In vivo and in vitro immunogenicity of novel MHC class I presented epitopes to confer protective immunity against chronic HTLV-1 infection.

Human T-cell leukemia virus type 1 (HTLV-1) has infected as many as 10 million people worldwide. While 90% are asymptomatic, 5% develop severe diseases including adult T-cell leukemia/lymphoka (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). No vaccine against HTLV-1 exists, and screening programs are not universal. However, patients with chronic HTLV-1 infection have high frequencies of HTLV-1-activated CD8+ T cells, and the two main HLA alleles (A2, A24) are present in 88% of infected individuals. We thus utilized an immunoproteomics approach to characterize MHC-I restricted epitopes presented by HLA-A2+, A24+ MT-2 and SLB-1 cell lines. Unlike traditional motif prediction algorithms, this approach identifies epitopes associated with cytotoxic T-cell responses in their naturally processed forms, minimizing differences in antigen processing and protein expression levels. Out of nine identified peptides, we confirmed six novel MHC-I restricted epitopes that were capable of binding HLA-A2 and HLA-A24 alleles and used in vitro and in vivo methods to generate CD8+ T cells specific for each of these peptides. MagPix MILLIPLEX data showed that in vitro generated epitope-specific CD8+ T cells secreted IFN-É£, granzyme B, MIP-1α, TNF-α, perforin and IL-10 when cultured in the presence of MT-2 cell line. Degranulation assay confirmed cytotoxic response through surface expression of CD107 on CD8+ T cells when cultured with MT-2 cells. A CD8+ T-cell killing assay indicated significant antiviral activity of CD8+ T cells specific against all identified peptides. In vivo generated CD8+ T cells similarly demonstrated immunogenicity on ELISpot, CD107 degranulation assay, and MagPix MILLIPLEX analysis. These epitopes are thus candidates for a therapeutic peptide-based vaccine against HTLV-1, and our results provide preclinical data for the advancement of such a vaccine.

A novel immunization approach for dengue infection based on conserved T cell epitopes formulated in calcium phosphate nanoparticles.

Dengue virus (DV) is the etiologic agent of dengue fever, the most significant mosquito-borne viral disease in humans. Most DV vaccine approaches are focused on generating antibody mediated responses; one such DV vaccine is approved for use in humans but its efficacy is limited. While it is clear that T cell responses play important role in DV infection and subsequent disease manifestations, fewer studies are aimed at developing vaccines that induce robust T cells responses. Potent T cell based vaccines require 2 critical components: the identification of specific T cell stimulating MHC associated peptides, and an optimized vaccine delivery vehicle capable of simultaneously delivering the antigens and any required adjuvants. We have previously identified and characterized DV specific HLA-A2 and -A24 binding DV serotypes conserved epitopes, and the feasibility of an epitope based vaccine for DV infection. In this study, we build on those previous studies and describe an investigational DV vaccine using T cell epitopes incorporated into a calcium phosphate nanoparticle (CaPNP) delivery system. This study presents a comprehensive analysis of functional immunogenicity of DV CaPNP/multipeptide formulations in vitro and in vivo and demonstrates the CaPNP/multipeptide vaccine is capable of inducing T cell responses against all 4 serotypes of DV. This synthetic vaccine is also cost effective, straightforward to manufacture, and stable at room temperature in a lyophilized form. This formulation may serve as an effective candidate DV vaccine that protects against all 4 serotypes as either a prophylactic or therapeutic vaccine.

Antibody blockade of CLEC12A delays EAE onset and attenuates disease severity by impairing myeloid cell CNS infiltration and restoring positive immunity.

The mechanism of dendritic cells (DCs) recruitment across the blood brain barrier (BBB) during neuroinflammation has been the least explored amongst all leukocytes. For cells of myeloid origin, while integrins function at the level of adhesion, the importance of lectins remains unknown. Here, we identified functions of one C-type lectin receptor, CLEC12A, in facilitating DC binding and transmigration across the BBB in response to CCL2 chemotaxis. To test function of CLEC12A in an animal model of multiple sclerosis (MS), we administered blocking antibody to CLEC12A that significantly ameliorated disease scores in MOG35-55-induced progressive, as well as PLP138-151-induced relapsing-remitting experimental autoimmune encephalomyelitis (EAE) mice. The decline in both progression and relapse of EAE occurred as a result of reduced demyelination and myeloid cell infiltration into the CNS tissue. DC numbers were restored in the spleen of C57BL/6 and peripheral blood of SJL/J mice along with a decreased TH17 phenotype within CD4+ T-cells. The effects of CLEC12A blocking were further validated using CLEC12A knockout (KO) animals wherein EAE disease induction was delayed and reduced disease severity was observed. These studies reveal the utility of a DC-specific mechanism in designing new therapeutics for MS.

