Article pubs.acs.org/ac
A Targeted Mass Spectrometry Assay for Detection of HIV Gag Protein Following Induction of Latent Viral Reservoirs Daniela Schlatzer,†,§ Aiman A. Haqqani,†,§ Xiaolin Li,† Curtis Dobrowolski,‡ Mark R. Chance,† and John C. Tilton*,† †
Center for Proteomics and Bioinformatics, Department of Nutrition, ‡Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, United States S Supporting Information *
ABSTRACT: During early infection, HIV-1 establishes a reservoir of latently infected cells that persist during antiretroviral therapy. These reservoirs are considered the primary obstacle to eradicating HIV-1 from patients, and multiple strategies are being investigated to eliminate latently infected cells. Measuring the reservoir size using an affordable and scalable assay is critical as these approaches move into clinical trials: the current “gold-standard” viral outgrowth assay is costly, labor-intensive, and requires large numbers of cells. Here, we assessed whether selective reaction monitoring-mass spectrometry (SRM-MS) is sufficiently sensitive to detect latent HIV reservoirs following reactivation of virus. The Gag structural proteins were the most abundant viral proteins in purified virus and infected cells, and tractable peptides for monitoring Gag levels were identified. We then optimized a Gag immunoprecipitation procedure that permitted sampling of more than 107 CD4+ T cells, a requirement for detecting exceedingly rare latently infected cells. Gag peptides were detectable in both cell lysates and supernatants in CD4+ T cells infected in vitro at frequencies as low as ∼1 in 106 cells and in cells from HIV-infected patients on suppressive antiretroviral therapy with undetectable viral loads. To our knowledge, this represents the first detection of reactivated latent HIV reservoirs from patients without signal amplification. Together, these results indicate that SRM-MS is a viable method for measuring latent HIV-1 reservoirs in patient samples with distinct advantages over current assays.
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to overestimations of the reservoir in PCR-based assays.14 Third, even potent stimulation of cells reactivates only a fraction of replication-competent latent proviruses in a stochastic manner, indicating that the viral outgrowth assay underreports the size of the latent reservoir.13 Therefore, assays that measure the latent reservoir will need exquisite sensitivity, the capacity to ignore or distinguish defective proviruses, and the ability to detect partial activation−represented by HIV gene or protein expression. Any assay used in clinical trials must also detect HIV despite the considerable sequence diversity expected between patients. Selective reaction monitoring-mass spectrometry (SRM-MS) is a widely used and validated adaptation of tandem mass spectrometry (MS/MS) to quantify low abundance proteins in complex samples.15 In traditional data-dependent (DDA) MS/ MS, precursor ions are automatically selected on the basis of their intensity in the first mass filter (Q1) of the mass spectrometer. Once fragmented in the collision cell (Q2), the fragments (product ions) are passed through the second mass filter (Q3), and product ions are detected. The mass information from both the precursor and product ions is
IV establishes a reservoir of latently infected cells during primary infection that persists during antiretroviral therapy (ART).1−3 As latently infected cells resume transcription and release infectious viruses when treatment is interrupted,4−7 life-long therapy with ART is required to prevent disease progression. These latent viral reservoirs are considered the primary barrier to eradicating HIV, and multiple strategies are being evaluated to eliminate latently infected cells.8−10 As these approaches move into clinical trials, an accurate and reproducible assay to measure HIV reservoirs is essential for assessing efficacy. The current gold-standard for measuring latent HIV reservoir size, the viral outgrowth assay,2,11,12 is labor-intensive, costly, and requires cells from multiple healthy donors, making it impractical for large clinical trials. Several factors make measuring the latent reservoir challenging. First, cells bearing replication-competent HIV proviruses are present at very low frequencies: ∼1 in 106−107 resting CD4+ T cells as measured by viral outgrowth assays.2,12 Second, there is an ∼100-fold higher frequency of cells containing “defective” proviruses that have integrated but are not capable of producing infectious viruses. These defective viruses frequently contain hypermutations, large deletions, frameshifts, or mutations in key viral regions such as the psi (Ψ) packaging or major splice donor (D1) sites13 and can lead © XXXX American Chemical Society
