An adjuvant-modulated vaccine response in human whole blood.

The restimulation of an immune memory response by in vitro culture of blood cells with a specific antigen has been used as a way to gauge immunity to vaccines for decades. In this commentary we discuss a less appreciated application to support vaccine process development. We report that human whole blood from pre-primed subjects can generate a profound adjuvant-modulated, antigen-specific response to several different vaccine formulations. The response is able to differentiate subtle changes in the quality of an immune memory response to vaccine formulations and can be used to select optimal conditions relating to a particular manufacture process step. While questions relating to closeness to in vivo vaccination remain, the approach is another big step nearer to the more relevant human response. It has special importance for new adjuvant development, complementing other preclinical in vivo and in vitro approaches to considerably de-risk progression of novel vaccines before and throughout early clinical development. Broader implications of the approach are discussed.

Screening Vaccine Formulations in Fresh Human Whole Blood.

Monitoring the immunological functionality of vaccine formulations is critical for vaccine development. While the traditional approach using established animal models has been relatively effective, the use of animals is costly and cumbersome, and animal models are not always reflective of a human response. The development of a human-based approach would be a major step forward in understanding how vaccine formulations might behave in humans. Here, we describe a platform methodology using fresh human whole blood (hWB) to monitor adjuvant-modulated, antigen-specific responses to vaccine formulations, which is amenable to analysis by standard immunoassays as well as a variety of other analytical techniques.

A high ratio of IC31(®) adjuvant to antigen is necessary for H4 TB vaccine immunomodulation.

A tuberculosis (TB) vaccine consisting of a recombinant fusion protein (H4) and a novel TLR9 adjuvant (IC31) is in clinical development. To better understand the H4-IC31 ratio, we measured the binding capacity of IC31 for H4 protein and immunized mice with formulations that contained limiting to excess ratios of IC31 to H4. An immunomodulated H4-specific IFNγ response was only observed when IC31 was present in excess of H4. Since TLR expression is species-specific and the vaccine is intended to boost BCG-primed immunity, we questioned whether data in mice would translate to humans. To address this question, we used the fresh human Whole Blood (hWB) recovered from BCG-vaccinated subjects to screen H4-IC31 formulations. We found IC31 modulation in hWB to be quite distinct from the TLR4-Adjuvant. Unlike TLR4-Adjuvant, IC31 formulations did not induce the pro-inflammatory cytokine TNFα, but modulated a robust H4-specific IFNγ response after 12 d of culture. We then re-stimulated the fresh hWB of 5 BCG-primed subjects with formulations that had excess or limiting IC31 binding for H4 protein and again found that an immunomodulated H4-specific IFNγ response needed an excess of IC31. Finally, we monitored the zeta (ζ) potential of H4-IC31 formulations and found that the overall charge of H4-IC31 particles changes from negative to positive once IC31 is in greater than 9-fold excess. Using two diverse yet mutually supportive approaches, we confirm the need for an excess of IC31 adjuvant in H4 TB vaccine formulations and suggest surface potential may be an important factor.

Screening vaccine formulations for biological activity using fresh human whole blood.

Understanding the relevant biological activity of any pharmaceutical formulation destined for human use is crucial. For vaccine-based formulations, activity must reflect the expected immune response, while for non-vaccine therapeutic agents, such as monoclonal antibodies, a lack of immune response to the formulation is desired. During early formulation development, various biochemical and biophysical characteristics can be monitored in a high-throughput screening (HTS) format. However, it remains impractical and arguably unethical to screen samples in this way for immunological functionality in animal models. Furthermore, data for immunological functionality lag formulation design by months, making it cumbersome to relate back to formulations in real-time. It is also likely that animal testing may not accurately reflect the response in humans. For a more effective formulation screen, a human whole blood (hWB) approach can be used to assess immunological functionality. The functional activity relates directly to the human immune response to a complete formulation (adjuvant/antigen) and includes adjuvant response, antigen response, adjuvant-modulated antigen response, stability, and potentially safety. The following commentary discusses the hWB approach as a valuable new tool to de-risk manufacture, formulation design, and clinical progression.

A sensitive flow cytometry-based cytotoxic T-lymphocyte assay through detection of cleaved caspase 3 in target cells.

We describe a highly sensitive flow cytometry-based CTL assay using the cleavage of caspase 3 in target cells as a readout. The assay involved labeling of cells with a cell tracker dye and staining permeabilized cells with an antibody recognizing cleaved caspase 3. The assay proved to be robust and reliable in measuring antigen-specific CTL activity in a number of human and murine systems, including MLR, human peptide-specific T-cell responses induced in vitro, and CTL responses following immunization of mice with viral and peptide vaccines. The assay was found to yield comparable results as 51Cr-release, but with markedly higher sensitivity. When compared to detection of antigen-specific T cells via HLA tetramer/pentamer-based methods of T-cell staining in HIV gag peptide-specific human T cell lines the caspase 3 cleavage readout assay exhibited a comparable level of sensitivity with detection of CTL function at antigen-specific T-cell frequencies of 1:15,000 or lower. A similar level of sensitivity was obtained when murine CTL assays were performed with MLR in which effector cells were highly diluted with naïve syngeneic spleen cells. Our results indicate that the caspase 3 cleavage assay may be a powerful tool to measure antigen-specific CTL responses in human vaccine trials and in pre-clinical animal models of CTL function at both high and low effector cell frequencies.

The gene associated with trichorhinophalangeal syndrome in humans is overexpressed in breast cancer.

A comprehensive differential gene expression screen on a panel of 54 breast tumors and >200 normal tissue samples using DNA microarrays revealed 15 genes specifically overexpressed in breast cancer. One of the most prevalent genes found was trichorhinophalangeal syndrome type 1 (TRPS-1), a gene previously shown to be associated with three rare autosomal dominant genetic disorders known as the trichorhinophalangeal syndromes. A number of corroborating methodologies, including in situ hybridization, e-Northern analysis using ORF EST (ORESTES) and Unigene EST abundance analysis, immunoblot and immunofluorescence analysis of breast tumor cell lines, and immunohistochemistry, confirmed the microarray findings. Immunohistochemistry analysis found TRPS-1 protein expressed in >90% of early- and late-stage breast cancer, including ductal carcinoma in situ and invasive ductal, lobular, and papillary carcinomas. The TRPS-1 gene is also immunogenic with processed and presented peptides activating T cells found after vaccination of HLA-A2.1 transgenic mouse. Human T cell lines from HLA-A*0201+ female donors exhibiting TRPS-1-specific cytotoxic T lymphocyte activity could also be generated.