A reductionist cell-free major histocompatibility complex class II antigen processing system identifies immunodominant epitopes

Immunodominance is defined as restricted responsiveness of T cells to a few selected epitopes from complex antigens. Strategies currently used for elucidating CD4(+) T cell epitopes are inadequate. To understand the mechanism of epitope selection for helper T cells, we established a cell-free antigen processing system composed of defined proteins: human leukocyte antigen-DR1 (HLA-DR1), HLA-DM and cathepsins. Our reductionist system successfully identified the physiologically selected immunodominant epitopes of two model antigens: hemagglutinin-1 (HA1) from influenza virus (A/Texas/1/77) and type II collagen (CII). When applied for identification of new epitopes from a recombinant liver-stage antigen of malaria falciparum (LSA-NRC) or HA1 from H5N1 influenza virus ('avian flu'), the system selected single epitopes from each protein that were confirmed to be immunodominant by their capacity to activate CD4(+) T cells from H5N1-immunized HLA-DR1-transgenic mice and LSA-NRC-vaccinated HLA-DR1-positive human volunteers. Thus, we provide a new tool for the identification of physiologically relevant helper T cell epitopes from antigens.     

Identification of major histocompatibility complex class II-restricted antigens and epitopes of the Epstein-Barr virus by a novel bacterial expression cloning approach

Epstein-Barr virus (EBV)-specific T cells have been successfully used to treat or prevent EBV-positive lymphoproliferative disease in hematopoietic stem cell transplant recipients, but the antigens recognized by the infused CD4+ T cells have remained unknown. Here, we describe a simple procedure that permits the identification of viral T-helper (TH)-cell antigens and epitopes. This direct antigen identification method is based on the random expression of viral polypeptides fused to chloramphenicol acetyltransferase (CAT) in bacteria, which are subsequently fed to major histocompatibility complex class II+ antigen-presenting cells and probed with antigen-specific T cells. The fusion of antigenic fragments to CAT offers several advantages. First, chloramphenicol treatment allows the selection of bacteria expressing antigen-CAT fusion proteins in frame, which greatly reduces the number of colonies to be screened. Second, antigenic fragments fused to CAT are expressed at high levels, even when derived from proteins that are toxic to bacteria. Third, the uniformly high expression level of antigen-CAT fusion proteins permits the establishment of large and representative pool sizes. Finally, antigen identification does not require knowledge of the restriction element and often leads directly to the identification of the T-cell epitope. Using this approach, the BALF4 and BNRF1 proteins were identified as targets of the EBV-specific T-helper-cell response, demonstrating that lytic cycle antigens are a relevant component of the EBV-specific TH-cell response.     

Double immunoenzymatic staining for the simultaneous detection of Epstein-Barr virus induced antigens

A double indirect immunoenzymatic staining was developed for the simultaneous visualization of Epstein-Barr virus-induced early antigens and virus capsid antigens in P3HR1 lymphoblastoid cell line. The double immunocytochemical staining was performed with a four-stage and a two-stage procedure employing human sera and monoclonal antibodies against Epstein-Barr virus-induced antigens, followed by the addition of specific alkaline phosphatase and peroxidase labeled antisera. The selection of substrates yielding reaction products of contrasting colours enabled the observer to distinguish cells expressing Epstein-Barr virus capsid antigens (blue) from cells expressing Epstein-Barr virus early antigens (brown).     

Double immunoenzymatic staining for the simultaneous detection of Epstein-Barr virus induced antigens

Summary A double indirect immunoenzymatic staining was developed for the simultaneous visualization of Epstein-Barr virus-induced early antigens and virus capsid antigens in P3HR1 lymphoblastoid cell line.The double immunocytochemical staining was performed with a four-stage and a two-stage procedure employing human sera and monoclonal antibodies against Epstein-Barr virus-induced antigens, followed by the addition of specific alkaline phosphatase and peroxidase labeled antisera.The selection of substrates yielding reaction products of contrasting colours enabled the observer to distinguish cells expressing Epstein-Barr virus capsid antigens (blue) from cells expressing Epstein-Barr virus early antigens (brown).     

