Protein kinase activity associated with polyoma virus middle T antigen in vitro.

A protein kinase activity can be detected in immunoprecipitates of extracts from polyoma virus (Py)-infected cells using antiserum raised against Py-transformed cells (anti-T serum). The activity is not detected in uninfected cells or when using control serum. Using rat anti-T serum both Py middle T and the heavy chain of rat IgG are phosphorylated, whereas using hamster anti-T serum only Py middle T is phosphorylated. Experiments using a number of different mutants of Py indicate that the kinase activity detected is under viral control and is associated with Py middle T. Consistent with this the kinase, like middle T, can be detected in purified preparations of plasma membranes. The kinase can also be detected in a large number of Py-transformed cells, but not in untransformed cells or in cells transformed by other viruses. Some of the Pytransformed cells which contain kinase activity lack full sized Py large T but all contain middle T. Kinase activity is not detected in a cell line (18.37) which contains integrated viral DNA of a nontransforming hr-t deletion mutant and which contains Py large T but not middle T or small t. These results show that Py middle T or a protein which specifically binds to it has protein kinase activity in vitro. Although these results raise the possibility that protein kinases play an essential role in Py-induced transformation, however, thus far we have no data which show unequivocally that the results are physiologically significant.     

Delineation of epitopes on the Py235 rhoptry antigen of Plasmodium yoelii YM

The 235-kDa antigenic rhoptry protein Py235 of Plasmodium yoelii is encoded by a large, highly polymorphic gene family. Monoclonal antibodies to some of these antigens have been shown to attenuate the virulence of the lethal YM strain of the parasite, converting a potentially fatal YM infection to a fulminating one typical of the nonlethal 17X strain, by inducing a switch in target cell preference from mature red blood cells to reticulocytes. The reason for this is not known but would suggest that antigenic determinants of Py235 may be useful in or as subunit vaccines. To identify such determinants, we constructed an epitope expression library of one Py235 variant and screened the library with the antibodies. Thus, we mapped 5- and 12-amino acid epitopes to the C-terminus of the antigen. Both epitopes were more reactive with protective than with nonprotective monoclonal antibodies. This may explain the differential protection conferred by these antibodies upon their passive transfer into mice.     

Untransformed rat cells containing free and integrated DNA of a polyoma nontransforming (Hr-t) mutant.

The polyoma virus hr-t deletion mutant A185, when compared to wild-type (Py) virus, is at least 10⁵ fold inhibited in its transforming ability. Total cellular DNA from 50 cell lines derived from individual colonies formed after infection of Rat-1 cells with A185 virus was analyzed for the presence of viral sequences by “blot” hybridization (Southern, 1975). Viral sequences were detected in two of these cellular DNAs. One positive cell line (18–37) was studied in detail. The viral sequences present in 18–37 cells as well as the viral sequences present in virus rescued from 18–37 after fusion with permissive mouse cells were identified as A185 and not Py sequences. The A185 viral sequences in 18–37 cells were found to exist both covalently linked to host DNA sequences (integrated) and as free forms. The integrated A185 viral sequences were present in a partial head-to-tail tandem array, as has been observed for Py sequences in transformed rat cells (Birg et al., 1979). Both integrated and free forms of A185 viral sequences were retained in subclones of the parental 18–37 cell line although a simplification of the integrated viral sequence was observed. In the 18–37 cells the 100K large T antigen was synthesized but the 55K middle and 22K small T antigen species were not detected. The 18–37 cells had a normal morphology, were density-sensitive, anchorage-dependent and did not form tumors when injected into syngeneic animals. This normal phenotype of the 18–37 cells was not a result of the inability of the cells to express the transformed phenotype, since the 18–37 cells could be transformed at a high frequency upon infection with Py virus. These results show that integration of viral sequences per se or the presence of the 100K large T antigen is not sufficient for the transformed phenotype to be expressed, and strongly suggest that Py-induced transformation is mediated by the 55K middle and/or 22K small T antigens.     

HIV-1, lipid rafts, and antibodies to liposomes: implications for anti-viral-neutralizing antibodies

The human immunodeficiency virus type 1 (HIV-1) is an enveloped virus with a lipid bilayer that contains several glycoproteins that are anchored in, or closely associated with, the membrane surface. The envelope proteins have complex interactions with the lipids both on the host cells and on the target cells. The processes of budding from host cells and entry into target cells occur at sites on the plasma membrane, known as lipid rafts, that represent specialized regions that are rich in cholesterol and sphingolipids. Although the envelope glycoproteins are antigenic molecules that potentially might be used for development of broadly neutralizing antibodies in a vaccine to HIV-1, the development of such antibodies that have broad specificities against primary field isolates of virus has been largely thwarted to date by the ability of the envelope proteins to evade the immune system through various mechanisms. In this review, the interactions of HIV-1 with membrane lipids are summarized. Liposomes are commonly used as models for understanding interactions of proteins with membrane lipids; and liposomes have also been used both as carriers for vaccines, and as antigens for induction of antibodies to liposomal lipids. The possibility is proposed that liposomal lipids, or liposome-protein combinations, could be useful as antigens for inducing broadly neutralizing antibodies to HIV-1.     

