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Identifying naturally occurring peptides bound to HLA class I molecules recognized by HLA-restricted cytotoxic T lymphocytes (CTL) is both relevant and central to the development of effective immunotherapeutic strategies against cancer. Several cancer-related genes have been reported for ovarian cancer, but very few are known to be naturally processed T cell epitopes. In the present study we used mass spectrometry to identify 16 novel HLA-A2-bound peptides from HLA-A2(+) ovarian cancer cell lines. All 16 peptides are derived from source proteins with diverse functions and marked homology to known proteins found in public databases. Synthetic peptide analogues of identified sequences were found to stabilize HLA-A2.1, albeit with varying affinities. The peptides were found to be antigenic in that a primary CD8(+) CTL response could be elicited from normal donor blood. The CTL generated were not only peptide specific, but failed to recognize targets pulsed with control peptides. In addition, recognition of shared HLA-A2-restricted epitopes by these CTL is suggested by their reactivity with a subset of HLA-A2(+) tumor lines and freshly isolated cancer cells or cell lines established from peritoneal ascites. These results were further corroborated by competitive inhibition of lysis of an otherwise susceptible cell line in the presence of cold peptide-pulsed targets. Furthermore, lack of recognition of several HLA-A2(+) control cell lines or cells isolated from normal ovaries suggests that these peptides are cancer related. These findings broaden the list of CTL-defined antigens that could lead to the development of multi-epitope vaccines for the treatment of ovarian cancer.

Glycosylphosphatidylinositol-anchored (GPI)-Db molecules are defective in mediating cytotoxic T lymphocytes (CTL) lysis of transfected lymphoma cells, compared to their transmembrane (TM) counterpart. This defect is manifest when antigenic peptide must be processed and presented through the endogenous pathway. These same transfectants can be lysed by allospecific CTL, or by antigen-specific Db-restricted CTL when pulsed with appropriate exogenous synthetic peptide, demonstrating that they can bind and present peptide for CTL-mediated lympholysis. The defect apparently results from differences between GPI-Db and TM-Db assembly and transport, or from differences in membrane topology that affect CD8+ CTL recognition of major histocompatibility complex/peptide complex.

Vaccines are most commonly administered using a needle and syringe; however, their use is associated with numerous drawbacks such as needlestick injuries to health care workers and the costs and logistical challenges associated with the safe disposal of sharps in the medical waste stream. The seriousness of these issues, the need to simplify global immunization programs, and the development of needle-free vaccine delivery have become a global priority. One needle-free vaccine approach being developed is the use of a vaccine patch which delivers the vaccine through the skin. Referred to as transcutaneous immunization (TCI), the topical application of a vaccine formulation on the skin targets the skin as an immunologically active site. In this chapter, we describe the development of the vaccine patch system and its evaluation in both preclinical and clinical studies. These studies demonstrate that the vaccine patch is a simple to use, needle-free vaccine system which induces robust immune responses. With its ability to be used outside the cold chain, it represents a solution to one of the major challenges facing global health care today.

Identification ofMycobacterium tuberculosisproteins that can provide immunological protection against tuberculosis is essential for the development of a more effective vaccine. To identify new vaccine targets, we have used immunoaffinity chromatography to isolate class I HLA-A*0201-peptide complexes fromM. tuberculosis-infected cells and sequenced the isolated peptides by mass spectrometry. From this material, we have identified three peptides derived from a singleM. tuberculosisprotein that is encoded by theM. tuberculosisRv0341 gene. Although no known protein encoded by the Rv0341 gene has been described, it is predicted to give rise to a 479-amino-acid protein with a molecular mass of 43.9 kDa. The three peptides identified are all nested and were found to be antigenic, in that they were capable of inducing peptide-specific, CD8+T cells from healthy blood donors in vitro and capable of recognizing and lysingM. tuberculosis-infected dendritic cells. This methodology provides a powerful tool for the identification ofM. tuberculosisproteins that can be evaluated as potential vaccine candidates.