Characterization of tau antigens isolated from uninfected and simian virus 40-infected monkey cells and papovavirus-transformed cells.

Tau antigens (also known as cellular or nonviral tumor antigens) were detected in uninfected and simian virus 40-infected monkey cells after immunoprecipitation with serum from hamsters bearing simian virus 40-induced tumours (anti-T serum). These two proteins (56,000 daltons) were digested to similarly sized peptides with various amounts of Staphylococcus aureus V8 protease. The Tau antigen isolated from infected monkey cells was closely related but was not identical to the corresponding protein from human cells transformed by simian virus 40, as determined by two-dimensional mapping of their methionine-labeled tryptic peptides. Hamster cells transformed by various primate papovaviruses (simian virus 40, BK virus, and JC virus) synthesized indistinguishable Tau antigens, as determined by two-dimensional peptide mapping. When tested by the same procedure, these proteins and the ones made in monkey and human cells were found to be related to the Tau antigens isolated from simian virus 40-transformed mouse and rat cells. Based on these results, an "evolutionary tree" was constructed to show the relationship among the methionine-containing tryptic peptides of all of these proteins.     

Herpes simplex virus type 1-specific cytotoxic T lymphocytes recognize virus nonstructural proteins.

The specificity of herpes simplex virus type 1-specific cytotoxic T cells was examined with target cells expressing either input viral structural antigens or antigens resulting from permissive infection or cells from an interrupted infection in which they expressed predominantly nonstructural immediate-early proteins. These studies indicated that only an insignificant minority of cytotoxic T cells recognized the input viral antigens, whereas a significant proportion (20 to 35%) recognized target cells that expressed the immediate-early proteins despite the absence of serologically detectable viral antigens upon the infected cell surface. The finding that a significant proportion of cytotoxic T-cell populations obtained from the draining lymph nodes of mice acutely infected with herpes simplex virus type 1 also recognized immediately-early gene-expressing target cells indicates the importance of nonstructural herpes simplex virus proteins to antiviral immunity in vivo.     

Independence of H-2 and viral antigens on the cell surface and absence of H-2 antigens on murine leukemia virus and mouse mammary tumor virus particles

By indirect immunoelectron microscopy we tested for the presence of H-2 antigens on murine mammary tumor virus (MMTV) and murine leukemia virus (MuLV) particles. The association of H-2 antigens and viral antigens on the virus-infected cell surface was investigated with antibody-induced redistribution. Mammary tumor cells and leukemia cell lines with different H-2 genotypes and carrying different MuMTV or MuLV were used. No H-2 antigens could be demonstrated on the envelope of MMTV and MuLV particles, even after the permeabilization of their envelopes with saponin. On the surface of virus-infected cells antibody-induced patching or capping of the viral antigens did not result in copatching or cocapping of the H-2 antigens. In the reciprocal tests no co-redistribution of viral antigens with H-2 antigens was seen. Our experiments failed to show any physical association between H-2 antigens and MMTV or MuLV antigens on the cell surface.Abbreviations used in this paper MMTV mammary tumor virus - MuLV murine leukemia virus - MHC major histocompatibility complex - IEM immunelectron microscopy     

Rhabdovirus Replication in Enucleated Host Cells

Infection of enucleated TC-7 monkey cells with rabies virus resulted in the synthesis of virus-directed RNA and the production of rabies antigens but not of infectious virus. The yield of infectious vesicular stomatitis virus from enucleated TC-7 cells, on the other hand, was almost as high as that from intact cells. Inhibition of the mitochondrial functions of enucleated cells by treatment with ethidium bromide did not influence the development of rabies antigens or the production of infectious vesicular stomatitis virus.     

Crossed immunoelectrophoretic studies of the solubility immunogenicity of herpes simplex virus antigens.