IFN-α-Induced Downregulation of miR-221 in Dendritic Cells: Implications for HCV Pathogenesis and Treatment.

Although interferon (IFN)-α is known to exert immunomodulatory and antiproliferative effects on dendritic cells (DCs) through induction of protein-coding IFN-stimulated genes (ISGs), little is known about IFN-α-regulated miRNAs in DCs. Since several miRNAs are involved in regulating DC functions, it is important to investigate whether IFN-α's effects on DCs are mediated through miRNAs as well. In this study, we examined miRNA expression patterns in myeloid DCs (mDCs) and plasmacytoid DCs after exposing them to IFN-α. We report that IFN-α downregulates miR-221 in both DC subsets via inhibition of STAT3. We validated proapoptotic proteins BCL2L11 and CDKN1C as miR-221 targets suggesting that IFN-α can induce DC apoptosis via miR-221 downregulation. In addition, we validated another miR-221 target, SOCS1, which is known to be a negative regulator of JAK/STAT signaling. Consistent with this, miR-221 overexpression in mDCs enhanced the secretion of proinflammatory cytokines IL-6 and TNF-α. In peripheral blood mononuclear cells (PBMCs) of HIV-1/HCV co-infected individuals undergoing IFN-α-based treatment the baseline miR-221 expression was lower in non-responders compared with responders; and miR-221 expression directly correlated with DC frequency and IL-6/TNF-α secretion. In addition to PBMCs, we isolated total liver cells and kupffer cells from HCV-infected individuals and individuals with alcoholic cirrhosis. We found that both total liver cells and kupffer cells from HCV-infected individuals had significantly higher miR-221 levels compared with individuals with cirrhosis. Overall, we demonstrate that IFN-α exerts both antiproliferative and immunomodulatory effects on mDCs via miR-221 downregulation; and IFN-miR-221 axis can play important role in HCV pathogenesis and treatment.

MHC Class I Presented T Cell Epitopes as Potential Antigens for Therapeutic Vaccine against HBV Chronic Infection.

Approximately 370 million people worldwide are chronically infected with hepatitis B virus (HBV). Despite the success of the prophylactic HBV vaccine, no therapeutic vaccine or other immunotherapy modality is available for treatment of chronically infected individuals. Clearance of HBV depends on robust, sustained CD8(+) T activity; however, the limited numbers of therapeutic vaccines tested have not induced such a response. Most of these vaccines have relied on peptide prediction algorithms to identify MHC-I epitopes or characterization of T cell responses during acute infection. Here, we took an immunoproteomic approach to characterize MHC-I restricted epitopes from cells chronically infected with HBV and therefore more likely to represent the true targets of CD8(+) T cells during chronic infection. In this study, we identified eight novel MHC-I restricted epitopes derived from a broad range of HBV proteins that were capable of activating CD8(+) T cells. Furthermore, five of the eight epitopes were able to bind HLA-A2 and A24 alleles and activated HBV specific T cell responses. These epitopes also have potential as new tools to characterize T cell immunity in chronic HBV infection and may serve as candidate antigens for a therapeutic vaccine against HBV infection.

MHC class I antigen presentation and implications for developing a new generation of therapeutic vaccines.