Alterations in expression of major histocompatibility complex (MHC) antigens on tumor cells clearly correlate with the tumorgenicity and metastatic potential of those cells. These changes in the biological behavior of the tumor cells are presumably secondary to resulting changes in their susceptibility to immune recognition and destruction. Murine leukemia viruses (MuLV) exert regulatory effects on class I genes of the MHC locus. MuLV infection results in substantial increases in cell surface expression of all three class I MHC antigens. These viral effects on MHC antigen expression profoundly influence immune-mediated interaction with the infected cells, as assessed by cytotoxic T lymphocyte recognition and killing. Control of class I MHC and beta-2 microglobulin genes by MuLV takes place via a trans-acting molecular mechanism. MuLV controls expression of widely separated endogenous cellular MHC genes, transfected xenogeneic class I MHC genes, and unintegrated chimeric genes consisting of fragments of class I MHC genes linked to a bacterial reporter gene. These findings indicate that MuLV exerts its effects on MHC expression via a trans mechanism. The MuLV-responsive sequences on the MHC genes appear to lie within 1.2 kilobases upstream of the initiation codon for those genes.

Two distinct antigenic sites were demonstrated on SV40 small t antigen using sera from hamsters bearing tumors induced by SV40-transformed cells. One of the antigenic sites on small t antigen cross-reacts with antigenic sites on large T antigen as rabbit antisera prepared against denatured large T antigen immunoprecipitated both proteins and its activity could be absorbed with extracts prepared from cells infected with the 0.54 0.59 deletion mutant dl2005 which synthesized large T antigen but not small t antigen. The second antigenic site was found to be unique for small t antigen and was identified using serum from a hamster bearing an SV40 lymphoma which preferentially immunoprecipitated small t antigen. Its activity to small t antigen could not be removed by absorption with dl 2005-infected cell extracts. Activity against unique antigenic sites on small t antigen was also found in sera from animals bearing another SV40-induced tumor (TSV5). The unique antigenic sites on small t antigen were located on the C-terminal half of the protein since shortened small t polypeptide induced by d1891 (which lacks C-terminal sequences) was not immunoprecipitated by the lymphoma serum. Unique antigenic sites on large T antigen were also demonstrated as certain sera from tumor-bearing hamsters immunoprecipitated only the large T antigen. These results indicate that animals bearing SV40 tumors immunologically distinguish the large T and small t antigens and produce antibodies specific for the common and unique antigenic sites on the two proteins.

Moloney murine leukemia virus (M-MuLV) and Moloney murine sarcoma virus (M-MSV) exert a regulatory effect on the class I genes of the murine major histocompatibility complex (MHC). We have previously shown that M-MuLV infection of mouse fibroblasts results in a substantial increase in cell surface expression of H-2K, H-2D, and H-2L proteins, whereas M-MSV, upon coinfection of the same cells, is apparently able to override the MuLV-induced increase in H-2 expression. As a result of this modulation, immune recognition of the infected cells is profoundly altered. Our efforts have been directed toward elucidating the molecular basis for this phenomenon. We report here that stimulation of interferon production as a result of infection with MuLV does not occur and, therefore, is not the cause of MuLV-induced enhancement of MHC expression. Control of H-2 class I and beta 2-microglobulin gene expression by M-MuLV, and probably by M-MSV, takes place at the transcriptional level as indicated by nuclear runoff studies and analysis of steady-state mRNA levels. Our demonstration that M-MuLV controls expression of widely separated endogenous cellular genes (those coding for H-2D, H-2K, H-2L, and beta 2-microglobulin), transfected class I MHC genes, and unintegrated chimeric genes consisting of fragments of class I MHC genes linked to sequences encoding a procaryotic enzyme, chloramphenicol acetyltransferase, suggests that M-MuLV exerts its effect in trans and not by proviral integration in the vicinity of the H-2 gene complex. Finally, we show that the sequences of at least one MHC gene, which are responsive to trans regulation by M-MuLV, lie within 1.2 kilobases upstream of the initiation codon for that gene.

Simian virus 40 large T antigen (D2 protein), purified to homogeneity from HeLa cells infected with adenovirus 2-SV40 hybrid virus (Ad2+D2), was tested for the presence of SV40 transplantation rejection antigenic sites by the in vivo transplantation rejection assay and by the in vitro lymphocyte-mediated cytotoxicity assay. The results show that the D2 protein immunized BALB/c mice against a challenge of syngeneic SV40 tumor cells. The D2 protein, upon injection into C57B1/6 mice, also induced the generation of thymus-derived cytotoxic lymphocytes which specifically lysed SV40-transformed C57B1/6 mouse embryo fibroblasts. These results suggest that a product(s) coded by the early SV40 genome is present at the surface of SV40-transformed cells and acts as the transplantation rejection antigen.