The nonionic detergent Triton X-100 was capable of solubilizing 90% of the protein content in herpes simplex virus (HSV)-infected rabbit cornea cells. The solubilized HSV antigens formed well-characterized precipitates by crossed immunoelectrophoresis in Triton X-100-containing agarose gel, allowing both identification and relative quantitation. Water-soluble and detergent-requiring HSV antigens were identified by different solubilization procedures in buffer with and without detergent. Five glycoprotein antigens were solubilized only in the presence of detergent, indicating their membrane-bound state. One non-glycosylated antigen was present in both a water-soluble and a membrane-bound form. Based upon the crossed immunoelectrophoretic precipitating patterns of Triton X-100-solubilized HSV antigens, it has been estimated that infected cells yield an amount of virus-specific protein equivalent to 2,000 enveloped virions per cell. Rabbits inoculated intracutaneously with Triton X-100-solubilized HSV antigens developed neutralizing antibodies against HSV almost as effectively as rabbits with an active HSV infection. Precipitins against individual HSV antigens in sera from rabbits infected with HSV and immunized with the Triton X-100-solubilized HSV antigens were assayed by the crossed immunoelectrophoretic technique. Sera from infected rabbits reacted more strongly and with a higher number of HSV antigens than sera from immunized rabbits.     

The origins of alloreactivity: differentiation of prekiller cells to viral infection results in alloreactive cytolytic T lymphocytes

Mice maintained in our animal colony become primed to Sendai virus. This "environmental" priming is reflected in a shift in prekiller activity from the Ly 123 to Ly 23 T cell set and in increased virus-specific cytolytic activity. This transition is accompanied by the development of cytolytic activity against allogeneic targets (not expressing Sendai antigens). These findings are consistent with the view that continued stimulation of Ly 123 cells by autologous MHC antigens, associated with foreign antigens such as a virus, generate Ly 23 prekiller cells that respond to alloantigens as well as autologous cells infected with the relevant virus.     

Immunoelectron microscopy of human retroviruses

The ultrastructural detection and identification of human retroviruses--HTLV (human T-cell lymphotropic virus) and HIV (human immunodeficiency virus)--have become an everyday task for pathologists and virologists as well as for cell and molecular biologists. The development of better and conventionally available immunocytochemical techniques, such as pre- or postembedding immunocytochemical methods, cryofixation-variants and low temperature embeddings, have made it possible to use them in this field. With the help of these methods the structural proteins of HTLV-I and HIV have been identified in infected cells. The virus assembly at the cell membrane has also been described in detail. Using these methods the incorporation of human transplantation antigens into the envelope of these viruses can be followed. Future studies should establish the pathological significance of this process.     

Characteristics of human-mouse hybrid cell lines and the changes induced in them by the poliomyelitis virus

Cells of the four hybrid lines between continuous mouse cells Rag and human diploid embryonal fibroblasts were polymorphic and had mitotic activity in fully formed monolayers. Most of the these mitoses were pathological. Hybrid cells examined 8 months after hybridization were susceptible to the poliomyelitis virus infection with partial cytopathologic effect, they produced virus antigens and the infectious virus. Small hybrid cells displayed a more pronounced cytopathologic effect than did big, polynuclear and mitotic cells. Hybrid cells that were passaged 1.5 months after infection did not excrete any infectious poliovirus but contained poliovirus antigens.     

Autopresentation of hepatitis B virus envelope antigens by T cells.

Processing and presentation by T cells appear to be limited to antigens that can directly interact with the T-cell surface, thereby overcoming the T-cell inefficiency in antigen capture and internalization. Our study provides evidence that the hepatitis B virus (HBV) envelope proteins can also be efficiently processed and presented by CD4+ and CD8+ T cells to other T cells in a human leukocyte antigen class II-restricted fashion. This phenomenon suggests a receptor-mediated interaction between T cells and the HBV envelope and defines a system that can, we hope, be exploited for the identification of the receptor binding site within the HBV envelope and for the characterization of the putative cellular HBV receptor.     