Received: December 21, 2016 Accepted: April 18, 2017
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experiments, infected cells were mixed with autologous, uninfected cells to generate frequencies of infected cells of 0.1−0.0001%. Latent Reservoirs in Patients. 100 × 106 PBMCs from two HIV-infected patients with undetectable viral loads on antiretroviral therapy were a generous gift of Dr. Jonathan Karn. Cells were thawed and CD4+ T cells purified using a negative selection isolation kit (STEMCELL Technologies) and resuspended at 1 × 106 cells/ml in RPMI/10 with 10 μg/ mL concanavalin A and 100 nm efavirenz for 72 h at 37 °C. Supernatants were collected and cells washed with PBS. Cells and supernatants were frozen at −80 °C, thawed, Gag immunoprecipitated with antibody #24−4, digested, and analyzed by SRM-MS as described below. Immunoprecipitation Experiments. 160 × 106 cells were infected as described above and 100 million cells lysed in 50 μL lysis buffer (1% SDS, 150 mM NaCl, 50 mM Tris, 1 mM DTT + protease inhibitors) at 90 °C for 5 min. SDS was diluted out using 450 μL dilution buffer (150 mM NaCl, 50 mM Tris, 0.55% sodium deoxycholate, and 1.1% Triton-X-100). Cell lysates were sonicated and incubated in triplicate with sepharose beads prebound to 1.67 μg of monoclonal antibodies or 12.5 μL of polyclonal anti-p24 antibody (NIH AIDS Reagent Program) overnight at 4 °C on an orbital rocker. Beads were recovered by centrifugation and washed three times in dilution buffer, followed by two washes with water. Gag was eluted and samples run by Western blot with semiquantitative determination of band intensities using Image-J. A fraction of nonimmunoprecipitated cell lysate was run, and the initial virus input was calculated. Immunoprecipitation experiments comparing monoclonal antibody #24-4 and polyclonal antibody for mass spectrometry was performed as described above with the exception that Dynabeads were used due to the ability to perform on-bead digestion with trypsin and Lys-C. Supernatants were lysed in dilution buffer, incubated with Dynabeads overnight, washed, and processed for LC-MS/MS or SRM analysis as described below. Data-Dependent (DDA) LC-MS/MS Detection of Gag in Purified Viruses, Infected Cells, and Immunoprecipation Experiments. Sample Preparation for Purified Virus or Cells. Five independently prepared, purified virus samples or infected cells from five different donors were lysed in 200 μL of 2% SDS and protease inhibitor tablet (Roche Diagnostics). Samples were cleaned of detergent using a previously published filter-aided sample preparation protocol with a 10-kDa molecular weight cutoff filter (Millipore) and buffer exchanged with 8 M Urea in 50 mM Tris (pH 8) to a final volume of 50 μL.20 Digestion protocol for viruses and cell lysates can be found in supporting materials. Sample Preparation/Enzymatic Digestion for Gag Immunoprecipated Samples. Magnetic beads were washed three times with 100 mM Tris-EDTA (pH 8), and supernatant was removed. Fifty microliters of 100 mM Tris-EDTA (pH 8) was added to beads. The digestion protocol for immunoprecipated samples can be found in Supporting Information. Proteomic Analysis Using Reverse-Phase Data-Dependent (DDA) LC-MS/MS. Sample were analyzed by LC-MS/MS using a LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific) equipped with a nanoACQUITY Ultrahigh pressure liquid chromatography system (Waters). Additional analytical parameters can be found in supporting materials. The LC-MS/MS raw files were processed and search using Mascot (Matrix
used to determine the intensity and identity (peptide sequence) of the precursor ion. In contrast, SRM-MS uses predefined filters to select known precursor masses that correspond to a target peptide and its most tractable product ions. Compared to MS/MS, SRM-MS can improve sensitivity by more than 2 orders of magnitude, allowing routine quantification of 50 attomoles (1 attomole = 10−18 moles) of a peptide of interest.15 In this study, we developed an assay for detecting HIV-1 in infected cells and supernatants using SRM-MS. The structural Gag proteins were the most abundant viral proteins both in infected cells and concentrated viruses, and several tractable Gag peptides were identified. We next optimized a Gag immunoprecipitation protocol to reduce noise from cellular proteins and allow sampling upward of 107 cells as would be required in