Major histocompatibility complex class I (MHC-I) presented peptide epitopes provide a 'window' into the changes occurring in a cell. Conventionally, these peptides are generated by proteolysis of endogenously synthesized proteins in the cytosol, loaded onto MHC-I molecules, and presented on the cell surface for surveillance by CD8(+) T cells. MHC-I restricted processing and presentation alerts the immune system to any infectious or tumorigenic processes unfolding intracellularly and provides potential targets for a cytotoxic T cell response. Therefore, therapeutic vaccines based on MHC-I presented peptide epitopes could, theoretically, induce CD8(+) T cell responses that have tangible clinical impacts on tumor eradication and patient survival. Three major methods have been used to identify MHC-I restricted epitopes for inclusion in peptide-based vaccines for cancer: genetic, motif prediction and, more recently, immunoproteomic analysis. Although the first two methods are capable of identifying T cell stimulatory epitopes, these have significant disadvantages and may not accurately represent epitopes presented by a tumor cell. In contrast, immunoproteomic methods can overcome these disadvantages and identify naturally processed and presented tumor associated epitopes that induce more clinically relevant tumor specific cytotoxic T cell responses. In this review, we discuss the importance of using the naturally presented MHC-I peptide repertoire in formulating peptide vaccines, the recent application of peptide-based vaccines in a variety of cancers, and highlight the pros and cons of the current state of peptide vaccines.

In vivo immunogenicity of Tax(11-19) epitope in HLA-A2/DTR transgenic mice: implication for dendritic cell-based anti-HTLV-1 vaccine.

Viral oncoprotein Tax plays key roles in transformation of human T-cell leukemia virus (HTLV-1)-infected T cells leading to adult T-cell leukemia (ATL), and is the key antigen recognized during HTLV-associated myelopathy (HAM). In HLA-A2+ asymptomatic carriers as well as ATL and HAM patients, Tax(11-19) epitope exhibits immunodominance. Here, we evaluate CD8 T-cell immune response against this epitope in the presence and absence of dendritic cells (DCs) given the recent encouraging observations made with Phase 1 DC-based vaccine trial for ATL. To facilitate these studies, we first generated an HLA-A2/DTR hybrid mouse strain carrying the HLA-A2.1 and CD11c-DTR genes. We then studied CD8 T-cell immune response against Tax(11-19) epitope delivered in the absence or presence of Freund's adjuvant and/or DCs. Overall results demonstrate that naturally presented Tax epitope could initiate an antigen-specific CD8T cell response in vivo but failed to do so upon DC depletion. Presence of adjuvant potentiated Tax(11-19)-specific response. Elevated serum IL-6 levels coincided with depletion of DCs whereas decreased TGF-ß was associated with adjuvant use. Thus, Tax(11-19) epitope is a potential candidate for the DC-based anti-HTLV-1 vaccine and the newly hybrid mouse strain could be used for investigating DC involvement in human class-I-restricted immune responses.

Dengue virus specific dual HLA binding T cell epitopes induce CD8+ T cell responses in seropositive individuals.

Dengue virus infects an estimated 300 million people each year and even more are at risk of becoming infected as the virus continues to spread into new areas. Despite the increase in viral prevalence, no anti-viral medications or vaccines are approved for treating or preventing infection. CD8+ T cell responses play a major role in viral clearance. Therefore, effective vaccines that induce a broad, multi-functional T cell response with substantial cross-reactivity between all virus serotypes can have major impacts on reducing infection rates and infection related complications. Here, we took an immunoproteomic approach to identify novel MHC class I restricted T cell epitopes presented by dengue virus infected cells, representing the natural and authentic targets of the T cell response. Using this approach we identified 4 novel MHC-I restricted epitopes: 2 with the binding motif for HLA-A24 molecules and 2 with both HLA-A2 and HLA-A24 binding motifs. These peptides were able to activate CD8+ T cell responses in both healthy, seronegative individuals and in seropositive individuals who have previously been infected with dengue virus. Importantly, the dual binding epitopes activated pre-existing T cell precursors in PBMCs obtained from both HLA-A2+ and HLA-A24+ seropositive individuals. Together, the data indicate that these epitopes are immunologically relevant T cell activating peptides presented on infected cells during a natural infection and therefore may serve as candidate antigens for the development of effective multi-serotype specific dengue virus vaccines.

Host Genetic Factors and Dendritic Cell Responses Associated with the Outcome of Interferon/Ribavirin Treatment in HIV-1/HCV Co-Infected Individuals.

