Davin C. Dillon

Differential Immune Responses and Protective Efficacy Induced by Components of a Tuberculosis Polyprotein Vaccine, Mtb72F, Delivered as Naked DNA or Recombinant Protein

Key Ags of Mycobacterium tuberculosis initially identified in the context of host responses in healthy purified protein derivative-positive donors and infected C57BL/6 mice were prioritized for the development of a subunit vaccine against tuberculosis. Our lead construct, Mtb72F, codes for a 72-kDa polyprotein genetically linked in tandem in the linear order Mtb32C-Mtb39-Mtb32N. Immunization of C57BL/6 mice with Mtb72F DNA resulted in the generation of IFN-γ responses directed against the first two components of the polyprotein and a strong CD8+ T cell response directed exclusively against Mtb32C. In contrast, immunization of mice with Mtb72F protein formulated in the adjuvant AS02A resulted in the elicitation of a moderate IFN-γ response and a weak CD8+ T cell response to Mtb32c. However, immunization with a formulation of Mtb72F protein in AS01B adjuvant generated a comprehensive and robust immune response, resulting in the elicitation of strong IFN-γ and Ab responses encompassing all three components of the polyprotein vaccine and a strong CD8+ response directed against the same Mtb32C epitope identified by DNA immunization. All three forms of Mtb72F immunization resulted in the protection of C57BL/6 mice against aerosol challenge with a virulent strain of M. tuberculosis. Most importantly, immunization of guinea pigs with Mtb72F, delivered either as DNA or as a rAg-based vaccine, resulted in prolonged survival (>1 year) after aerosol challenge with virulent M. tuberculosis comparable to bacillus Calmette-Guérin immunization. Mtb72F in AS02A formulation is currently in phase I clinical trial, making it the first recombinant tuberculosis vaccine to be tested in humans.

Identification of genes overexpressed in head and neck squamous cell carcinoma using a combination of complementary DNA subtraction and microarray analysis.

Objectives/Hypothesis To discover unique genes specific for squamous cell carcinoma of the head and neck for eventual development as tumor markers and vaccine candidates.

T Cell Expression Cloning of a Mycobacterium tuberculosis Gene Encoding a Protective Antigen Associated with the Early Control of Infection

Infection of C57BL/6 mice with Mycobacterium tuberculosis results in the development of a progressive disease during the first 2 wk after challenge. Thereafter, the disease is controlled by the emergence of protective T cells. We have used this infection model in conjunction with direct T cell expression cloning to identify Ags involved with the early control of the disease. A protective M. tuberculosis-specific CD4 T cell line derived from mice at 3 wk postchallenge was used to directly screen an M. tuberculosis genomic expression library. This screen resulted in the identification of a genomic clone comprising two putative adjacent genes with predicted open reading frames of 10 and 41 kDa, MTB10 and MTB41, respectively (the products of Rv0916c and Rv0915c, respectively, in the TubercuList H37Rv database). MTB10 and MTB41 belong to the PE and PPE family of proteins recently identified to comprise 10% of the M. tuberculosis genome. Evaluation of the recombinant proteins revealed that MTB41, but not MTB10, is the Ag recognized by the cell line and by M. tuberculosis-sensitized human PBMC. Moreover, C57BL/6 mice immunized with MTB41 DNA developed both CD4- (predominantly Th1) and CD8-specific T cell responses to rMTB41 protein. More importantly, immunization of C57BL/6 mice with MTB41 DNA induced protection against infection with M. tuberculosis comparable to that induced by bacillus Calmette-Guérin. Thus, the use of a proven protective T cell line in conjunction with the T cell expression cloning approach resulted in the identification of a candidate Ag for a subunit vaccine against tuberculosis.

Use of multiepitope polyproteins in serodiagnosis of active tuberculosis.

