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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.

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. Whilst 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 prior to and throughout early clinical development. Broader implications of the approach are discussed.

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.

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.

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.

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, includingin situhybridization, 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 carcinomain situand 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.

The CAG repeat polymorphism of the androgen receptor gene has been associated with an increased prostate cancer risk, and the repeat length correlated with cancer stage and grade at presentation. Men with an allele length of/= 18 CAG repeats have a 2-fold increase in risk for high-stage or high-grade prostate cancer, compared with patients with a longer CAG repeat. We examined the significance of the CAG repeat polymorphism of the androgen receptor gene for predicting prostate cancer progression among 318 patients treated by radical prostatectomy for clinically localized prostate cancer between 1987 and 1994.Leukocyte DNA was collected and genotyping of the CAG repeat polymorphism was performed using a PCR-based direct sequencing method. Risk ratios were calculated for developing biochemical recurrence for patients associated with an allele length of/= 18 CAG repeats, compared with patients with an allele length of18 CAG repeats, controlling for grade, stage and serum PSA level at diagnosis using Cox proportional hazard modeling.Overall, the CAG repeat allele was not predictive of recurrence; tumor grade, stage and PSA level at diagnosis were the only predictors of recurrence in a multivariate analysis. However, for patients at low risk for recurrence (Gleason score 2 to 6, stage pT2, and PSA/= 10 ng./ml.), the relative risk of recurrence associated with an allele of/= 18 CAG repeats was 8.07 (95% C.I., 2.02 to 32.2, p = 0.004), compared with patients with an allele length of18 CAG repeats. In contrast, for patients at high risk of recurrence (Gleason score/= 7, stage pT3/4, or PSA10 ng./ml.), the relative risk associated with the/= 18 CAG repeat allele was 0.72 (95% C.I., 0.33 to 1.57, p = 0.41), compared with patients with the18 CAG repeat allele.The length of the CAG repeat polymorphism of the androgen receptor gene may be important for prostate cancer recurrence among patients who are otherwise at low risk for recurrence after radical prostatectomy. These findings have potential implications for patient selection for adjuvant treatment, and for the development of novel treatments.

Mutations of the BRCA1 and BRCA2 genes account for approximately 80% of hereditary breast/ovarian cancer families, but the size of these two genes makes mutation analysis time-consuming and technically challenging. In some populations such as the Ashkenazi Jewish and the French-Canadian, a small number of recurrent founder mutations account for the majority of mutations in cancer families. We have therefore developed two rapid genetic screening tests, which allow us to detect three frequent frameshift mutations in the Ashkenazi Jewish population and five frameshift mutations in the French-Canadian population. These fluorescent non-radioactive methods permit the simultaneous detection of multiple mutations by generating multiplexed PCR-amplified gene fragments, and by discriminating these on the basis of their size in a denaturing polyacrylamide gel. Using these methods, we were able to correctly identify all mutants in a blinded analysis of 276 DNA samples, including 30 derived from paraffin-embedded tumor samples and 10 from buccal-cell brushes, with no false positive or false negative results. These techniques designed for the direct detection of recurrent mutations in the BRCA1 and BRCA2 genes, have the advantages of being efficient, sensitive, cost-effective, and are applicable to large scale screening for epidemiologic studies.

Background. Uterine papillary serous carcinoma (UPSC) shares common pathologic, genetic, and clinical features with other serous cancers of mullerian origin. The most common histologic type of ovarian tumor associated with BRCA mutations is papillary serous. Because of these histologic similarities, we postulated that, in some cases, UPSC may be a manifestation of a field defect in BRCA1 carriers, which also includes ovarian carcinoma, fallopian tube carcinoma, and primary peritoneal carcinoma. Methods. Fifty-six living patients with UPSC were contacted through their treating physicians and agreed to a family history interview and to provide a blood specimen for BRCA testing. The protein truncation test was used to detect mutations in exons 10 and 11 of BRCA1 and in exon 11 of BRCA2. The presence of four common mutations was assessed by PCR-based specific assays. Results. A high proportion of patients had a past history of breast cancer (11%) or a first-degree relative with breast cancer (29%). Four patients were from families with site-specific hereditary breast cancer. However, there was no clear example of the hereditary breast–ovarian cancer syndrome, and none of the 56 patients was found to carry a BRCA1 or BRCA2 mutation. Conclusions. BRCA mutations do not appear to predispose to UPSC and this type of cancer does not appear to be a manifestation of the classical hereditary breast–ovarian cancer syndrome. The observed association between UPSC and breast cancer may be due to the presence of mutations in other cancer predisposing genes.