Activated B cells mediate efficient expansion of rare antigen-specific T cells.

Potent professional antigen-presenting cells (APC) are essential tools to activate and expand antigen-specific T cells in vitro for use in adoptive immunotherapy. CD40-activated B cells can be easily generated and propagated from human donors and have been successfully used to generate antigen-specific T-cell cultures. Here we show that CD40-activated B cells strongly and specifically expand rare populations of antigen-specific CD8 T cells, with frequencies of less than 1 in 20,000 CD8 T cells in peripheral blood. We focused on T cells recognizing an epitope from the human papillomavirus 16 (HPV-16) E7 protein. In 6 of 6 healthy donors, epitope-specific CD8+ T cells were found to be "rare" by this criterion, as shown by staining with human leukocyte antigen (HLA)/peptide multimers. Using peptide-loaded CD40-activated B cells, epitope-specific T cells could be selectively expanded in all donors up to 10(6) fold, and the resulting T-cell cultures contained up to 88% specific T cells. These results strongly encourage the use of CD40-stimulated B cells as APCs in immunotherapy.

Chemically synthesized protein as tumour-specific vaccine: immunogenicity and efficacy of synthetic HPV16 E7 in the TC-1 mouse tumour model.

Many successful candidate vaccines capable of combating tumours in animal models come to an untimely end because of the costs associated with the approval and production of the GMP-grade materials, which are usually of biological origin, for use in humans. We have used a GMP-compatible method to chemically synthesize a pure synthetic E7 protein of the human papillomavirus type 16 (HPV16-E7). This oncogen-derived protein is constitutively expressed in cervical cancer and its precursors and is thus considered as an excellent target for tumour-specific immunity. Injection of a mixture of the synthetic HPV16-E7 protein and the synthetic adjuvant CpG in mice resulted in strong functional HPV16-specific cytotoxic T-lymphocyte responses as measured by CD8+ MHC class I-tetramer staining, the detection of antigen-specific intracellular IFNgamma production and the ability to protect mice against a challenge with HPV16-E7+ TC-1 tumour cells in both prophylactic and therapeutic vaccination regimens. Our results demonstrate the potential use of pure synthetic vaccines that can be efficiently produced under GMP at low cost, which will stimulate the translation of new vaccination strategies into phase I/II clinical trials.

A synthetic E7 gene of human papillomavirus type 16 that yields enhanced expression of the protein in mammalian cells and is useful for DNA immunization studies.

A synthetic E7 gene of human papillomavirus (HPV) type 16 was generated that consists entirely of preferred human codons. Expression analysis of the synthetic E7 gene in human and animal cells showed levels of E7 protein 20- to 100-fold higher than those obtained with wild-type E7. Enhanced expression of E7 protein resulted from highly efficient translation, as well as increased stability of the E7 mRNA due to its codon optimization. Higher levels of E7 protein in cells transfected with synthetic E7 correlated with significant loss of cell viability in various human cell lines. In contrast, lower E7 protein expression driven by the wild-type gene resulted in a slight induction of cell proliferation. Furthermore, mice inoculated with plasmids expressing the synthetic E7 gene produced significantly higher levels of E7 antibodies than littermates injected with wild-type E7, suggesting that synthetic E7 may be useful for DNA immunization studies and the development of genetic vaccines against HPV-16. In view of these results, we hypothesize that HPVs may have retained a pattern of G + C content and codon usage distinct from that of their host cells in response to selective pressure. Thus, the nonhuman codon bias may have been conserved by HPVs to prevent compromising viability of the host cells by excessive viral early protein expression, as well as to evade the immune system.

Synthetic peptides induce a cytotoxic response against human papillomavirus type-18.