ABSTRACT Screening of genomic expression libraries from Mycobacterium tuberculosis with sera from tuberculosis (TB) patients or rabbit antiserum to M. tuberculosis led to the identification of novel antigens capable of detecting specific antibodies to M. tuberculosis. Three antigens, Mtb11 (also known as CFP-10), Mtb8, and Mtb48, were tested together with the previously reported 38-kDa protein, in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies in TB patients. These four proteins were also produced as a genetically fused polyprotein, which was tested with two additional antigens, DPEP (also known as MPT32) and Mtb81. Sera from individuals with pulmonary and extrapulmonary TB, human immunodeficiency virus (HIV)-TB coinfections, and purified protein derivative (PPD)-positive and PPD-negative status with no evidence of disease were tested. In samples from HIV-negative individuals, the ELISA detected antibodies in >80% of smear-positive individuals and >60% smear-negative individuals, with a specificity of ∼98%. For this group, smears detected 81.6% but a combination of smear and ELISA had a sensitivity of ∼93%. The antigen combination detected a significant number of HIV-TB coinfections as well as antibodies in patients with extrapulmonary infections. Improved reactivity in the HIV-TB group was observed by including the antigen Mtb81 that was identified by proteomics. The data indicate that the use of multiple antigens, some of which are in a single polyprotein, can be used to facilitate the development of a highly sensitive test for M. tuberculosis antibody detection.

Classically Restricted Human CD8+ T Lymphocytes Derived from Mycobacterium tuberculosis-Infected Cells: Definition of Antigenic Specificity

Previous studies in murine and human models have suggested an important role for HLA Ia-restricted CD8+ T cells in host defense to Mycobacterium tuberculosis (Mtb). Therefore, understanding the Ags presented via HLA-Ia will be important in understanding the host response to Mtb and in rational vaccine design. We have used monocyte-derived dendritic cells in a limiting dilution analysis to generate Mtb-specific CD8+ T cells. Two HLA-Ia-restricted CD8+ T cell clones derived by this method were selected for detailed analysis. One was HLA-B44 restricted, and the other was HLA-B14 restricted. Both were found to react with Mtb-infected, but not bacillus Calmette-Guérin-infected, targets. For both these clones, the Ag was identified as culture filtrate protein 10 (CFP10)/Mtb11, a 10.8-kDa protein not expressed by bacillus Calmette-Guérin. Both clones were inhibited by the anti-class I Ab and anti-HLA-B,C Abs. Using a panel of CFP10/Mtb11-derived 15-aa peptides overlapping by 11 aa, the region containing the epitopes for both clones has been defined. Minimal 10-aa epitopes were defined for both clones. CD8+ effector cells specific for these two epitopes are present at high frequency in the circulating pool. Moreover, the CD8+ T cell response to CFP10/Mtb11 can be largely accounted for by the two epitopes defined herein, suggesting that this is the immunodominant response for this purified protein derivative-positive donor. This study represents the first time CD8+ T cells generated against Mtb-infected APC have been used to elucidate an Mtb-specific CD8+ T cell Ag.

Transcriptional complementarity in breast cancer: application to detection of circulating tumor cells.

AbstractBackground: We used a combination of genetic subtraction, silicon DNA micro-array analysis, and quantitative PCR to identify tissue-and tumor-specific genes as diagnostic targets for breast cancer. Methods and Results: From a large number of candidate antigens, several specific subsets of genes were identified that showed concordant and complementary expression profiles. Whereas transcriptional profiling of mammaglobin resulted in the detection of 70% of tumors in a panel of 46 primary and metastatic breast cancers, the inclusion of three additional markers resulted in detection of all 46 specimens. Immunomagnetic epithelial cell enrichment of circulating tumor cells from the peripheral blood of patients with metastatic breast cancer, coupled with RT-PCR-based amplification of breast tumor—specific transcripts, resulted in the detection of anchorage-independent tumor cells in the majority of patients with breast cancer with known metastatic disease. Conclusion: Complementation of mammaglobin with three additional genes in RT-PCR increases the detection of breast cancers in tissue and circulating tumor cells.

Serological expression cloning and immunological evaluation of MTB48, a novel Mycobacterium tuberculosis antigen