Cell-Cycle Dependent Appearance of Murine Leukemia-Sarcoma Virus Antigens

Normal rat kidney (NRK) cells, NRK cells infected with Rauscher murine leukemia virus, and NRK cells infected with Kirsten murine sarcoma-leukemia virus (NRK-K) were synchronized by a double thymidine block. At intervals after release from thymidine blockage, the cells were examined for the presence of viral antigens in the cytoplasm and on the cell surface by immunofluorescent microscopy by using goat anti-Rauscher murine leukemia virus and goat anti-Moloney leukemia virus (Tween-ether disrupted) sera. Detection of viral antigens in the cytoplasm was periodic during the cell cycle. Antigens were detected first during the S phase, increased during the G2 phase, and disappeared during the M and G1 phases. A similar pattern of surface immunofluorescence was observed. Infectious virus was detected in culture fluids from synchronized cells during the M phase. Surface immunofluorescence was detected in NRK-K cells with anti-Rauscher murine leukemia virus and may represent the presence of group-specific antigens on the cell surface. Control, uninfected NRK cells, which did not normally fluoresce, showed weak immunofluorescence during the S and G2 phases after synchronization. Synchronization can be used to amplify latent oncornavirus expression.     

Analysis of murine major histocompatibility complex class II-restricted T-cell responses to the flavivirus Kunjin by using vaccinia virus expression.

The present paper analyzes the influence of major histocompatibility complex (MHC) class II (Ir) genes on MHC class II-restricted T-cell responses to West Nile virus (WNV) and recombinant vaccinia virus-derived Kunjin virus antigens and identifies the immunodominant Kunjin virus antigens. Generally, mice were primed by intravenous infection with WNV or Kunjin virus, and their CD4+ T cells were stimulated in vitro 14 days later with WNV or Kunjin virus antigens to pulse macrophage or B-cell antigen-presenting cells (APC). WNV-specific in vitro T-cell responses from H-2b mice were higher than those from H-2d, H-2k, and H-2q mice. When recombinant vaccinia virus-derived Kunjin virus antigen preparations were tested in vitro, Kunjin virus-immune T cells of H-2b haplotype responded most strongly to structural (prM, C, E) and membrane-associated nonstructural (NS1) proteins encoded by VKV 1031 and showed weaker responses to cytosolic nonstructural protein NS5 (VKV 1022), whereas the responders of H-2k haplotype responded most strongly to the antigens encoded by VKV 1022 and gave lesser responses to VKV 1031. H-2d T cells gave weaker responses than either H-2b or H-2k cells, with responses to VKV 1031 generally being higher than those to VKV 1022. Responses to VKV 1023 or VKV 1024 encoding all of the NS3 to NS5 gene sequence or to VKV 1023 encoding all of NS3 were weak or absent. Within a given inbred strain, B cells and macrophages differed in their abilities to present recombinant vaccinia virus-derived Kunjin virus antigens, both in terms of magnitude of T-cell responses induced and the particular Kunjin virus protein presented. T cells from different non-MHC genetic backgrounds varied in their requirements of macrophage numbers as APC for maximum reactivity, suggesting that the concentration of class II MHC antigens and other molecules affecting APC-T-cell interaction varied in mice with different genetic backgrounds. Regardless of MHC haplotype, responses to VKV 1024, which encompasses VKV 1023 and VKV 1022, were either absent or lower than those to VKV 1022, possibly reflecting differences in the processing requirements of these two proteins. When mice were primed intravenously with recombinant vaccinia virus and when their CD4+ T cells were stimulated in vitro with native Kunjin virus antigens, VKV 1031 primed more efficiently than Kunjin virus and VKV 1022 primed similarly to Kunjin virus.     