patient samples. Gag protein was detected both in infected cells as well as in supernatant. Detecting Gag protein within cells facilitates analysis of partial reactivation, whereas monitoring supernatant virus increases stringency by measuring only fully activated cells harboring HIV capable of releasing intact viruses. Using in vitro infections, we found that SRM-MS improved the limit of detection of Gag peptides by more than an order of magnitude compared to DDA LC-MS/MS and increased the limit of quantification nearly 2 orders of magnitude, allowing quantification of Gag at infections as low as ∼1 in 106 cells. Finally, Gag peptides were identified in samples from three HIV-infected patients with fully suppressed virus on antiretroviral therapy, with a fourth patient right at or slightly below the limit of detection. To our knowledge, this represents the first demonstration that HIV can be detected directly from reactivated cells without requiring sample amplification by multiple rounds of viral replication or PCR, illustrating the sensitivity of SRM-MS in a clinically relevant setting.
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METHODS Cells. This study was conducted according to the principles specified in the Declaration of Helsinki and under local ethical guidelines (Case Western Reserve University Institutional Review Board (IRB)). Normal donor samples were obtained from ALLCELLS, LLC. All donors were negative for HBV, HCV, and HIV. CD4+ T cells were isolated using RosetteSep CD4+ T cell enrichment kit antibodies (STEMCELL Technologies) prior to ficoll gradient separation. Cells were cryopreserved and treated with benzonase upon thawing, prior to infection. Production of Viruses. 2.5 × 106 293T/17 cells were plated into 10 cm plates and transfected after 24 h with 10 μg of pNL4−3-deltaE-EGFP core (NIH AIDS Reagent Program (cat. no. 11100) from Drs. Haili Zhang, Yan Zhou, and Robert Siliciano), 7.5 μg bla-Vpr plasmid (obtained from Dr. Robert Doms), and 6.0 μg HIV Env JOTO.TA1.224716,17 (obtained from Drs. Beatrice Hahn and George Shaw) using calcium phosphate methods.18 Media was replaced after 6 h and supernatant harvested at 72 h. Supernatants were filtered, concentrated through 20% sucrose, and the viral pellet resuspended in PBS and frozen according to published protocols.19 Viral concentrations were determined by p24 ELISA (Cell Biolabs). In Vitro Viral Infections. CD4+ T cells from healthy controls were infected with 50−100 ng p24 equiv/106 cells of HIV, spinoculated at 1200g × 2 h, and then incubated at 37 °C for 72 h. An aliquot was analyzed by flow cytometry to determine the percentage of infected cells. For dilution B
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Analytical Chemistry Science London, version 2.1) and searched against the Uniprot database (550 552 sequences; 196 472 675 residues). Mascot search settings were as follow: trypsin enzyme specificity; mass accuracy window for precursor ion, 8 ppm; mass accuracy window for fragment ions, 0.8 Da; carbamidomethylation of cysteines as fixed modifications; oxidation of methionine as variable modification; and one missed cleavage. Searched files were then imported into Scaffold (Proteome Software Inc., version 4.6.2) for peptide validation and quantification by spectral count. Peptide identifications were accepted if there were >95% probability by the Peptide Prophet algorithm.21 Spectral counts were reported using total spectrum count for each peptide identified. Liquid Chromatography and Selected Reaction Monitoring (SRM) Mass Spectrometry. Samples were analyzed using a Waters NanoACQUITY Ultra performance liquid chromatography system (Waters) and a TSQ Quantum Ultra mass spectrometry (Thermo). The criteria for peptide selection for SRM were detection in the previous DDA LC-MS/MS analysis of CD4+ T cells infected with reporter virus or purified virus. Three of the 5 peptides selected were detected in both infected cells and purified virus. Two additional peptides were included in the SRM assay that were only identified in the purified virus sample but were selected due to their abundance in virus sample. Additional analytical parameters can be found in supporting materials. A total of 4 transitions per peptide were monitored for quantification. The precursor and product ion pairs are included in Table S-1. Pinpoint software (Thermo) was used to process the SRM data and signal intensities of each endogenous peptide was extracted. A limit of detection was determined for each peptide as well linearity of response for each peptide using extracted peak areas.