ABSTRACT Improved diagnostics are needed for the detection ofMycobacterium tuberculosis, especially for patients with smear-negative disease. To address this problem, we have screenedM. tuberculosis (H37Rv and Erdman strains) genomic expression libraries with pooled sera from patients with extrapulmonary disease and with sera from patients with elevated reactivity withM. tuberculosis lysate. Both serum pools were reactive with clones expressing a recombinant protein referred to here as MTB48. The genomic sequence of the resulting clones was identical to that of the M. tuberculosis H37Rv isolate and showed 99% identity to the Mycobacterium bovis and M. bovis BCG isolate sequences. The genomic location of this sequence is 826 bp upstream of a region containing theesat-6 gene that is deleted in the M. bovis BCG isolate. The mtb48 1,380-bp open reading frame encodes a predicted 47.6-kDa polypeptide with no known function. Southern and Western blot analyses indicate that this sequence is present in a single copy and is conserved in the M. tuberculosis and M. bovis isolates tested but not in other mycobacterial species tested, includingMycobacterium leprae and Mycobacterium avium. In addition, the native protein was detected in the cytoplasm, as was a processed form that was also shed into the medium during culture. Serological analysis of recombinant MTB48 and theM. tuberculosis 38-kDa antigen with a panel of patient and control sera indicates that the inclusion of recombinant MTB48 in a prototype serodiagnostic test increases assay sensitivity for M. tuberculosis infection when it is combined with other known immunodominant antigens, such as the 38-kDa antigen.

Characterization of KLK4 expression and detection of KLK4-specific antibody in prostate cancer patient sera.

The ability to identify prostate tumor or prostate tissue specific genes that are expressed at high levels and use their protein products as targets could greatly aid in the diagnosis and treatment of prostate cancer. Using a polymerase chain reaction (PCR)-based subtraction technique, we have recovered the recently described KLK4 (prostase) gene from human prostate cDNA. In this study, KLK4 gene expression in human prostate tumors was further characterized using cDNA quantitative PCR and immunohistochemistry, demonstrating that the gene is specifically expressed at both the mRNA and protein levels in normal human prostate tissue, and in both primary and metastatic prostate tumor samples. Quantitative mRNA analysis also demonstrated low level expression including adrenal gland, salivary gland and thyroid. Finally, it was demonstrated that prostate cancer patient sera contain antibodies that bind specifically to recombinant KLK4 protein. This antibody has been used to detect KLK4-specific peptides in epitope mapping experiments. The relatively specific expression profile and elevated level of KLK4 mRNA and protein in both tumor and normal prostate tissues, in addition to detectable KLK4-specific antibody in cancer patient sera, supports additional efforts to determine if KLK4 can play a role in the diagnosis of prostate cancer, the monitoring of residual disease, or act as a target for immunotherapy.

Detection of Mammaglobin in the Sera of Patients with Breast Cancer

Current procedures for the diagnosis of breast cancer are cumbersome and invasive, making detection of this disease difficult. A rapid screening test for early detection of breast cancer would allow for better management of this deadly disease. In this report, we show that, with the exception of the skin, mammaglobin mRNA is specifically expressed in mammary tissue and commonly overexpressed in breast cancer. Mammaglobin is not expressed in other types of cancer including colon, lung, ovarian, and prostate cancer. Breast-specific expression of mammaglobin protein was shown using immunohistochemical methods. Mammaglobin is secreted from both established breast cancer cell lines and primary breast carcinoma cells cultured in vitro. Using a monoclonal antibody-based assay for monitoring the presence of mammaglobin in serum, elevated levels of mammaglobin were detected in sera of patients with breast cancer, but not in healthy women. Thus, mammaglobin, which is overexpressed and secreted from breast carcinoma cells, is detectable in sera of patients with breast cancer and may provide a rapid screening test for the diagnosis and management of breast cancer.

Identification of Mycobacterium tuberculosis vaccine candidates using human CD4+ T-cells expression cloning.

To identify Mycobacterium tuberculosis (Mtb) antigens as candidates for a subunit vaccine against tuberculosis (TB), we have employed a CD4+ T-cell expression screening method. Mtb-specific CD4+ T-cell lines from nine healthy PPD positive donors were stimulated with different antigenic substrates including autologous dendritic cells (DC) infected with Mtb, or cultured with culture filtrate proteins (CFP), and purified protein derivative of Mtb (PPD). These lines were used to screen a genomic Mtb library expressed in Escherichia coli and processed and presented by autologous DC. This screening led to the recovery of numerous T-cell antigens, including both novel and previously described antigens. One of these novel antigens, referred to as Mtb9.8 (Rv0287), was recognized by multiple T-cell lines, stimulated with either Mtb-infected DC or CFP. Using the mouse and guinea pig models of TB, high levels of IFN-gamma were produced, and solid protection from Mtb challenge was observed following immunization with Mtb9.8 formulated in either AS02A or AS01B Adjuvant Systems. These results demonstrate that T-cell screening of the Mtb genome can be used to identify CD4+ T-cell antigens that are candidates for vaccine development.