Immunofluorescent Cell-Counting Assay for Lymphocytic Choriomeningitis Virus

A quantitative assay for lymphocytic choriomeningitis virus was developed and standardized. The assay is based on direct immunofluorescent staining of infected L-929 cell monolayers and enumeration of cells containing fluorescent viral antigens. Maximal adsorption of virus to cells occurred within 1 h. Observations on the sequential development of viral antigens within cells showed that specific cytoplasmic fluorescence appeared within 10 h. The optimal time for enumerating fluorescent cells was from 18 to 20 h after addition of virus. A linear relationship was demonstrated between the number of infected cells and the relative virus concentration. Fluorescent cells were distributed randomly in infected cover slip cell monolayers. The immunofluorescent cell-counting assay for lymphocytic choriomeningitis virus was highly precise and reproducible.     

Relationship Among Tau Antigens Isolated from Various Lines of Simian Virus 40-Transformed Cells

In addition to the virus-specified tumor antigens, simian virus 40-transformed cells contain at least one other protein which can be immunoprecipitated with serum from animals bearing simian virus 40-induced tumors. This protein, which is designated Tau antigen, has an apparent molecular weight of 56,000 as determined by electrophoresis on acrylamide gels. The relationship among Tau antigens isolated from different lines of simian virus 40-transformed cells was examined by comparing the methionine-labeled tryptic peptides of these proteins by two-dimensional fingerprinting on thin-layer cellulose plates. In this fashion, we initially determined that the Tau antigens isolated from three different lines of transformed mouse cells were very similar. Second, we found that Tau antigen isolated from a line of rat transformants was closely related, but not identical, to the mouse cell Tau antigens. Approximately 70% of their methionine peptides comigrated in two dimensions. Finally, we showed that Tau antigen isolated from a line of transformed human cells was only partially related to the mouse and rat proteins. About 40% of the methionine peptides of the human protein were also contained in the Tau antigens from the other two species. These results strongly indicate that the Tau antigens isolated from these various simian virus 40-transformed cell lines contain common amino acid sequences.     

Virus-specific changes in mouse cells inoculated with a strain of human cytomegalovirus.

Cytopathic changes and virus-specific antigens developed in, then disappeared from, mouse fibroblasts infected by a strain of human cytomegalovirus (CMV), but their disappearance was delayed in cells treated with idoxuridine prior to infection. The replication of vesicular stomatitis virus and herpes simplex virus was restricted in human CMV-infected mouse cells as long as human CMV-specific antigens were present. Virus-specific antigens could be induced by treatment with idoxuridine or arginine deficiency in mouse cells which had previously turned "negative".     

Antibody-induced capping of measles virus antigens on plasma membrane studied by electron microscopy.

Antibodies specific for measles virus could redistribute ("cap") virus antigens on infected HeLa cells as shown by transmission and scanning electron microscopy. Using an indirect immunoperoxidase technique, infected cells showed diffuse, circumferential distribution of virus antigens over the cell surface when mixed with antibody at 4 C. At 37 C, virus-coated microvilli concentrated on one pole of the cell, leaving the remainder of the plasma membrane devoid of both viral antigens and microvillus projections. Whereas extreme polar displacement of virus-antibody complexes frequently occurred, endocytosis was rarely seen. The findings indicate that antiviral antibodies can move and cluster virus on plasma membranes and suggest that virus-antibody complexes are stripped and shed from the cell surface.     

Destruction of L Cells by Mengo Virus: Use of Interferon to Study the Mechanism

Interferon, when added to L cells, inhibited the synthesis of infectious Mengo viral ribonucleic acid, hemagglutinins, and infectious virus by 85 to 95%. Serum-blocking antigens were also reduced by the action of interferon, but threefold excess amounts of these antigens accumulated in interferon-treated cultures above the amounts expected for the quantity of infectious virus that was produced in these cultures. Radioautographic analysis showed that 28 to 36% of the cells of an interferon-treated population synthesized viral ribonucleic acid and 36 to 47% produced viral antigens as determined by an immunofluorescence technique. Despite the reductions in synthesis of viral components, all cells in an interferon-treated culture underwent cytopathic effects at the same time as cells in infected cultures which had not been treated with interferon. The results are compatible with the hypothesis that the cell destruction which results from the infection of L cells with Mengo virus is due to a protein which is coded for by the virus but is not a component of the mature virion.