Whole cell ELISA for detection of tumor antigen expression in tumor samples.

We have developed a whole cell enzyme-linked immunosorbent assay (ELISA) that detects the melanoma antigens gp100 and S-100 in tumor samples from patients with metastatic melanoma and the antigen CA 125 in tumor samples from patients with ovarian carcinoma. The assay is relatively simple to perform and interpret without specialized expertise in pathology. It is both sensitive and selective and is amenable to being automated. The results correlate very well with those obtained by flow cytometry and are in good accord with published values obtained by immunohistochemistry. We believe that this assay should be very helpful for characterizing autologous cell cancer vaccines and may also represent a useful alternative to immunohistochemistry for cancer diagnosis. It should be adaptable to other types of cancer where tumor antigens have been identified and good antibodies are available.

CD40 activation enhances the magnitude of cellular immune responses against p53 but not the avidity of the effectors.

Engagement of CD40 on the surface of antigen-presenting cells (APC) has been shown to substitute for T cell help in activating APC to stimulate cytotoxic T lymphocytes (CTL). We explored whether this powerful non-specific signal could enhance the CTL response to a self epitope from a tumor-associated antigen. We immunized mice with a lipopeptide covering the H-2Kd-restricted epitope, amino acids 232-240 of murine wild-type p53, followed by treatment with an activating anti-CD40 monoclonal antibody. Anti-CD40 antibody, given subcutaneously or intravenously, significantly enhanced effector activity against targets pulsed with non-lipidated 232-240 nonamer epitope peptide, as assessed both by a CTL lysis assay and an enzyme-linked immunospot (ELISPOT) assay for interferon-gamma-secreting cells. However, despite this enhancement, we could not detect activity against targets expressing p53 endogenously by either assay. This most likely reflects the low avidity of the effectors as determined by a titration of peptide on the target cells. The implications of this work for cancer immunotherapy based on specific responses directed against tumor-associated antigens are discussed.

OspA lipoprotein of Borrelia burgdorferi is a mucosal immunogen and adjuvant.

The outer surface protein A (OspA) lipoprotein of Borrelia burgdorferi, like cholera toxin and the heat-labile enterotoxin of Escherichia coli, induces pro-inflammatory cytokines. This suggested that, like those toxins, OspA might be a mucosal immunogen and adjuvant. OspA, administered intranasally (i.n.) or intragastrically, induced strong serum IgG and salivary gland IgA responses. The serum IgG isotypes were indicative of a mixed T helper 1 and T helper 2 response, the latter being more pronounced. The N-terminal tripalmitoyl-S-glyceryl-cysteine (Pam3Cys) lipid moiety was absolutely required. OspA strongly enhanced the serum IgG and salivary gland IgA responses to jack bean urease co-administered by the i.n. route. OspA also enhanced the response to tetanus toxoid and induced limited protection against challenge. A synthetic lipopeptide also adjuvanted the response to urease by the i.n. route, but was ca 500-fold less potent on a molar basis than OspA. These results suggest that OspA or other lipoproteins may be useful in mucosal vaccines.

Biological activities of native and recombinant Borrelia burgdorferi outer surface protein A: dependence on lipid modification.

Borrelia burgdorferi lipoproteins are 50- to 500-fold more active as cytokine inducers and B-cell mitogens than Escherichia coli lipoproteins and synthetic peptides containing the tripalmitoyl-S-glyceryl-cysteine moiety. To investigate the source of this unique potency, we compared native OspA from B. burgdorferi with recombinant lipidated OspA produced in E. coli. As little as 10 ng of either protein per ml stimulated B-cell proliferation and production of cytokines and nitric oxide by macrophages. The two proteins induced comparable antibody responses in mice. Nonlipidated OspA made in E. coli had no stimulatory activity. Thus, lipid modification is essential both in vivo and in vitro for the immunological properties of OspA. The lipid moiety appears equally active whether produced in B. burgdorferi or in E. coli.

Safety and immunogenicity of a recombinant outer surface protein A Lyme vaccine.

