Chimpanzee-origin adenovirus vectors as vaccine carriers.

Vaccines based on replication-defective adenoviral vectors are being developed for infectious agents and tumor-associated antigens. Early work focused on vaccines derived from a common human serotype of adenovirus, that is, adenovirus of the serotype 5 (AdHu5). Neutralizing antibodies against AdHu5 virus, present in a large percentage of the human population, dampen the efficacy of vaccines based on this carrier. To circumvent this problem, we generated vectors derived from chimpanzee adenoviruses. Here we describe some basic parameters of vectors derived from chimpanzee adenoviruses C68 and C7, including growth characteristics, yields of infectious particles, effects of additional deletions in E3 and E4 and lengths of the inserted foreign sequence as they relate to the suitability for their eventual development as vaccine carriers for clinical use.

Generation and characterization of monoclonal antibodies against the E6 and E7 oncoproteins of HPV.

Generation of three monoclonal antibodies (MAbs) to the major oncoproteins of human papillomavirus (HPV) was accomplished by an intense prime/boost regimen. Mice were primed with expression vectors expressing either the E6 or E7 oncoproteins of HPV-16 followed by boosting with a vaccinia virus construct and a replication-defective E1-deleted adenoviral recombinant of the human strain 5, and last, with baculovirus-derived HPV-16 E6 and E7 proteins in incomplete Freunds' adjuvant. Splenocytes were then fused with a myeloma cell line. The vaccination protocol generated one anti-E7 MAb of the IgM isotype and two anti-E6 MAbs of the IgG1 subisotype. The MAbs were tested for functionality in standard laboratory assays and found to detect the E6 and E7 proteins, respectively. The E7 MAb cross-reacted with the HPV-1a E7 oncoprotein. The binding sites of the MAbs were mapped to defined regions of each viral protein.

A method that allows easy characterization of tumor-infiltrating lymphocytes.

A method was developed to compare the lymphocytic infiltrates in regressing vs. progressing experimental mouse tumors using a model for human papillomavirus-16 (HPV-16) oncoprotein-linked cancer. Tumor cells mixed with matrigel, composed of natural matrix substances that provide a basement membrane structure for adherent cells, were inoculated into mice vaccinated with an efficacious vaccine to the E7 oncoprotein or a vaccine to a control antigen. The tumor cells remained within the solidified gel and recruited a cellular infiltrate that could readily be analyzed upon removal of the gelatinous mass containing progressing or regressing tumors. The results show that tumors recruit activated CD8(+) T cells regardless of their antigen specificity. In regressing tumors expressing an appropriate target antigen for the vaccine-induced CD8(+) T cells, a strong increase of the tumor antigen-specific T cell population was observed over time. Progressing tumors that lacked the target antigen for the activated CD8(+) T cell population did not show this selective enrichment.

Staining of antigen activated lymphocytes (SAAL): a highly specific method for flow cytometric quantitation of tumor-specific CD8(+) T cells.

A novel method for quantitative analysis of tumor-specific CD8(+) T lymphocytes was developed. Lymphocytes from mice vaccinated with tumor-associated antigens (TAAs) were expanded for 5 days in tissue culture and then stimulated in vitro for 5 h with tumor cells. They were subsequently surface-stained for CD8 and for intracellular interferon gamma (IFN-gamma) and analyzed by flow cytometry. The specificity and sensitivity of this assay, staining of antigen-activated lymphocytes (SAAL), was comparable to that of surface staining with major histocompatibily class (MHC) I-peptide tetramers or of staining of peptide re-stimulated CD8(+) T cells for intracellular IFN-gamma. The assay did not exhibit the high background activity of traditional 51Cr-release assays that without elaborate effector cell purifications commonly fail to distinguish between T cell-mediated antigen-specific cytolysis and non-specific lysis by lymphokine-activated killer (LAK) cells. The described method, which does not require prior identification of individual TAAs and their T cell epitopes nor access to specific reagents such as MHC-peptide tetramers, represents a simple yet useful technique for studying tumor-specific cytolytic T cell responses.