OBJECTIVES: Over 90% of cervical carcinomas express human papillomavirus (HPV) E6 and E7 proteins. These unique antigens are ideal targets for the development of cytotoxic T-lymphocytes (CTL) for antitumor immunotherapy. In this study we identify peptides from HPV-18 E6 and E7 proteins that bind to HLA class I molecules. We further show that these peptides are able to induce peptide-specific CTL from an HLA-A2-positive (+) peripheral blood donor in vitro. METHODS: A computer-assisted algorithm was devised to identify peptides from HPV-18 E6 and E7 proteins that bind to HLA-A2 molecules. Peptides that were predicted to bind were synthesized and their binding activity was determined. HLA-A2(+) irradiated stimulator cells pulsed with HPV-18 peptides were incubated with HLA-A2(+) peripheral blood mononuclear cells. Cytotoxicity assays were performed to assess specific cell lysis. RESULTS: Of 295 possible sequences, the computer-assisted algorithm predicted 10 peptides that would have a high probability of binding to HLA-A2. The 4 strongest binding peptides were analyzed for their ability to induce cytotoxic cells against HPV-18 peptide-pulsed targets. Two of the peptides induced significant lysis. CONCLUSIONS: There are limited data on peptide-based immunotherapy for HPV-18(+) tumors. The combination of our computer-assisted algorithm and binding assay permits rapid selection of potential CTL epitopes. We identified two peptides that were able to induce peptide-specific lysis. These two epitopes are candidates for a peptide-based vaccine against HPV-18(+) tumors. The model described has broad applications and can be used in the development of immunotherapy for other types of cancers.

Synthetic peptides of human papillomavirus type 18 E6 harboring HLA-A2.1 motif can induce peptide-specific cytotoxic T-cells from peripheral blood mononuclear cells of healthy donors.

To identify cytotoxic T-cell (CTL) epitopes against human papillomavirus type 18 (HPV 18) E6 protein that might be useful for developing peptide-based vaccine against HPV 18 infection, 18 peptides which possibly contain CTL epitopes were selected on the basis of previously described human leukocyte antigen (HLA)-A2.1-binding motif and chemically synthesized. In the binding assay of the synthetic peptides, 8 out of 18 synthetic peptides enhanced the expression of HLA-A2.1 molecules on T2 cell surface, which implies that these peptides were able to bind the HLA molecules. Those peptides having good binding affinity to HLA-A2.1 were tested for their ability to activate CTLs which were isolated from peripheral blood mononuclear cells (PBMCs) of healthy blood donors and to kill the target T2 cells pulsed with the same peptide. Five out of eight tested peptides activated CTLs and killed the target cells.

T cell proliferative responses against human papillomavirus type 16 E7 oncoprotein are most prominent in cervical intraepithelial neoplasia patients with a persistent viral infection.

T cell proliferative responses against human papillomavirus type 16 (HPV-16) E7 protein were studied in relation to HPV status over time in 51 women originally diagnosed with abnormal cervical cytology and participating in a follow-up study. HPV-16-positive patients were grouped as having either a persistent, a cleared or a fluctuating HPV-16 infection as determined by PCR in consecutive cervical smears up until the moment of testing. Positive proliferative responses against HPV-16 E7 were found in 15/26 patients with a persistent, cleared or fluctuating HPV-16 infection (57.7%). In contrast, 0/15 patients who had been typed HPV-negative during follow-up showed positive responses (P = 0.0005). Further analysis showed positive responses to be more frequent in patients with persistent HPV-16 infections and stable or progressing cervical lesions (8/9 patients reactive, 88.9%) as compared to patients with cleared or fluctuating HPV-16 infections and stable or regressing cervical lesions (7/17, 41.1%, P = 0.04). The relatively strong T cell proliferative responses against HPV-16 E7 observed in patients with a persistent HPV-16 infection and progressive cervical lesions indicate that the effectivity of such responses cannot be predicted and apparently depends on additional factors.

Proliferative T cell responses to human papillomavirus type 16 L1 peptides in patients with cervical dysplasia.