HIV-1/HCV co-infection is a significant health problem. Highly active antiretroviral treatment (HAART) against HIV-1 has proved to be fairly successful. On the other hand, direct acting antiviral drugs against HCV have improved cure rates but high cost and development of drug resistance are important concerns. Therefore PEGylated interferon (PEG-IFN) and ribavirin (RBV) still remain essential components of HCV treatment, and identification of host factors that predict IFN/RBV treatment response is necessary for effective clinical management of HCV infection. Impaired dendritic cell (DC) and T cell responses are associated with HCV persistence. It has been shown that IFN/RBV treatment enhances HCV-specific T cell functions and it is likely that functional restoration of DCs is the underlying cause. To test this hypothesis, we utilized an antibody cocktail (consisting of DC maturation, adhesion and other surface markers) to perform comprehensive phenotypic characterization of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in a cohort of HIV-1/HCV co-infected individuals undergoing IFN/RBV treatment. Our results show that pre-treatment frequencies of mDCs are lower in non-responders (NRs) compared to responders (SVRs) and healthy controls. Although, the treatment was able to restore the frequency of mDCs in NRs, it downregulated the frequency of CCR7+, CD54+ and CD62L+ mDCs. Pre-treatment frequencies of pDCs were lower in NRs and decreased further upon treatment. Compared to SVRs, NRs exhibited higher ratio of PD-L1+/CD86+ pDCs prior to treatment; and this ratio remained high even after treatment. These findings demonstrate that enumeration and phenotypic assessment of DCs before/during therapy can help predict the treatment outcome. We also show that before treatment, PBMCs from SVRs secrete higher amounts of IFN-γ compared to controls and NRs. Upon genotyping IFNL3 polymorphisms rs12979860, rs4803217 and ss469415590, we found rs12979860 to be a better predictor of treatment outcome. Collectively, our study led to identification of important correlates of IFN/RBV treatment response in HIV-1/HCV co-infected individuals.

Autoantibody biomarkers identified by proteomics methods distinguish ovarian cancer from non-ovarian cancer with various CA-125 levels.

PURPOSE: CA-125 has been a valuable marker for detecting ovarian cancer, however, it is not sensitive enough to detect early-stage disease and not specific to ovarian cancer. The purpose of our study was to identify autoantibody markers that are specific to ovarian cancer regardless of CA-125 levels. METHODS: Top-down and iTRAQ quantitative proteomics methods were used to identify high-frequency autoantibodies in ovarian cancer. Protein microarrays comprising the recombinant autoantigens were screened using serum samples from various stages of ovarian cancer with diverse levels of CA-125 as well as benign and healthy controls. ROC curve and dot blot analyses were performed to validate the sensitivity and specificity of the autoantibody markers. RESULTS: The proteomics methodologies identified more than 60 potential high-frequency autoantibodies in ovarian cancer. Individual serum samples from ovarian cancer stages I-IV compared to control samples that were screened on a microarray containing native recombinant autoantigens revealed a panel of stage I high-frequency autoantibodies. Preliminary ROC curve and dot blot analyses performed with the ovarian cancer samples showed higher specificity and sensitivity as compared to CA-125. Three autoantibody markers exhibited higher specificity in various stages of ovarian cancer with low and normal CA-125 levels. CONCLUSIONS: Proteomics technologies are suitable for the identification of protein biomarkers and also the identification of autoantibody biomarkers when combined with protein microarray screening. Using native recombinant autoantigen arrays to screen autoantibody markers, it is possible to identify markers with higher sensitivity and specificity than CA-125 that are relevant to early detection of ovarian cancer.

MHC class I-presented T cell epitopes identified by immunoproteomics analysis are targets for a cross reactive influenza-specific T cell response.