Figure 1. Detection of HIV proteins in purified viral particles and infected cells. (A) Amino acid sequence of Gag with shaded gray bars denoting the p17 (matrix), p24 (capsid), p7 (nucleocapsid) and p6 subunits. Amino acids detected in concentrated virus samples by LCMS/MS are shown in bold text; coverage of the Gag protein was 84%. (B) Gag residues detected in infected CD4+ T cells shown in bold text. Sequence coverage was 10%.
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RESULTS HIV-1 Gag Structural Proteins Are the Most Abundant Viral Protein in Purified Particles and Infected CD4+ T Cells. Five independently prepared, concentrated HIV viral samples and infected cell samples were analyzed by LC-MS/ MS. In concentrated viral samples, the HIV Gag, Pol, Env, Nef, and Vpr proteins were detectable in all samples (5/5), with Rev and Vif detected in 4/5 and 3/5 samples, respectively. Tat and Vpu were not detected in any of the purified viral samples. Gag was the most abundant total protein, as expected on the basis of its critical structural role (Table S-2). Mass spectrometry coverage of the Gag polyprotein reached 84% (Figure 1A). To determine the most abundant viral proteins in infected cells, 10 × 106 CD4+ T cells were infected with 50−100 ng of p24 equiv/106 cells of an EGFP-expressing reporter virus. Using flow cytometric detection of EGFP, the percentage of infected cells was determined to be 2%, and samples were prepared for data-dependent acquisition (DDA) LC-MS/MS. The viral Gag and Gag-Pol proteins were the only viral proteins detected in all samples. Gag was the 1042nd most abundant protein in the sample (Table S-2); however, protein coverage declined to only 10% of Gag (Figure 1B). Gag Immunoprecipitation Improves Detection of Peptides by DDA LC-MS/MS. The 10 × 106 CD4+ cells infected with reporter viruses yielded ∼60 μg of total protein per sample. However, only 0.5 μg−corresponding to ∼80 000 cells−could be loaded onto the liquid chromatography column. We reasoned that enrichment of Gag by immunoprecipitation would enable sampling of more CD4+ T cells and might improve detection by reducing background from cellular
proteins. As the majority of antibodies and assays for HIV Gag proteins recognize the p24 capsid subunit, we obtained eight monoclonal and one polyclonal anti-p24 antibodies from the NIH AIDS Reagent Program. In total, 160 × 106 CD4+ T cells were infected with an HIV reporter virus22 and cultured for 3 days, yielding 2.3% EGFP+ cells by flow cytometry. Cells were harvested, washed, lysed, sonicated, mixed, and then redivided equally to make 10 experimental conditions. One of these was reserved as a non-IP control for the initial virus to calculate the percent recovery of immunoprecipitations; the remaining nine cell lysate conditions were incubated overnight at 4 °C in the presence of buffers containing different anti-p24 monoclonal antibodies or polyclonal antibody, followed by Western blot analysis and semiquantitative measurements using ImageJ. The percent recovery of Gag protein following immunoprecipitation with monoclonal antibodies ranged from 19.4−57.2%, with a mean of 36.5% recovery (Figure 2). The polyclonal antibody and monoclonal antibody #24-4 demonstrated the highest Gag recovery. Anti-p24 Monoclonal Antibodies Have Reduced Breadth but Increased Intensity of Gag Detection by DDA LC-MS/MS. Next, we compared monoclonal antibody #24-4 and the polyclonal antibody for MS/MS detection. 40 × 106 CD4+ T cells were infected with HIV, and lysed after 72 h of incubation. Lysates were incubated with Dynabeads bound to #24-4 or the polyclonal antibody overnight at 4 °C, followed by washes and on-bead digestion and analyzed by LC-MS/MS. While the polyclonal antibodies provided more robust sequence coverage of Gag (37% sequence coverage for the polyclonal Ab C