Viral recombinant vaccines to the E6 and E7 antigens of HPV-16.

Most cancerous lesions of the uterine cervix are linked to persistent infections with human papillomaviruses (HPV), most notably HPV-16 or -18. Vaccine-induced immune responses to the HPV early antigens E6 and E7, which contribute to cell transformation and are thus expressed in these cervical cancers, could potentially eradicate malignant cells. We generated recombinant vaccines based on E1-deleted adenovirus human strain 5 or on vaccinia virus strain Copenhagen expressing either the E6 or E7 oncoproteins of HPV-16. The different vaccines were compared in two experimental mouse tumor models employing Balb/c or C57Bl/6 mice. Data presented here demonstrate that depending on the model either CD4(+) or CD8(+) T cells provide protection to tumor cell challenge, resulting in striking differences in the efficacy of the four vaccines under investigation.

In situ stimulation of a T helper cell hybridoma with a cellulose-bound peptide antigen.

Many enzyme-linked immunosorbent assays take advantage of immobilized antigens for the identification of antibody binding sites. Generally, the analysis of cellulose membrane-bound B-cell epitopes is currently considered of high utility. We adapted this methodology for the stimulation of a T helper cell hybridoma with known specificity. Forty overlapping peptides corresponding to the entire rabies virus nucleoprotein were synthesized in duplicates on a single sheet of 90x130 mm size amino-modified paper. The efficacy of the peptide assembly was monitored by color staining of the unreacted amino groups. After completion of the synthesis, the side-chain protecting groups were removed, and the membrane was thoroughly cleaned of all organic and inorganic contaminants. The membrane was cut into pieces, and a standard lymphokine release assay was performed directly from the paper-bound antigens. From all the 40 peptide spots only peptide 31D stimulated the proliferation of the 9C5.D8-H T-cell hybridoma, known to react to this peptide. By using this protocol, as little as 0.4 microgram (approximately 200 pmole) of peptide could be detected. According to mass spectrometry the T-cell stimulation proceeded as a true solid-phase assay. The peptide neither leached from the membrane nor was cleaved by the medium-splenocyte mixture. Additionally, tryptic digestion of the cellulose membrane released the expected peptide fragments.

Genetic adjuvants.

In 1992, the era of DNA vaccines began with the report of antibody production upon intradermal injection of mice with a plasmid vector expressing a foreign antigen (1). A rapid succession of subsequent manuscripts showed stimulation of immune responses, including cytolytic T cells, upon inoculation of expression-vectors specific for antigens derived from viruses, bacteria, protozoa and tumor-associated antigens (2-7). Plasmid DNA can be applied through various routes of injection including: intradermal, intramuscular, subcutaneous, intravenous, or directly on mucosal membranes (1,2,8,9). The most commonly used methods of inoculation involve the use of DNA-coated gold beads propelled into the skin by a gene gun or intramuscular inoculation of the vector in saline solution.

The effect of CpG sequences on the B cell response to a viral glycoprotein encoded by a plasmid vector.

The effect of palindromic CpG sequences on the B cell response to plasmid vectors expressing a highly immunogenic viral glycoprotein was investigated. Methylation of the CpG sequences of bacterial expression vectors abolished their ability to induce an antibody response to the transgene product in mice. The antibody response could be rescued by concomitant injection of oligonucleotides carrying immunostimulatory sequences. The B cell response to two plasmid vectors, both expressing the same viral glycoprotein but containing a different content of the highly stimulatory AACGTT motif, was compared. Comparable B cell responses were induced to the two constructs given at an optimal vaccine dose while the vector containing additional palindromic sequences resulted in higher antibody titers at a suboptimal dose. These data indicate that deletion of CpG motifs or methylation of such sequences in plasmid DNA can abrogate the immune response to the vector encoded antigen and might thus enhance their usefulness as gene therapy vehicles.