Influenza virus infection and the resulting complications are a significant global public health problem. Improving humoral immunity to influenza is the target of current conventional influenza vaccines, however, these are generally not cross-protective. On the contrary, cell-mediated immunity generated by primary influenza infection provides substantial protection against serologically distinct viruses due to recognition of cross-reactive T cell epitopes, often from internal viral proteins conserved between viral subtypes. Efforts are underway to develop a universal flu vaccine that would stimulate both the humoral and cellular immune responses leading to long-lived memory. Such a universal vaccine should target conserved influenza virus antibody and T cell epitopes that do not vary from strain to strain. In the last decade, immunoproteomics, or the direct identification of HLA class I presented epitopes, has emerged as an alternative to the motif prediction method for the identification of T cell epitopes. In this study, we used this method to uncover several cross-specific MHC class I specific T cell epitopes naturally presented by influenza A-infected cells. These conserved T cell epitopes, when combined with a cross-reactive antibody epitope from the ectodomain of influenza M2, generate cross-strain specific cell mediated and humoral immunity. Overall, we have demonstrated that conserved epitope-specific CTLs could recognize multiple influenza strain infected target cells and, when combined with a universal antibody epitope, could generate virus specific humoral and T cell responses, a step toward a universal vaccine concept. These epitopes also have potential as new tools to characterize T cell immunity in influenza infection, and may serve as part of a universal vaccine candidate complementary to current vaccines.

Unique and differential protein signatures within the mononuclear cells of HIV-1 and HCV mono-infected and co-infected patients.

BACKGROUND: Pathogenesis of liver damage in patients with HIV and HCV co-infection is complex and multifactorial. Although global awareness regarding HIV-1/HCV co-infection is increasing little is known about the pathophysiology that mediates the rapid progression to hepatic disease in the co-infected individuals. RESULTS: In this study, we investigated the proteome profiles of peripheral blood mononuclear cells from HIV-1 mono-, HCV mono-, and HIV-1/HCV co-infected patients. The results of high-resolution 2D gel electrophoresis and PD quest software quantitative analysis revealed that several proteins were differentially expressed in HIV-1, HCV, and HIV-1/HCV co-infection. Liquid chromatography-mass spectrometry and Mascot database matching (LC-MS/MS analysis) successfully identified 29 unique and differentially expressed proteins. These included cytoskeletal proteins (tropomyosin, gelsolin, DYPLSL3, DYPLSL4 and profilin-1), chaperones and co-chaperones (HSP90-beta and stress-induced phosphoprotein), metabolic and pre-apoptotic proteins (guanosine triphosphate [GTP]-binding nuclear protein Ran, the detoxifying enzyme glutathione S-transferase (GST) and Rho GDP-dissociation inhibitor (Rho-GDI), proteins involved in cell prosurvival mechanism, and those involved in matrix synthesis (collagen binding protein 2 [CBP2]). The six most significant and relevant proteins were further validated in a group of mono- and co-infected patients (n = 20) at the transcriptional levels. CONCLUSIONS: The specific pro- and anti- apoptotic protein signatures revealed in this study could facilitate the understanding of apoptotic and protective immune-mediated mechanisms underlying HIV-1 and HCV co-infection and their implications on liver disease progression in co-infected patients.

Investigation of ovarian cancer associated sialylation changes in N-linked glycopeptides by quantitative proteomics.

BACKGROUND: In approximately 80% of patients, ovarian cancer is diagnosed when the patient is already in the advanced stages of the disease. CA125 is currently used as the marker for ovarian cancer; however, it lacks specificity and sensitivity for detecting early stage disease. There is a critical unmet need for sensitive and specific routine screening tests for early diagnosis that can reduce ovarian cancer lethality by reliably detecting the disease at its earliest and treatable stages. RESULTS: In this study, we investigated the N-linked sialylated glycopeptides in serum samples from healthy and ovarian cancer patients using Lectin-directed Tandem Labeling (LTL) and iTRAQ quantitative proteomics methods. We identified 45 N-linked sialylated glycopeptides containing 46 glycosylation sites. Among those, ten sialylated glycopeptides were significantly up-regulated in ovarian cancer patients' serum samples. LC-MS/MS analysis of the non-glycosylated peptides from the same samples, western blot data using lectin enriched glycoproteins of various ovarian cancer type samples, and PNGase F (+/-) treatment confirmed the sialylation changes in the ovarian cancer samples. CONCLUSION: Herein, we demonstrated that several proteins are aberrantly sialylated in N-linked glycopeptides in ovarian cancer and detection of glycopeptides with abnormal sialylation changes may have the potential to serve as biomarkers for ovarian cancer.

Quantitative immunoproteomics analysis reveals novel MHC class I presented peptides in cisplatin-resistant ovarian cancer cells.

