No abstract

The Ark and Mass serotype-specific epitopes of infectious bronchitis virus were studied by immunofluorescence and immunoprecipitation of mutant and recombinant spike glycoproteins (S protein) expressed in mouse L cells. Serotype-specific monoclonal antibodies could bind to the recombinant proteins of Ark99 and Mass41 expressed from the chimeras in which the N-terminal thirds of the S1 sequences were reciprocally exchanged. Therefore, it appears that the respective serotype-specific epitopes of both strains were localized within the C-terminal two-thirds of the S1 proteins. Deletion and insertion of a five-amino-acid fragment on the S1 proteins of Ark99 and Mass41 altered the serotype-specific epitopes. This result implies that the five-aminoacid insertion on the S1 protein of the Ark serotype was involved in determining the conformation of the protein, probably acting as a spacer. In addition, it appears that an interaction between sequences of the N-terminal third and the remaining portion of the S1 protein determines the tertiary structure of the protein as well as the conformation of the serotype-specific epitope.

BACKGROUND: Women with TNBC who do not achieve a pathologic complete response (pCR) with preoperative (preop) chemotherapy have a high risk of recurrence and death from BC. Immunotherapy is an attractive strategy as human BCs can be immunogenic, and enhancing the immune effector function may augment the cytotoxic effects of standard therapies. CLINICAL TRIAL: Following IRB-approved informed consent, 10 pts with locally advanced TNBC received preop dose-dense doxorubicin/cyclophosphamide (AC) followed by paclitaxel and carboplatin (TCb) chemotherapy, combined with antigen-loaded (TNBC antigens: Cyclin B1, WT1, and control viral antigens: CEF) autologous monocyte-derived DC vaccinations administered intratumorally and subcutaneously. DCs were generated with GM-CSF and type I interferon, loaded with antigen in the form of long peptides and activated with innate ligands (LPS and Clo75) and CD40 ligand. Vaccines were given at 4 time points prior to definitive surgery, and 3 times post-surgery, pre- and post-radiation therapy (RT). Safety was the primary study endpoint, and pCR rate in breast and axilla was a secondary endpoint. Correlative studies included assessment of immune response via ELISpot and transcriptional profiling of blood samples collected over time. RESULTS: All pts received the 4 vaccines during preop chemotherapy, and 7/10 received all 7 vaccines. At the time of definitive surgery, 4 pts achieved a pCR, 3 pts had macroscopic residual disease in the breast and axillary lymph nodes, and 3 pts had residual cancer burden scores of 1. As of June 1, 2017, all pts have been in follow-up for at least 1 year s/p completion of all vaccines, and 7/10 patients have no evidence of disease. To assess immune signatures with IFN-γ-ELISpot, PBMCs from baseline (BL) and several time points during vaccine treatment were cultured with control peptides or with peptide libraries covering vaccine antigens. Using a linear mixed model to account for repeated and missing observations we found statistically significant (α = 0.05) increases in Cyclin B1, WT1, and CEF ELISpots in at least 1 time point post-DC vaccination and in follow-up. Compared to BL, Cyclin B1 and WT1 increased at 3 day pre-RT in 8/10 and 7/10 pts, respectively. To assess transcriptional signatures, a linear mixed model was utilized to determine statistically significant differences in fold-change over time compared to the BL and healthy controls. Modular analysis of differentially expressed transcripts at BL revealed downregulation of transcripts related to the monocyte lineage in 7/10 pts. Longitudinal analysis revealed profound transcriptional changes during AC with downregulation of lymphocyte modules and upregulation of innate and inflammation modules. While the latter ones have normalized during TCb and follow-up, T cell module remained substantially downregulated throughout treatment and follow-up. CONCLUSIONS: Combination of preop chemotherapy and intratumoral and subcutaneous autologous DC vaccination is safe in locally advanced TNBC pts and is linked with profound changes in immune transcription signatures and with expansion of antigen-specific immune responses that can be detected in IFN-γ ELISpot. Citation Format: Palucka AK, Roberts LK, Zurawski SM, Tarnowski J, Turner J, Wang X, Blankenship D, Smith JL, Levin MK, Finholt JP, Burkeholder SB, Timis R, Muniz LS, Dao T, Grant M, Banchereau J, Zurawski G, Pascual V, O'Shaughnessy JA. Immune and transcriptional signatures of dendritic dell (DC) vaccination combined with chemotherapy in locally advanced, triple-negative breast cancer (TNBC) patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-01.