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in patients; therefore, we continued the SRM-MS assay development using monoclonal antibody clone #24-4. Gag from Both Infected Cells and Viruses Released into the Supernatant Can Be Immunoprecipitated and Detected by LC-MS/MS. One of the challenges in identifying latently infected cells containing replication-competent HIV is the preponderance of cells infected with defective viruses. We hypothesized that analyzing viruses released into the supernatant could help the assay in two important ways. First, any cells that produce Gag but do not release viral particles would be eliminated from analysis, increasing the stringency of the assay. Second, cell-free viruses might improve detection as noise from cellular proteins should be markedly reduced. To test this hypothesis, we harvested supernatants in parallel with cell lysates from the experiment comparing #24-4 and the polyclonal antibody. Immunoprecipitations from supernatants with the monoclonal antibody provided both better breadth of detection (24% sequence coverage for the polyclonal Ab vs 39% from the monoclonal Ab, Figure 3C) and enhanced intensity of detected peptides (25 total spectra for the polyclonal Ab vs 38 for the monoclonal Ab, Figure 3D). These data indicate that detection of virus in the supernatant is possible using our assay and that the monoclonal antibody again demonstrated superior sampling depth of Gag. SRM-MS Improves the Limits of Detection and Quantification of Gag Peptides Compared with LC-MS/ MS. To assess the sensitivity of LC-MS/MS and SRM-MS at detecting and quantifying rare frequencies of infected cells, we infected CD4+ T cells with reporter virus and an aliquot of
Figure 2. Recovery of Gag proteins following immunoprecipitation with anti-Gag monoclonal or polyclonal antibodies. CD4+ T cells infected at a 2.32% rate were divided equally into replicates, and Gag was immunoprecipitated using monoclonal antibodies (clone numbers listed in figure) or anti-p24 polyclonal antibody. Percent recovery of Gag protein was calculated by semiquantitative comparison of Western blot bands using ImageJ with a nonimmunopreciptated control sample (initial virus).
vs 28% for the monoclonal Ab, focused on p24 as expected, Figure 3A), the monoclonal antibody condition generated more total Gag spectral counts (24 spectra detected with the polyclonal Ab vs 36 with the monoclonal Ab, Figure 3B). Following immunoprecipitation, Gag was the 18th most abundant protein in the samples (Table S-2). These results suggest that immunoprecipitation with polyclonal antibodies results in slightly enhanced breadth of Gag detection but that monoclonal antibodies provide superior sampling intensity. We reasoned that sampling depth will be particularly important in detecting the low frequency of latently infected cells expected
Figure 3. Comparison of the breadth (sequence coverage) and intensity (total spectral counts) of Gag detection by LC-MS/MS. (A) Gag protein and peptides detected in cell lysate samples following immunoprecipitation with monoclonal antibody #24-4 or polyclonal antibody. (B) Total Gag spectral counts for the polyclonal and monoclonal antibody immunoprecipitations from cell lysate samples. (C) Gag protein and peptides detected in supernatants following immunoprecipitation. (D) Total Gag spectral counts from supernatant samples immunoprecipitated with monoclonal or polyclonal antibodies. D
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Figure 4. Assessing the limit of detection of LC-MS/MS for identifying and quantifying Gag peptides. Infected CD4+ T cells were mixed with autologous uninfected cells to generate aliquots with 0.1%, 0.01%, 0.001%, and 0.0001% infection rates. Cell lysates were immunoprecipitated with monoclonal Gag antibody #24-4 and analyzed by DDA LC-MS/MS (left column) or SRM-MS (right column).