Platinum-based chemotherapy is widely used to treat various cancers including ovarian cancer. However, the mortality rate for patients with ovarian cancer is extremely high, largely due to chemo-resistant progression in patients who respond initially to platinum based chemotherapy. Immunotherapy strategies, including antigen specific vaccines, are being tested to treat drug resistant ovarian cancer with variable results. The identification of drug resistant specific tumor antigens would potentially provide significant improvement in effectiveness when combined with current and emerging therapies. In this study, using an immunoproteomics method based on iTRAQ technology and an LC-MS platform, we identified 952 MHC class I presented peptides. Quantitative analysis of the iTRAQ labeled MHC peptides revealed that cisplatin-resistant ovarian cancer cells display increased levels of MHC peptides derived from proteins that are implicated in many important cancer pathways. In addition, selected differentially presented epitope specific CTL recognize cisplatin-resistant ovarian cancer cells significantly better than the sensitive cells. These over-presented, drug resistance specific MHC class I associated peptide antigens could be potential targets for the development of immunotherapeutic strategies for the treatment of ovarian cancer including the drug resistant phenotype.

Cytotoxic T-lymphocyte immunotherapy for ovarian cancer: a pilot study.

The objective was to evaluate the toxicity and feasibility of intraperitoneal infusion of tumor-specific cytotoxic T lymphocytes (CTL) as therapy for recurrent ovarian cancer, and to determine if repetitive cycles of CTL generation and infusion measurably increases the host's ovarian cancer immune response. In this study, 7 subjects with recurrent ovarian cancer confined to the peritoneal cavity underwent up to 4 cycles, each cycle beginning with a leukapheresis for collection of precursor lymphocytes, which were stimulated in vitro with mucin 1, a tumor-specific antigen found commonly in ovarian cancer cells. The resulting new CTL for each cycle were reintroduced into the host by intraperitoneal infusion. Immunologic parameters (killer cells, cytokine production, memory T lymphocytes, and natural killer cells) were studied. Toxicity, CA-125, and survival data were also evaluated. The tumor marker CA-125 was nonstatistically significantly reduced after the first month of immunotherapy. However, after that it rose. Killer cells, cytokine production, and memory T lymphocytes increased after the first cycle of stimulation, but plateaued or reduced thereafter. The percent of natural killer cells inversely correlated with other immune parameters. Median survival was 11.5 months. One subject is free of disease since December, 2000. Multiple cycles, beyond 1 cycle, of T-cell stimulation followed by adoptive T-cell infusion, may not enhance the in vivo immune response.

Conserved MHC class I-presented dengue virus epitopes identified by immunoproteomics analysis are targets for cross-serotype reactive T-cell response.

Dengue fever and dengue hemorrhagic fever are significant global public health problems, and understanding the overall immune response to infection will contribute to appropriate management of the disease and its potentially severe complications. Live attenuated and subunit vaccine candidates, which are under clinical evaluation, induce primarily an antibody response to the virus and minimal cross-reactive T-cell responses. Currently, there are no available tools to assess protective T-cell responses during infection or after vaccination. In this study, we utilize an immunoproteomics process to uncover novel HLA-A2-specific epitopes derived from dengue virus (DV)-infected cells. These epitopes are conserved, and we report that epitope-specific cytotoxic lymphocytes (CTLs) are cross-reactive against all 4 DV serotypes. These epitopes have potential as new informational and diagnostic tools to characterize T-cell immunity in DV infection and may serve as part of a universal vaccine candidate complementary to current vaccines in trial.

Role of T-cell epitope-based vaccine in prophylactic and therapeutic applications.

Prophylactic and therapeutic vaccines against viral infections have advanced in recent years from attenuated live vaccines to subunit-based vaccines. An ideal prophylactic vaccine should mimic the natural immunity induced by an infection, in that it should generate long-lasting adaptive immunity. To complement subunit vaccines, which primarily target an antibody response, different methodologies are being investigated to develop vaccines capable of driving cellular immunity. T-cell epitope discovery is central to this concept. In this review, the significance of T-cell epitope-based vaccines for prophylactic and therapeutic applications is discussed. Additionally, methodologies for the discovery of T-cell epitopes, as well as recent developments in the clinical testing of these vaccines for various viral infections, are explained.