Early viable deletion (0.54/0.59) mutants were tested for their ability to induce SV40 transplantation antigen (TrAg) in infected or transformed nonpermissive mouse cells in an attempt to determine the requirement for small t antigen in the expression of TrAg at the cell surface. The results indicate that dl (0.54/0.59) mutants are as efficient as wild-type SV40 in generating specific cytotoxic lymphocytes in C57BU6 mice and in immunizing BALB/c mice against an SV40 tumor cell challenge. Mouse (C57BU6) cells transformed by these mutants were also susceptible to lysis by the specifically sensitized lymphocytes. It can, therefore, be concluded that the synthesis of small t antigen is not an absolute requirement for expression of SV40 TrAg in SV40-infected or -transformed nonpermissive cells.

Primary mouse embryo fibroblasts of C57B1/6 origin and cells derived from a tumor induced by polyoma virus in a C57B1/6 mouse were transformed with SV40. The tumorigenic potential of these cells in normal adult and SV40-immunized mice was correlated with the synthesis of SV40 tumor antigens including the transplantation rejection antigen (TrAg) and with the presence of SV40 early region DNA sequences. Primary cells transformed by SV40 (B6/WT-3) induced tumors in immunocompetent adult syngeneic mice after adaptation in the immunosuppressed host. Passage of these tumor cells (B6/WT-3-T) through SV40-immunized mice resulted in the retention of both T and t antigens and TrAg. However, passage of SV40-transformed polyoma tumor cells through SV40-immunized immunocompetent ;adult mice but not: in nonimmunized mice resulted in the loss of expression of SV40 tumor antigens including TrAg. This loss correlated with the loss of SV40 early region sequences from these double transformed cells. These results demonstrate that the establishment of in vitro SV40-transformed primary mouse cells into a tumor capable of progressive growth in high responder mice does not lead to the selection of variants which have lost the expression of early region DNA sequences.

Simian virus 40 (SV40) large tumor (T) antigen was isolated from SV40-infected permissive and -transformed cells by immunoprecipitation with anti-T sera and purified by electrophoresis on solubilizable SDS-polyacrylamide gels. This method proved to be a rapid and efficient procedure for purifying T antigen as 74% of the immunoprecipitated T antigen was subsequently recovered. The purified, SDS-denatured large T antigen retained its ability to react with anti-T antibody and to immunize mice against a challenge of syngeneic SV40 tumor cells, indicating that the antigenic sites present on the large T antigen molecule that induce a tumor rejection response in vivo are resistant to denaturation by SDS.

The lysis of murine sarcoma virus–murine leukaemia virus (MSV–MuLV)-induced tumour cells by cytotoxic T lymphocytes (CTL) appears to require that an antigen specified by MSV–MuLV, or induced by the infection, be presented in association with class I major histocompatibility complex antigens1,2. The viral proteins of the tumorigenic MuLV seem to be a part of the antigens recognized by these dually restricted anti-MuLV CTL, but the precise nature of the putative viral antigen(s) recognized by CTL is unknown. Studies using recombinant viruses have suggested that a product of the viral envelope gene (env gene), perhaps the glycoprotein gp70, is the viral antigen recognized by CTL3. Attempts to use purified gp70 or anti-gp70 antibodies to block CTL recognition of retrovirus-induced tumour cells, however, have yielded contradictory results4–6. To examine more closely the role of gp70 in the CTL response to MuLV infections, we have constructed murine cell lines which express only the env gene of the Moloney murine leukaemia virus (M-MuLV). We show here that BALB/c-3T3 cells expressing the M-MuLV envelope gene products on their cell surface are susceptible to lysis by M-MuLV-specific CTL.

During viral transformation and/or infection, antigenic changes occur on the surface of transformed and infected cells that initiate a variety of cellular as well as humoral immune responses. A major component of this cellular immune response is the generation of cytotoxic T lymphocytes (CTL) which are capable of specifically recognizing and then lysing transformed or infected cells. Analysis of the specificity of these CTL has shown that immune recognition of virus-encoded antigens expressed as integral membrane proteins on the surface of these cells is in association with the class I major histocompatibility antigens H-2K, D, and L.(1–3) Target cells must not only express the appropriate viral antigen but must also express the same class I major histocompatibility antigen (H-2K, D, and L) as the effector CTL. This phenomenon is referred to as H-2-restricted, CTL recognition. This associative recognition (viral + H-2 antigens) required for CTL recognition contrasts with the recognition of antigen alone by antibody and has made it difficult to precisely define the antigenic determinants recognized by H-2-restricted, virus-specific CTL.