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

Hum Vaccin Immunother. 2014;10(12):3531-43. doi: 10.4161/21645515.2014.980210.

Comber JD1, Karabudak A, Huang X, Piazza PA, Marques ET, Philip R.

1a Immunotope Inc ; Doylestown , PA USA.

Abstract

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.

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

Hepat Res Treat. 2014;2014:860562. doi: 10.1155/2014/860562. Epub 2014 May 26.

Comber JD1, Karabudak A1, Shetty V2, Testa JS3, Huang X1, Philip R1.

1Immunotope, Inc., Doylestown, PA 18902, USA. 2Baylor College of Medicine, Houston, TX 77030, USA. 3Celldex Therapeutics, Hampton, NJ 08827, USA.

 

Abstract

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.

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

PLoS One. 2012;7(11):e48484. doi: 10.1371/journal.pone.0048484. Epub 2012 Nov 7.

Testa JS1, Shetty V, Hafner J, Nickens Z, Kamal S, Sinnathamby G, Philip R.

1Immunotope, Inc., Doylestown, PA, USA.

Abstract

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.

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

Clin Proteomics. 2012 Aug 2;9(1):10. doi: 10.1186/1559-0275-9-10.

Shetty V1, Hafner J, Shah P, Nickens Z, Philip R.

1Immunotope, Inc,, 3805 Old Easton Road, Doylestown, PA, 18902, USA.

Abstract

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.

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

J Proteomics. 2012 Jun 18;75(11):3270-90. doi: 10.1016/j.jprot.2012.03.044. Epub 2012 Apr 3.

Shetty V1, Nickens Z, Testa J, Hafner J, Sinnathamby G, Philip R.

1Immunotope, Inc., 3805 Old Easton Road, Doylestown, PA 18902, United States.

 

Abstract

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.

Copyright © 2012 Elsevier B.V. All rights reserved.

 

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

J Proteomics. 2011 May 1;74(5):728-43. doi: 10.1016/j.jprot.2011.02.020. Epub 2011 Mar 11.

Shetty V1, Sinnathamby G, Nickens Z, Shah P, Hafner J, Mariello L, Kamal S, Vlahović G, Lyerly HK, Morse MA, Philip R.

1Immunotope Inc., 3805 Old Easton Road, Doylestown, PA 18902, USA.

Abstract

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.

Copyright © 2011 Elsevier B.V. All rights reserved.

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Investigation of sialylation aberration in N-linked glycopeptides by lectin and tandem labeling (LTL) quantitative proteomics.

Anal Chem. 2010 Nov 15;82(22):9201-10. doi: 10.1021/ac101486d. Epub 2010 Oct 5.

Shetty V1, Nickens Z, Shah P, Sinnathamby G, Semmes OJ, Philip R.

1Immunotope, Inc., 3805 Old Easton Road, Doylestown, Pennsylvania 18902, USA.

Abstract

The accuracy in quantitative analysis of N-linked glycopeptides and glycosylation site mapping in cancer is critical to the fundamental question of whether the aberration is due to changes in the total concentration of glycoproteins or variations in the type of glycosylation of proteins. Toward this goal, we developed a lectin-directed tandem labeling (LTL) quantitative proteomics strategy in which we enriched sialylated glycopeptides by SNA, labeled them at the N-terminus by acetic anhydride ((1)H(6)/(2)D(6)) reagents, enzymatically deglycosylated the differentially labeled peptides in the presence of heavy water (H(2)(18)O), and performed LC/MS/MS analysis to identify glycopeptides. We successfully used fetuin as a model protein to test the feasibility of this LTL strategy not only to find true positive glycosylation sites but also to obtain accurate quantitative results on the glycosylation changes. Further, we implemented this method to investigate the sialylation changes in prostate cancer serum samples as compared to healthy controls. Herein, we report a total of 45 sialylated glycopeptides and an increase of sialylation in most of the glycoproteins identified in prostate cancer serum samples. Further quantitation of nonglycosylated peptides revealed that sialylation is increased in most of the glycoproteins, whereas the protein concentrations remain unchanged. Thus, LTL quantitative technique is potentially an useful method for obtaining simultaneous unambiguous identification and reliable quantification of N-linked glycopeptides.

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Shared immunoproteome for ovarian cancer diagnostics and immunotherapy: potential theranostic approach to cancer.

J Proteome Res. 2007 Jul;6(7):2509-17. Epub 2007 Jun 5.

Philip R1, Murthy S, Krakover J, Sinnathamby G, Zerfass J, Keller L, Philip M.

1Immunotope Inc., The Pennsylvania Biotechnology Center, 3805 Old Easton Road, Doylestown, Pennsylvania 18902, USA.

Abstract

Elimination of cancer through early detection and treatment is the ultimate goal of cancer research and is especially critical for ovarian and other forms of cancer typically diagnosed at very late stages that have very poor response rates. Proteomics has opened new avenues for the discovery of diagnostic and therapeutic targets. Immunoproteomics, which defines the subset of proteins involved in the immune response, holds considerable promise for providing a better understanding of the early-stage immune response to cancer as well as important insights into antigens that may be suitable for immunotherapy. Early administration of immunotherapeutic vaccines can potentially have profound effects on prevention of metastasis and may potentially cure through efficient and complete tumor elimination. We developed a mass-spectrometry-based method to identify novel autoantibody-based serum biomarkers for the early diagnosis of ovarian cancer that uses native tumor-associated proteins immunoprecipitated by autoantibodies from sera obtained from cancer patients and from cancer-free controls to identify autoantibody signatures that occur at high frequency only in cancer patient sera. Interestingly, we identified a subset of more than 50 autoantigens that were also processed and presented by MHC class I molecules on the surfaces of ovarian cancer cells and thus were common to the two immunological processes of humoral and cell-mediated immunity. These shared autoantigens were highly representative of families of proteins with roles in key processes in carcinogenesis and metastasis, such as cell cycle regulation, cell proliferation, apoptosis, tumor suppression, and cell adhesion. Autoantibodies appearing at the early stages of cancer suggest that this detectable immune response to the developing tumor can be exploited as early-stage biomarkers for the development of ovarian cancer diagnostics. Correspondingly, because the T-cell immune response depends on MHC class I processing and presentation of peptides, proteins that go through this pathway are potential candidates for the development of immunotherapeutics designed to activate a T-cell immune response to cancer. To the best of our knowledge, this is the first comprehensive study that identifies and categorizes proteins that are involved in both humoral and cell-mediated immunity against ovarian cancer, and it may have broad implications for the discovery and selection of theranostic molecular targets for cancer therapeutics and diagnostics in general.

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