Human papillomavirus type 16 (HPV-16) can cause genital warts, cervical dysplasias and carcinoma of the cervix. Cell-mediated immunity is thought to be important in protection against the virus and in its elimination, but little is known about the mechanisms involved. In a cross-sectional study we have demonstrated proliferative T cell responses to peptides representing the HPV-16 L1 capsid protein (aa 199-409) in the peripheral blood of 63% of patients (n = 41) with histological evidence of cervical dysplasia and in 45% of healthy age-matched controls (n = 11). This was achieved by generating short-term T cell lines (STLs) from each individual in vitro against a beta-galactosidase-HPV- 16 L1 (aa 199-409) fusion protein for 2 weeks, and then identifying the HPV epitopes they recognized with overlapping synthetic peptides (15-mers) spanning this region in 3 day specificity assays. Histological grading and HPV typing by PCR were performed on patients' cervical biopsies taken at the same clinical visit as the peripheral blood samples. An immunogenic region was identified between aa 311-345 in 73% of patients (18% in controls) who responded to HPV-16 L1 (aa 199-409). The number of responders to this region was significantly higher in patients with HPV-16-positive biopsies when compared to those with HPV-16-negative biopsies (P = 0.006), as was the number of responders to individual peptides 311-325 (NLASSNYFPTPSGSM; p = 0.04) and 321-335 (PSGSMVTSDAQIFNK; P = 0.004) representing this region. The mean level of response to each individual peptide was also higher in the patient group than the controls (P < 0.05). The most significant finding was that all patients with evidence of a current HPV-16 infection responded to one or more L1 peptides (P = 0.0004) and 92% had high grade cervical intraepithelial neoplasia (CIN III). We also found that the CIN III group was more likely to respond to any L1 peptide than either the atypical group (P = 0.04) or the controls (P = 0.05). Data from four individuals showed that the majority of peptide-specific STLs were CD4+ but some CD8+ STLs were also detected.

Sequence variation in the capsid protein genes of human papillomavirus type 16

We have cloned and sequenced the L1 and L2 genes from human papillomavirus type 16 (HPV16) DNA-containing cervical cytology samples collected from the U.K. and Trinidad. Samples containing high copy numbers of HPV16 DNA were selected as being likely to contain fully functional virus DNA molecules in an episomal state, rather than in an integrated and possibly altered state. In comparison with the perviously published sequence of HPV16 isolated from an invasive cancer a variety of differences were detected in both L1 and L2. The pattern of changes appears to be different in samples from the two geographic regions. One of the differences (resulting in D at position 202 of the L1 protein) reported recently to be functionally important for virus particle assembly was found to occur in all the samples examined. Variations in L1 found within known immunoreactive regions or hydrophobic domains should be taken into account in design of prophylactic vaccines for HPV16 based on virus-like particles. All variations within L2 protein were found in hydrophilic domains in the carboxy-terminal half of L2. These positions were highly variable among other types of papillomavirus and are located outside the known L2 immunoreactive region. (AU)

Direct analysis of peptide binding to cell-associated MHC class I molecules.

Exogenously added synthetic peptides can mimic endogenously produced antigenic peptides recognized on target cells by MHC class I-restricted cytolytic T lymphocytes. While it is assumed that exogenous peptides associate with class I molecules on the target cell surface, direct binding of peptides to cell-associated class I molecules has been difficult to demonstrate. Using a newly developed binding assay based on photoaffinity labeling, we have investigated the interaction of two antigenic peptides, known to be recognized in the context of H-2Kd or H-2Db, respectively, with 20 distinct class I alleles on living cells. None of the class I alleles tested, with the exception of H-2Kd or H-2Db, bound either of the peptides, thus demonstrating the exquisite specificity of peptide binding to class I molecules. Moreover, peptide binding to cell-associated H-2Kd was drastically reduced when metabolic energy, de novo protein synthesis or protein egress from the endoplasmic reticulum was inhibited. It is thus likely that exogenously added peptides do not associate with the bulk of class I molecules expressed at the cell surface, but rather bind to short-lived molecules devoid of endogenous peptides.