OBJECTIVE: To evaluate the safety and immunogenicity of a recombinant outer surface lipoprotein A (OspA) Lyme vaccine in healthy adults. DESIGN: Randomized, double-blind, placebo-controlled trial. SETTING: Clinical research unit of a medical center. PARTICIPANTS: Thirty-six healthy adult volunteers aged 18 through 65 years. INTERVENTIONS: Volunteers were randomly assigned to receive two 10-micrograms doses of OspA Lyme vaccine, OspA Lyme vaccine adsorbed to alum, or a buffer placebo. Subjects in the OspA Lyme vaccine group received a third dose. Patients were assessed after each vaccination for a total follow-up period of 1 year. Serum samples for antibody determination were drawn at baseline, 2 and 3 weeks after dose 1, once per week for 4 weeks after dose 2, 20 weeks after dose 2, and 1 month after dose 3. MAIN OUTCOME MEASURES: Local and systemic adverse reactions and antibody levels specific for OspA. RESULTS: The most common reactions were local pain and tenderness at the injection site. Adverse events did not increase following the second or third dose. Two doses of both vaccine formulations elicited high-titer antibodies that inhibited replication of Borrelia burgdorferi in vitro. No differences were noted in antibody levels elicited by the adsorbed and nonadsorbed formulations. CONCLUSION: Two or three doses of OspA Lyme vaccine are safe and immunogenic in adults.

Cloning, overexpression, and genomic mapping of the 14-kDa subunit of human replication protein A.

Replication protein A (RPA) is a three-subunit protein that plays a central role in eukaryotic DNA replication, recombination, and repair. We have previously reported the cloning and bacterial expression of the 70- and 32-kDa subunits of human RPA (hRPA). We have now cloned the 14-kDa subunit (hRPA3) from a HeLa cell cDNA library. The hRPA3 cDNA is a 692-base pair sequence that contains an open reading frame encoding a protein of 121 amino acids with a calculated molecular mass of 13.6 kDa. The deduced amino acid sequence shows only limited similarity to the small subunit of yeast RPA and is unrelated to any other protein in the current data banks. A recombinant protein containing a short histidine tag at the NH2 terminus has been purified in good yield from Escherichia coli by metal-chelate affinity chromatography. Antibodies prepared against recombinant hRPA3 recognize the native protein and inhibit SV40 DNA replication in vitro. We have localized the genes for the 70-, 32-, and 14-kDa subunits to chromosomes 17, 1, and 7, respectively, using polymerase chain reaction amplification of genomic DNA from rodent-human hybrid cell lines. Since RPA appears to be involved in several fundamental cellular processes, the physical mapping of the RPA genes may be useful in identifying possible human genetic defects associated with RPA deficiency or dysfunction.

Role of attached lipid in immunogenicity of Borrelia burgdorferi OspA.

OspA is a protective antigen of the Lyme disease spirochete Borrelia burgdorferi. Expression of the full-length B. burgdorferi B31 OspA gene in Escherichia coli produces a protein that is processed posttranslationally by signal peptidase II and contains an attached lipid moiety. The recombinant OspA lipoprotein has been purified by detergent extraction and ion-exchange chromatography. Priming and boosting with OspA lipoprotein, either with no adjuvant or adsorbed to alum, elicited a strong, dose-dependent immunoglobulin G response. Serum from vaccinated mice inhibited spirochetal growth in vitro. Mice immunized twice with as little as 0.4 micrograms of OspA lipoprotein were protected against an intradermal challenge with 10(4) infectious spirochetes. The ability of the purified recombinant lipoprotein to induce a strong protective response in the absence of toxic adjuvants makes it an excellent candidate antigen for a human vaccine against Lyme disease. By contrast, no serum immunoglobulin G or growth inhibitory response to OspA nonlipoprotein was seen at any dose. The difference in immunogenicities of the lipoprotein and nonlipoprotein forms of OspA is not due to any difference in the antigenicities of the two proteins. These results suggest that posttranslational lipid attachment is a critical determinant of the immunogenicity of the OspA protein.

Interaction of DNA polymerase alpha-primase with cellular replication protein A and SV40 T antigen.

The purified human single-stranded DNA binding protein, replication protein A (RP-A), forms specific complexes with purified SV40 large T antigen and with purified DNA polymerase alpha-primase, as shown by ELISA and a modified immunoblotting technique. RP-A associated efficiently with the isolated primase, as well as with intact polymerase alpha-primase. The 70 kDa subunit of RP-A was sufficient for association with polymerase alpha-primase. Purified SV40 large T antigen bound to intact RP-A and to polymerase-primase, but not to any of the separated subunits of RP-A or to the isolated primase. These results suggest that the specific protein-protein interactions between RP-A, polymerase-primase and T antigen may play a role in the initiating of SV40 DNA replication.

Characterization of a cDNA encoding the 70-kDa single-stranded DNA-binding subunit of human replication protein A and the role of the protein in DNA replication.