A ubiquitin independent degradation pathway utilized by a hepatitis B virus envelope protein to limit antigen presentation.

Hepatitis B virus envelope glycoproteins Large (L), Middle (M) and Small (S) are targets of the host cellular immune system. The extent to which the host recognizes viral antigens presented by infected cells is believed to play a decisive role in determining if an infection will be resolved or become chronic. As with other antigens, HBV envelope polypeptides must be degraded, presumably by cellular proteasomes, to be presented by the MHC I pathway. We have used M as a model to study this process and determine how ER quality control monitors these foreign polymeric proteins and disposes of them through the ER-associated degradation (ERAD) pathway. Using both wild type and mutant HBV M protein, we found that unlike most ERAD substrates, which require ubiquitination for retrotranslocation and degradation, the HBV M protein, which only contains two lysine residues, can undergo rapid and complete, ubiquitin independent, proteasome dependent degradation. The utilization of this pathway had a functional consequence, since proteins degraded through it, were poorly presented via MHC I. To test the hypothesis that the level of ubiquitination, independent of protein degradation, controls the level of antigen presentation, we inserted two additional lysines into both the wild type and mutant M protein. Amazingly, while the addition of the lysine residues dramatically increased the level of ubiquitination, it did not alter the rate of degradation. However and remarkably, the increased ubiquitination was associated with a dramatic increase in the level of antigen presentation. In conclusion, using the HBV surface protein as a model, we have identified a novel ubiquitin independent degradation pathway and determined that this pathway can have implications for antigen presentation and potentially viral pathogenesis.

Investigation of plasma biomarkers in HIV-1/HCV mono- and coinfected individuals by multiplex iTRAQ quantitative proteomics.

The analysis of plasma samples from HIV-1/HCV mono- and coinfected individuals by quantitative proteomics is an efficient strategy to investigate changes in protein abundances and to characterize the proteins that are the effectors of cellular functions involved in viral pathogenesis. In this study, the infected and healthy plasma samples (in triplicate) were treated with ProteoMiner beads to equalize protein concentrations and subjected to 4-plex iTRAQ labeling and liquid chromatography/mass spectrometry (LC-MS/MS) analysis. A total of 70 proteins were identified with high confidence in the triplicate analysis of plasma proteins and 65% of the proteins were found to be common among the three replicates. Apolipoproteins and complement proteins are the two major classes of proteins that exhibited differential regulation. The results of quantitative analysis revealed that APOA2, APOC2, APOE, C3, HRG proteins were upregulated in the plasma of all the three HIV-1 mono-, HCV mono-, and coinfected patient samples compared to healthy control samples. Ingenuity pathway analysis (IPA) of the upregulated proteins revealed that they are implicated in the hepatic lipid metabolism, inflammation, and acute-phase response signaling pathways. Thus, we identified several differentially regulated proteins in HIV-1/HCV mono and coinfected plasma samples that may be potential biomarkers for liver disease.

MHC class I-presented lung cancer-associated tumor antigens identified by immunoproteomics analysis are targets for cancer-specific T cell response.

The development of potent cancer vaccines for common malignancies such as lung cancer requires identification of suitable target antigens. We hypothesized that peptide epitopes naturally presented by MHC class I molecules on the surface of cancer cells would be the most relevant targets. We used LC/MS/MS analysis and identified 68 MHC class I-presented peptides from lung cancer cells. Using the criteria of strong consensus for HLA-A2 binding and relevance of the source proteins to malignant phenotype, we selected 8 peptides for functional characterization. These peptides, with a range of binding affinities, were confirmed to stabilize HLA-A2 molecules and were used to activate peptide-specific CTLs that efficiently recognized lung tumor cells. No correlation between the transcript levels of the source proteins and the extent of peptide-specific T cell recognition of lung cancer cells was observed. Furthermore, the peptide specific CTLs failed to recognize HLA-A2+ normal lung cells despite expression of the mRNA encoding the source proteins from which the peptides were derived. We conclude that MHC class I associated peptide epitopes are a more relevant source of authentic tumor antigens than over-expressed proteins and the identified peptides may be used as antigens for therapeutic vaccine strategies to treat lung cancer.