infected cells was analyzed by flow cytometry to determine the percentage of EGFP+ cells. Infected cells were then mixed with autologous uninfected CD4+ T cells to generate 20 × 106 cell aliquots with infection rates of 0.1%, 0.01%, 0.001%, and
0.0001% and cell lysates were immunoprecipitated with the #24-4 monoclonal antibody and analyzed for detection and intensity of HIV peptides. By LC-MS/MS, three peptides K.ETINEEAAEWDR.L, R.FAVNPGLLETSEGCR.Q, and E
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To determine if SRM-MS could identify HIV-1 peptides in patient samples, we isolated CD4+ T cells from four HIVinfected patients with undetectable viral loads. Approximately 20 million CD4+ T cells were recovered and stimulated with 10 μg/mL concanavalin A for 72 h in the presence of 100 nm efavirenz to block further viral spread. Supernatants and cell lysates were immunoprecipitated as described above. The viral reservoirs from these patients have previously been characterized using an Envelope mRNA Detection by Ion Torrent Sequencing (EDITS) assay (Jonathan Karn, personal communication). In contrast to the viral outgrowth assay, the EDITS assay maps high-throughput sequencing reads and compares them to a standard curve generated by sorting HIV-1 infected CD4+ cells and titrating them into uninfected PBMC. The reservoir size of patients 2293, 1570, 2511, and 2546 were determined by EDITS to be 75.2, 58.9, 58.6, and 31.7 infected cell equivalents/106 cells, respectively. Importantly, assays measuring viral RNA have been reported to have ∼50-fold higher infected cell frequencies compared to viral outgrowth assays;23 thus, these patients would be expected to have reservoir sizes near the range of 0.5−2.0 infected cells per million using a traditional viral outgrowth assay. Using SRM, the R.WIILGLNK.I peptide was detected in the supernatant of reactivated CD4+ T cells from patient 2511, and its identity was confirmed by analysis of fragment ions using Pinpoint software (Figure 6A). The R.WIILGLNK.I peptide was also detected in cell lysates, but the noise was substantially higher because of the large number of cellular proteins in the sample (data not shown). The 478.8 → 657.439 transition was the most intense for all four patients (Figure 6B). A peptide corresponding to R.WIILGLNK.I was detected in all samples with appropriate transitions by Pinpoint; however, in patient 2546 the signal-to-noise ratio was only 1.5, suggesting that we are slightly below the accepted limit of detection in this sample. Interestingly, we found an excellent correlation between the intensity of the monitored R.WIILGLNK.I transition by SRMMS and the frequency of infected cells as measured by EDITS (R2 = 0.96, p = 0.019) (Figure 6C); if patient 2546 is removed from the analysis, a strong correlation is maintained (R2 = 0.92), but significance is lost with only 3 data points. Further investigation of the assay with additional patient samples will be necessary to more precisely define detectable reservoir size using SRM-MS. Nevertheless, the detection of Gag protein directly from reactivated latently infected cells and supernatant from patient samples is remarkable as most current assays rely on amplification of signal through multiple rounds of viral replication or PCR of viral nucleic acids.
