less than 1.9 copies per million cells. Collagenase-digested rectal biopsy-derived cells were positive for very low levels of HIV DNA but not RNA; no sequence for confirmatory studies could be obtained. HIV antibodies levels were low and declined over the course of approximately 18 months.
Conclusion. Although the subject has had intermittent evidence for HIV persistence in some assays in some laboratories, the extremely low levels of virus which were detected, while pushing the limits of sensitivity and specificity, and the inability to match sequence with the subject's pre-therapy virus make it impossible to conclude that the subject remains HIV infected.
from Discussion with study author:
The presentation at the Sitges Resist Wksp is getting attention and the interpretation by some appears to be controversial, so this report is intended to capture the facts.
"We applied a series of measurements of HIV persistence to the study of the "Berlin Patient," who has achieved an apparent cure after a hematopoietic stem cell transplant from a donor who was homozygous for the CCR5 delta-32 deletion. Our objectives were: (1) to determine if HIV had been fully eradicated as a consequence of the transplant; and (2) to define the potential role of various reservoir measurements in cure research."
The study results reported, as with all studies, are open to interpretation by anyone, so you might hear other interpretations, some of which may get the facts of the studies correct but interpret the findings as they choose, while others may get the facts incorrect. Some observers may interpret the results as false positives (due to contamination), while others may believe they are true positives. However, this study is the first planned in a series to try to understand exactly what is the case with the Berlin patient. The purposes of this study, as told to me by one of the investigators, Steven Yukl, were to see if they could detect any virus and to better understand the roles and limits of various measurements in evaluating potentially curative interventions. The Berlin Patient could have noninfectious HIV, which would be a "sterilizing cure", or it could be that some of it is infectious but his immune system is resistant to infection and/or controls the virus. The Berlin patient had 97% CCR5-dependent virus before the transplant & the the donor cells do not produce CCR5, so even if he has some residual virus, most of it would be unable to infect his donor immune system even if it were "infectious" to another person. Yukl said it's also possible there was contamination associated with the assays and that there is no HIV present; the findings reported from this study are inconclusive. But these researchers plan to continue studying this to try to find out if the Berlin patient received a sterilizing cure or if the immune system he has is controlling HIV, which we would be called a "functional cure".
The facts are that 3 labs were able to detect HIV sequence from 2 different sites (plasma and gut) at 4 different time points, but no sequence data are available from any of these samples. some or all of them could be real, or they could all be false positives from contamination. if "real," i think that the virus is more likely to be noninfectious, as only a small percentage of HIV is fully infectious, and 2 labs were not able to detect any infectious virus in co-cultures from blood cells. however, it is worth noting that the co-cultures assess for infectious virus from blood cells (where HIV was not detected in the quantitative PCR assays) rather than plasma or gut (the places where HIV was detected in the PCR assays), and that the culture assays to detect infectious virus are much less sensitive than the quantitative PCR assays. he could certainly have intermittent production of very low levels of virus, and it could even be infectious R5 virus, which would not be expected to able to infect his donor immune system, or (much less likely) infectious X4 virus that is controlled by his immune system before it has a chance to spread. the bottom line is that we can't be certain that the detectable HIV is real, and we don't know whether all of it is noninfectious or whether some of it could be relication-competent. additional studies are needed. however, he is still able to control virus without medicines ("functional cure"), and he may still have had a "sterilizing cure" (no infectious virus), so i think he is still an example of a very successful treatment.
from poster: Results (Summary)
·CD4+ T cell counts remained fairly stable and within the normal range (Figure 2).
·In plasma, HIV RNA was detected in 2 (of 4) labs from 3 different time points (Table 1).
·No HIV DNA or RNA could be detected in PBMC (4 and 3 labs, respectively; Table 2-3).
·No replication-competent virus could be detected in 2 labs with extensive experience with co-cultures, even with 1.4 billion CD4+T cells from leukapheresis (Table 4).
·HIV DNA was detected in the rectum in 1 lab (Table 5).
·No HIV could be detected in CSF (2 labs) or in cells circulating in CSF (1 lab) (Table 6).
·Three different labs detected HIV (2 from plasma, 1 from gut) at 4 different time points. Levels were lower than typical ART-suppressed patients (Table 7) and close to the limit of the most sensitive assays. Sequence data are not available from plasma or gut.
·Two labs cloned and sequenced HIV from PBMC. However, these sequences bear little resemblance to each other or the pre-transplant variant, and one of the two sequences exhibits near perfect homology to a common lab strain, suggesting contamination.
·HIV-specific Ab levels were readily detectable but tended to decrease over time (Figure 3).
Summary from slides
1) Most assays for HIV were negative:
-No HIV DNA or RNA was detected in PBMC
-No infectious virus was isolated from peripheral CD4+T cells
-No HIV was detected in CSF fluid or cells
2) However, 3 different labs were able to detect HIV (2 from plasma, 1 from gut) at 4 different time points:
-Levels were lower than typical ART-suppressed patients and close to the limit of the most sensitive assays.
-Sequence data are not available from plasma or gut.
3) HIV-specific Ab levels were readily detectable, but detuned assays tended to show a decrease in Ab over time.
·Reconstitution with an R5-resistant but effective immune system has led to viral control ("functional cure") and possibly eradication of replication-competent HIV ("sterilizing cure"), although the intermittent detection of low-level virus raises the possibility that there has not been complete eradication.