Replication protein A (RP-A) is a three-subunit single-stranded DNA-binding protein that has been isolated from human cells. RP-A is essential for SV40 DNA replication and may also be important in genetic recombination. The sequence of a cDNA encoding the 70-kDa subunit of human RP-A is reported. The 616-amino acid predicted open reading frame of the human protein is 31% identical with the 621-amino acid open reading frame of the 70-kDa subunit of RP-A from the yeast Saccharomyces cerevisiae. Both proteins share a highly conserved putative metal binding domain of the 4-cysteine type. The human cDNA directs production in Escherichia coli of a 70-kDa protein that reacts with a monoclonal antibody directed against the 70-kDa subunit of human RP-A. The recombinant 70-kDa subunit, purified from bacteria, exhibits single-stranded DNA binding activity comparable to that of the complete RP-A complex. The 70-kDa subunit is able to substitute for the complete human RP-A complex in stimulating the activity of DNA polymerase alpha-primase on a poly(dA).oligo(dT) template. However, the 70-kDa subunit alone cannot substitute for the complete RP-A complex in SV40 DNA replication in vitro, suggesting an important functional role for the other subunits.

An essential Saccharomyces cerevisiae single-stranded DNA binding protein is homologous to the large subunit of human RP-A.

Single-stranded DNA binding proteins (SSBs) are known to play a role in DNA replication and recombination in prokaryotes. An SSB was previously purified from the yeast Saccharomyces cerevisiae. This SSB stimulated the activity of a cognate strand exchange protein (SEP1) in vitro suggesting a role in recombination. We have cloned and functionally analyzed the gene encoding this protein. DNA sequencing of the cloned DNA revealed a 621 amino acid open reading frame with a coding potential for a Mr 70,269 polypeptide. Highly significant amino acid homology was detected between this S.cerevisiae gene and the Mr 70,000 subunit polypeptide of human RP-A, a cellular protein essential for SV40 DNA replication in vitro. Therefore, we named the S.cerevisiae gene RPA1. RPA1 encodes an essential function in this organism as shown by tetrad analysis of heterozygous insertion mutants and is continuously required for mitotic growth. Cells lacking RPA1 accumulate as multiply budded cells with a single nucleus suggesting a defect in DNA replication.

The primary structure of the 32-kDa subunit of human replication protein A.

Replication protein A (RP-A) is a complex of three polypeptides of molecular mass 70, 32, and 14 kDa, which is absolutely required for simian virus 40 DNA replication in vitro. We have isolated a cDNA coding for the 32-kDa subunit of RP-A. An oligonucleotide probe was constructed based upon a tryptic peptide sequence derived from whole RP-A, and clones were isolated from a lambda gt11 library containing HeLa cDNA inserts. The amino acid sequence predicted from the cDNA contains the peptide sequence obtained from whole RP-A along with two sequences obtained from tryptic peptides derived from sodium dodecyl sulfate-polyacrylamide gel-purified 32-kDa subunit. The coding sequence predicts a protein of 29,228 daltons, in good agreement with the electrophoretically determined molecular mass of the 32-kDa subunit. No significant homology was found with any of the sequences in the GenBank data base. The protein predicted from the cDNA has an N-terminal region rich in glycine and serine along with two acidic and two basic segments. Monoclonal antibodies have been raised against the 70- and 32-kDa subunits of RP-A. The cloned cDNA has been overexpressed in bacteria using an inducible T7 expression system. The protein made in bacteria is recognized by a monoclonal antibody that is specific for the 32-kDa subunit of RP-A. This monoclonal antibody against the 32-kDa subunit inhibits DNA replication in vitro.

Physical evidence for the temporal transition of transcription in bacteriophage lambda.

A high proportion of intracellular lambda DNA molecules are found to have D-loops, when isolated under four different conditions: (1) lambda Ots after 7 min at 31 degrees C in the presence of chloramphenicol; (2) lambda Ots after 7 min at 31 degrees C without chloramphenicol; (3) lambda Ots after 30 min at 42 degrees C; and (4) lambda cIIcIII after 50 min at 37 degrees C. The great majority of these D-loops contain RNA and are produced by E. coli RNA polymerase. In the presence of chloramphenicol, D-loops are mostly limited to the immediate early regions of the major leftward and rightward operons. At early times, with no chloramphenicol present, D-loops map primarily within the delayed early regions of the two major operons. At late times, D-loops are found mostly within the major late operon of the bacteriophage DNA. This physical evidence corroborates evidence of the temporal transition in lambda transcription obtained by other means. Chloramphenicol is shown to block the transition from immediate early to delayed early transcription.