Chemical synthesis of human papillomavirus type 16 E7 oncoprotein: autonomous protein domains for induction of cellular DNA synthesis and for trans activation.

The human papillomavirus type 16 E7 protein belongs to a family of nuclear oncoproteins that share amino acid sequences and functional homology. To localize biochemical activities associated with E7, we chemically synthesized the full-length 98-amino-acid polypeptide and several deletion mutant peptides. We show that the E7 polypeptide is biologically active and possesses at least two functional domains; the first induces cellular DNA synthesis in quiescent rodent cells, and the second trans activates the adenovirus E1A-inducible early E2 promoter and binds zinc. Further, each domain is autonomous and can function on separate peptides. DNA synthesis induction activity maps within the N-terminal portion of the molecule, which contains sequences related to adenovirus E1A conserved domains 1 and 2 required for cell transformation and binding of the retinoblastoma gene product. trans-Activation and Zn-binding activities map within the C-terminal portion of the molecule, a region which contains Cys-X-X-Cys motifs. trans Activation does not require protein synthesis, implying a mechanism that involves interaction with a preexisting cellular factor(s). E7 trans activates the adenovirus E2 promoter but not other E1A-inducible viral promoters, suggesting the possibility that E7 trans activation involves interaction, directly or indirectly, with cellular transcription factor E2F.

In vitro trans-activation by chemically synthesized adenovirus region 3 peptides. Reaction properties and mutational analysis.

The adenovirus E1A gene encodes a protein that transcriptionally activates viral early genes. We have reported that a 49-amino acid chemically synthesized adenovirus type 2 E1A region 3 peptide, PD3 (residues 140-188 of the 289-amino acid protein), can stimulate transcription in vitro from the adenovirus major late promoter. Here we describe reaction properties of E1A trans-activation in vitro with the major late promoter and the early gene 3 promoter and the structural requirements for activity. Stimulation of transcription by PD3 is highest with low levels of DNA template and nuclear extract, and the presence of PD3 eliminates the need to preincubate template with nuclear extract to achieve optimal transcription. These findings suggest that PD3 facilitates a rate-limiting step in the formation of a promoter complex. Analysis of deletion and cysteine substitution mutant PD3 peptides indicates that the C-terminal 70% of the peptide is sufficient for trans-activation in vitro and supports the hypothesis that PD3 contains two functional subregions. The function of one region (residues 140 to about 152) can be overridden under conditions used for in vitro transcription. The second region (residues 153-188) is essential and may function both as a promoter-binding region and as an activating region in vitro.

Solid-phase synthesis of a nonadecapeptide coded for by the v-myb oncogene.

A nonadecapeptide comprising a predicted B-cell determinant from the v-myb oncoprotein was synthesized by Merrifield's solid-phase method. Hydrogen chloride in dichloromethane was used for protective t-butyloxycarbonyl group removal; the deprotection was monitored using a new qualitative deprotection test. The nonadecapeptide coupled to a carrier elicited a high titre of protein-reactive antipeptide antibodies.

Demonstration that a chemically synthesized BPV1 oncoprotein and its C-terminal domain function to induce cellular DNA synthesis.

Bovine papillomavirus type 1 contains the smallest known oncogene (ORF E5), encoding a hydrophobic 44 amino acid protein. To study the biochemical functions of the E5 oncoprotein, we have chemically synthesized it and several deletion mutant peptides. We demonstrate induction of cellular DNA synthesis in growth-arrested cells by microinjection of E5 oncoprotein. This activity can be broken down into two functionally distinguishable domains. Remarkably, the first domain, which alone is sufficient to induce cellular DNA synthesis, contains only the C-terminal 13 amino acids. This is the smallest known protein fragment that can autonomously activate cellular DNA synthesis. The second domain is the hydrophobic middle region, which by itself fails to induce cellular DNA synthesis but confers a 1000-fold increase in specific activity. The N-terminal one-third of the molecule is dispensable for induction of DNA synthesis.