·The collective data suggest alternative possibilities: (1) the 4 positive samples may be false positives due to PCR contamination at the extreme limits of assay sensitivity; or (2) HIV DNA may persist in tissue if not blood cells, and the infected cells may make HIV RNA that can be intermittently detected in plasma.
·Given the nature of the transplant, it is theoretically possible that any persistent virus will be found in non-T cell populations.
·The gradual decline in HIV-specific Ab suggests a slow loss of host Ab-producing cells and lack of progressive infection of the R5-resistant donor immune system.
·The combination of plasma, blood, and mucosal measures of HIV nucleic acid may not be sufficient to prove or disprove cure. Other measures, including HIV antibody levels, may be required.
Conclusions from slides:
1) Reconstitution with an R5-resistant immune system has led to viral control ("functional cure") and possibly eradication of infectious HIV ("sterilizing cure"). However, the intermittent detection of low-level virus makes it impossible to conclude definitively that a full cure was achieved.
2) The collective data suggest alternative possibilities:
A. The 4 positive samples may be false positives due to PCR contamination at the limits of assay sensitivity
B. HIV DNA may persist in tissue if not blood cells, and these infected cells may make HIV RNA that can be intermittently detected in plasma.
Further corroboration requires sequencing.
3) Given the nature of the transplant, it is theoretically possible that any persistent virus will be found in non-T cell populations.
BACKGROUND from poster:
We applied a series of measurements of HIV persistence to the study of the "Berlin Patient," who has achieved an apparent cure after a hematopoietic stem cell transplant from a donor who was homozygous for the CCR5 delta-32 deletion. Our objectives were: (1) to determine if HIV had been fully eradicated as a consequence of the transplant; and (2) to define the potential role of various reservoir measurements in cure research.
Case Report (Figure 1)
·Heterozygous for CCR5Δ32
·HIV for ≥10yrs (on ART for 4 yrs) prior to diagnosis of AML (FAB M4). 2.9% X4 or dual-tropic
Transplant (Hutter et al NEJM 2009):
·Feb 2007: Treated with chemo, total body irradiation (TBI), conditioning (ATG, cyclosporin, mycophenolate), and allogeneic stem cell transplant (SCT) (day 0) from HLA-matched, homozygous CCR5Δ32 donor.
·Achieved 100% chimerism.
·ART stopped at time of transplant
·AML relapsed; treated with chemo, TBI, and second allogeneic SCT (March 2008) from the same donor
Post-transplant studies (Hutter et al NEJM 2009; Allers et al Blood 2011):
·HIV RNA undetectable in plasma (45mo) and CSF (17 mo)
·HIV DNA undetectable in PBMC (45mo), bone marrow (40mo), brain (17mo), and colon (29 mo)
·Loss of HIV-specific T cell responses
·Loss of Ab to Pol and Gag but not Env
·Increase in peripheral and colonic CD4+T cell numbers to ≥ normal (24mo)
·No CCR5 expression or wild type gene in colonic CD4+T cells (24, 29mo)
·CCR5 detected on colonic macrophages at 5.5 mo but not at 24-29mo.
·No CCR5 expressed in liver (12mo) or brain (17mo)
Study Rationale and Hypotheses
Several theoretical reasons suggest HIV might persist or recur, including:
·Presence of X4 variant prior to transplant
·After transplant, his CD4+ T cells could be infected ex vivo with X4 (but not R5) virus
·Presence of CCR5+ target cells post-transplant (5.5 mo)
·Possibility of long-lived HIV reservoirs or CCR5+ cells not susceptible to conditioning
·HIV may persist in rare long-lived cells in tissues that are more resistant to chemo, radiation, and graft-replacement.
·Latent R5 virus may be intermittently expressed at low levels, but will likely not be able to establish a spreading infection.
·If X4 virus persists, it may eventually emerge and spread.
Procedures to obtain specimens included multiple large volume blood draws, leukapheresis, flexible sigmoidoscopy with biopsies from rectum (30 biopsies), and a lumbar puncture.
Samples (plasma, PBMC, gut, CSF) were sent to 6 different labs with expertise in detecting extremely low levels of virus: (1) Blood Systems Research Institute (Michael Busch), (2) NIH (Tae-Wook Chun), (3) Solna, Sweden (Sarah Palmer), (4) UCSD/VASD (Doug Richman), (5) Johns Hopkins (Robert Siliciano), and (6) UCSF/SFVAMC (Joseph Wong).
Cell-associated HIV DNA in PBMC was measured in 4 labs using qPCR or digital droplet PCR.
Plasma HIV RNA was measured in 4 different labs using different techniques: (1) Roche AmpliPrep assay, (2) Single Copy Assay (SCA), (3) Transcription-Mediated Amplification (TMA), and (4) modified Abbott assay that involves pelleting virus from 30ml of plasma.
Cell-associated HIV RNA in PBMC was measured in 3 labs using qRT-PCR or TMA.
Latently-infected peripheral CD4+ T cells (in infectious units per million cells, IUPM) were measured in 2 labs by co-culture.
Cell-associated HIV DNA and RNA were measured from both bulk rectal biopsies and collagenase-digested rectal cells in 1 lab using qPCR and qRT-PCR.
Cell-free HIV RNA in CSF was measured in 2 labs; 1 lab also measured HIV DNA in CSF cells
Blood and gut cells were also sent for various immunology studies, including CD4/CD8 T cell counts, markers of T cell activation, coreceptor usage, HIV-specific T cell responses, and levels of HIV-specific antibodies.