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Abstract Background Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). Results Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results (CD4+cytokine+ cells and CD8+cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17–44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5–20%, depending upon the experiment. The inter-lab C.V. was lowest (18–24%) for samples with a mean of >0.5% IFNγ + T cells, and highest (57–82%) for samples with a mean of Conclusion ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.

It is known that physical linkage of TLR ligands and vaccine antigens significantly enhances the immunopotency of the linked antigens. We have used this approach to generate novel influenza vaccines that fuse the globular head domain of the protective hemagglutinin (HA) antigen with the potent TLR5 ligand, flagellin. These fusion proteins are efficiently expressed in standard E. coli fermentation systems and the HA moiety can be faithfully refolded to take on the native conformation of the globular head. In mouse models of influenza infection, the vaccines elicit robust antibody responses that mitigate disease and protect mice from lethal challenge. These immunologically potent vaccines can be efficiently manufactured to support pandemic response, pre-pandemic and seasonal vaccines. [ABSTRACT FROM AUTHOR]

The majority of untreated human immunodeficiency virus (HIV) type 1-infected individuals ultimately develop uncontrolled viremia and progressive disease. Cytotoxic T lymphocytes (CTLs) are known to play an important role in controlling HIV-1 replication, which has led to an increasing interest in augmenting conventional antiretroviral therapy with therapeutic vaccination. The successful development of a therapeutic vaccine will rely on the ability to correlate an aspect of the immune response with clinical outcome. In this study, the CD8[SUP+] T cell maturation status of antigen-specific cells in models of well and poorly controlled virus infections were compared, to show that a memory phenotype predominates when antigen loads are absent or low. In HIV-1 infection, the emergence of memory CD8[SUP+] T cells was found to occur only in individuals with highly suppressed viral replication for an extended duration. Such assessments of the immune response may provide a refined measure of virus control. [ABSTRACT FROM AUTHOR]

Influenza virus is a global health concern due to its unpredictable pandemic potential. This potential threat was realized in 2009 when an H1N1 virus emerged that resembled the 1918 virus in antigenicity but fortunately was not nearly as deadly. 5J8 is a human antibody that potently neutralizes a broad spectrum of H1N1 viruses, including the 1918 and 2009 pandemic viruses. Here, we present the crystal structure of 5 J8 Fab in complex with a bacterially expressed and refolded globular head domain from the hemagglutinin (HA) of the A/California/07/2009 (H1N1) pandemic virus. 5J8 recognizes a conserved epitope in and around the receptor binding site (RBS), and its HCDR3 closely mimics interactions of the sialic acid receptor. Electron microscopy (EM) reconstructions of 5J8 Fab in complex with an HA trimer from a 1986 H1 strain and with an engineered stabilized HA trimer from the 2009 H1 pandemic virus showed a similar mode of binding. As for other characterized RBS-targeted antibodies, 5J8 uses avidity to extend its breadth and affinity against divergent H1 strains. 5J8 selectively interacts with HA insertion residue 133a, which is conserved in pandemic H1 strains and has precluded binding of other RBS-targeted antibodies. Thus, the RBS of divergent HAs is targeted by 5J8 and adds to the growing arsenal of common recognition motifs for design of therapeutics and vaccines. Moreover, consistent with previous studies, the bacterially expressed H1 HA properly refolds, retaining its antigenic structure, and presents a low-cost and rapid alternative for engineering and manufacturing candidate flu vaccines. [ABSTRACT FROM AUTHOR]

There is considerable interest in developing immunotherapeutic approaches to elicit tumor-specific CTL responses in cancer patients. Epitope-based approaches aim to deliver the antigenic peptides or epitopes recognized by CTLs rather than the intact tumor antigen. Many tumor-associated proteins are nonmutated self proteins for which the dominant peptide epitopes are usually poorly immunogenic. The subdominant epitopes, however, can elicit robust T cell responses if optimized for their ability to bind to class I MHC molecules. Only the epitopes for a few tumor antigens expressed in human cancers have been defined to this level, mainly for technical reasons. The means to rapidly screen and characterize the binding of epitopes derived from complex tumor-associated antigens is an important enabling technology. Here, we have used the high-throughput technology iTopia to identify those peptides derived from the tumor-associated antigen survivin that bind 8 class I alleles. A library of overlapping nonamers spanning the length of the survivin protein was initially screened for peptides capable of binding each allele. Nineteen HLA-A*0201, zero HLA-A*0101, seven HLA-A*0301, twelve HLA-A*1101, twenty-four HLA-A*2402, six HLA-B*0702, six HLA-B*0801, and eight HLA-B*1501 binding peptides were identified based on an arbitrary cutoff. Peptides capable of binding a given allele were further characterized by their affinity for MHC class I molecules and by the rate of dissociation of the complex. This information should help guide functional studies and future epitope-based immunotherapies.