K.NWMTETLLVQNANPDCK.Twere detected from dilutions as low as 1 infected cell in 104 uninfected cells (0.01%) and twoR.WIILGLNK.I and R.MYSPTSILDIR.Qwere detectable at dilutions of 1:105 (0.001%) (Figure 4). Peptides were linear across detectable infection rates with R2 values of >0.95 (Table S-3). By SRM, all peptides were detectable at dilutions of 1:105 (0.001%), and two peptidesK.ETINEEAAEWDR.L and R.WIILGLNK.Iwere detectable at 1:106 (0.0001%), representing a 1−2 order of magnitude increase in the limit of detection using SRM-MS (Figure 4). SRM-MS peptides were confirmed by transitions. While these results estimate the sensitivities of the LC-MS/MS and SRMMS assays, several caveats must be noted. First, the reporter viruses used here have EGFP in place of Env and expression of the reporter may not correlate perfectly with Gag protein production. Second, experiments comparing untreated and efavirenz-treated samples indicated that input virus accounted for up to 30−50% of the signal (data not shown). Nevertheless, these data suggest that the K.ETINEEAAEWDR.L and R.WIILGLNK.I peptides can be detected at very low frequencies of infected CD4+ T cells. Detection of Reactivated Latent HIV-1 in CD4+ T Cells from Patients on Effective Antiretroviral Therapy. Reactivation of latent HIV from patients poses additional challenges including sequence variation, the resting memory profile of the cells harboring latent HIV, and stochastic reactivation by latency reversing agents.13 While the ability of SRM-MS to select for peptides with specific mass/charge (M/ Z) characteristics dramatically improves the sensitivity to detect target peptides, it introduces an additional problem: amino acid substitutions alter the massand potentially the chargeof the peptide and preclude detection. Without detailed consideration of HIV diversity, it is extremely likely that a proportion of viruses could go undetected. Importantly, SRMMS is not limited to the detection of a single precursor peptide and its fragments, but rather, it can detect over a hundred parental peptides and their fragments. Using the subtype B frequency of five Gag peptides that were identified by mass spectrometry, we estimate that >99.99% of subtype B viruses would be detected using only the most common ten variants of these five peptides (Figure 5).
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CONCLUSIONS As strategies to eliminate HIV reservoirs move into clinical trials, an assay to monitor reservoir size in patients is critical to evaluate their efficacy. The viral outgrowth assay is expensive, labor-intensive, and requires cells from multiple healthy donors. A variety of alternative assays have been developed, including quantification of total or integrated viral DNA24−27 and viral mRNA transcripts.23 However, PCR-based DNA and RNA assays show approximately 100-fold and 50-fold, respectively, higher levels of HIV infected cells14,23 than the viral outgrowth assays, likely due to the presence of defective proviruses that may not form infectious particles.13 More recently, two novel approaches for detection of Gag have been developed. First, Martrus and colleagues reported a flow-cytometry-based assay detecting gag-pol mRNA and intracellular Gag p24 protein that
Figure 5. Five HIV-1 subtype B consensus Gag peptides detected by MS and the frequency of the most common subtype B variants. Using the most common 10 variants of each peptide that do not alter trypsin digestion sites, >99.99% of subtype B viruses will contain one detectable peptide. Using only the R.WIILGLNK.I peptide that was detected in patient samples, ∼97% of subtype B HIV-1 viruses would be detectable using the 10 most common variants. F
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Figure 6. Detection of Gag peptides from reactivated latent reservoirs from patient samples. (A) CD4+ T cells from a patient with undetectable HIV-1 on antiretroviral therapy were stimulated with 10 μg Concanavalin A to reactivate latent HIV in the presence of 100 nM efavirenz to block viral spread. The Gag peptide R.WIILGLNK.I was detected in immunoprecipitated supernatant samples with peptide identity confirmed by Pinpoint software. The peptide eluted at 36 min (highlighted region). The transitions monitored were 478.8 → 431.261 (green), 478.8 → 544.345 (purple), 478.8 → 657.429 (blue), and 478.8 → 770.513 (yellow). (B) SRM-MS chromatograms of peptide R.WIILGLNK.I monitoring the most intense (478.8 → 657.429) transition in immunoprecipitated supernatant samples from four patients with undetectable HIV on antiretroviral therapy. (C) Correlation between the intensity of the monitored R.WIILGLNK.I transition and reservoir size as determined by EDITS (infected cell equivalents per million cells).
tion. Partial reactivation can be detected via Gag expression within cells, while any mutations precluding particle release will be excluded from supernatant detection. Mutations that lead to the release of noninfectious particles, however, will likely only be discriminated using viral outgrowth assays. The SRM-MS assay can easily be adapted to viral outgrowth if this becomes a major confounder of reservoir size measurements in clinical trials. The ability to flexibly monitor reservoirs with increasing stringency: infected cells, viruses released into supernatant, and viral outgrowth is a major strength of the SRM-MS latency assay. Finally, the sequence diversity of HIV can be addressed by expanding SRM-MS to monitor not only the consensus peptide sequences but also common variants. Even using only the R.WIILGLNK.I peptide that was identified in patient samples, adding 9 other common variants of this peptide increases detection to cover over 97% of subtype B viruses. Other HIV subtypes can be detected by adding or replacing peptides; however, we anticipate that clinical trials examining HIV eradication strategies will be performed in settings where subtype B is most common. Together, these results demonstrate the remarkable sensitivity of SRM-MS in a clinically relevant application, namely, the detection of reactivated HIV-1 Gag proteins from latently infected cells from patients. Continued optimization and testing of the SRM-MS protocol described here could make this highly flexible assay ideal for monitoring clinical trials of viral eradication strategies.
enables detection of reactivated latent HIV-1 in primary cells infected in vitro but has not been demonstrated on patient samples.28 Second, a high-sensitivity p24 ELISA developed by Quanterix, Inc. has been used to measure reactivation of latent reservoirs; however, two studies examining this assay used culture conditions without antiretroviral drugs, making it difficult to determine whether viral replication is amplifying the signal.29,30 Here, we sought to characterize whether selective reaction monitoring mass spectrometry (SRM-MS) was sufficiently sensitive to detect reactivation of virus in rare latently infected cells. Monitoring the viral reservoir is complicated by multiple factors including: (1) the low frequency of latently infected cells,2,11,12 (2) an approximately 100-fold higher frequency of cells containing defective proviruses,13 (3) incomplete reactivation of latent cells even with potent mitogens,13 and (4) sequence diversity of HIV-1. Nevertheless, Gag immunoprecipitation and SRM-MS was able to detect Gag from patients with approximately 30−80 HIV mRNA-expressing cells per 106 CD4+ T cells, corresponding to roughly 0.5−2.0 infectious units per million (IUPM) by traditional viral outgrowth assays.23 To our knowledge, this is the first assay to successfully detect viral proteins or nucleic acids from reactivated latent reservoirs from patient samples without subsequent signal amplification by viral replication or PCR. We expect correlations between signal and the initial reservoir size will become less reliable if viral replication is required to amplify signal, as patient- and virus-specific differences will likely lead to differences in viral replication efficiency. Although we are at or slightly below the limit of detection with patient 2546, further optimization of the methods described here, along with future advances in instrumentation, will continue to advance the sensitivity of SRM-MS at detecting extremely rare peptide epitopes. For instance, simply incubating reactivated cells for longer periods of time, even in the presence of ART, can boost detection as more infected cells are recruited to produce virus and more virus accumulates in the supernatant. As demonstrated here, the SRM-MS latency reactivation assay enables detection of HIV-1 Gag protein both in infected cells and released virions. This helps address complications stemming from defective proviruses and incomplete reactiva-
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b05070. Precursor and product ion pairs for selected reaction monitoring (SRM) (PDF) Identified proteins (XLSX) Peptide sequence (XLSX) Materials and methods (PDF) G
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
John C. Tilton: 0000-0002-4218-5870 Author Contributions §
(D.S. and A.A.H.) These authors contributed equally to this work. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We would like to thank Jonathan Karn for his generous gift of PBMCs from HIV-infected patients. This work was supported by NIH Grant R21 AI113148. A.A.H., D.S., X.L., and C.D. performed experiments. D.S., M.C, and J.C.T. designed the study, supervised the project, and wrote the manuscript.
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DOI: 10.1021/acs.analchem.6b05070 Anal. Chem. XXXX, XXX, XXX−XXX