HIV-1, lipid rafts,and antibodies to liposomes: implications for anti-viral-neutralizing antibodies (Review)

The human immunodeficiency virus type 1 (HIV-1) is an enveloped virus with a lipid bilayer that contains several glycoproteins that are anchored in, or closely associated with, the membrane surface. The envelope proteins have complex interactions with the lipids both on the host cells and on the target cells. The processes of budding from host cells and entry into target cells occur at sites on the plasma membrane, known as lipid rafts, that represent specialized regions that are rich in cholesterol and sphingolipids. Although the envelope glycoproteins are antigenic molecules that potentially might be used for development of broadly neutralizing antibodies in a vaccine to HIV-1, the development of such antibodies that have broad specificities against primary field isolates of virus has been largely thwarted to date by the ability of the envelope proteins to evade the immune system through various mechanisms. In this review, the interactions of HIV-1 with membrane lipids are summarized. Liposomes are commonly used as models for understanding interactions of proteins with membrane lipids; and liposomes have also been used both as carriers for vaccines, and as antigens for induction of antibodies to liposomal lipids. The possibility is proposed that liposomal lipids, or liposome-protein combinations, could be useful as antigens for inducing broadly neutralizing antibodies to HIV-1.     

A polyoma mutant that encodes small T antigen but not middle T antigen demonstrates uncoupling of cell surface and cytoskeletal changes associated with cell transformation.

The hr-t gene of polyoma virus encodes both the small and middle T (tumor) antigens and exerts pleiotropic effects on cells. By mutating the 3' splice site for middle T mRNA, we have constructed a virus mutant, Py808A, which fails to express middle T but encodes normal small and large T proteins. The mutant failed to induce morphological transformation or growth in soft agar, but did stimulate postconfluent growth of normal cells. Cells infected by Py808A became fully agglutinable by lectins while retaining normal actin cable architecture and normal levels of extracellular fibronectin. These properties of Py808A demonstrated the separability of structural changes at the cell surface from those in the cytoskeleton and extracellular matrix, parameters which have heretofore been linked in the action of the hr-t and other viral oncogenes.     

Plasmodium yoelii: combinatorial expression of variants of the 235 kDa rhoptry antigen during infection

The 235 kDa rhoptry protein Py235 of Plasmodium yoelii, has been implicated in erythrocyte invasion by the merozoite forms of the parasite. Py235 is encoded by a large, highly polymorphic gene family, members of which appear to be differentially transcribed. However, it is not clear how many variants are expressed at the protein level during an infection cycle and whether or not these variants are expressed selectively or combinatorially. Certain monoclonal antibodies to Py235 have been shown to attenuate parasite virulence upon passive transfer into mice, suggesting that this antigen or its derivatives may be useful vaccine candidates. To provide a basis for this, we sought to identify those variants that are recognised by the host immune system, and to establish the pattern of expression of the antigen in mice during infection. Using Py235 monoclonal antibodies as probes, we isolated distinct antigenic variants from an expression library, suggesting that the antigen repertoire is potentially large and that different Py235 variants may be produced during infection. The implications of these observations are discussed with respect to the ability of a cloned parasite line to express distinct antigenic variants in vivo.     

Development of a cytotoxic T-lymphocyte-based, broadly protective influenza vaccine

The current vaccination strategy against influenza is to induce production of antibodies directed against the surface antigens of these viruses. However, frequent changes in the surface antigens of influenza viruses allow them to avoid antibody-mediated immunity. On the other hand, it is known that cytotoxic T-lymphocyte (CTL) populations directed against internal antigens of influenza A virus are broadly cross-reactive to influenza virus subtypes. The present authors have previously demonstrated that antigens chemically coupled to the surface of liposomes made using unsaturated fatty acids are cross-presented by APCs via MHC class I to CD8(+) T cells and induce antigen-specific CTLs. Based on this finding, a liposome vaccine that is capable of inducing CTL response against internal antigens of influenza viruses and removing virus-infected cells in the host has been developed. The CTL-based liposomal technique might be applicable for developing vaccines against influenza and other viruses, such as hepatitis C, HIV, and severe acute respiratory syndrome corona virus, which frequently change their surface antigenic molecules.     

Characterization of K virus and its comparison with polyoma virus.

The antigenic relationship between the two murine papovaviruses, K virus and polyoma virus, was examined by serological techniques to determine whether they shared any antigenic components. No cross-reactivity was found associated with the viral (V) antigens by the indirect immunofluorescence, neutralization, or hemagglutination-inhibition tests. The tumor (T) antigens expressed in transformed cells or cells productively infected by either K or polyoma virus did not cross-react by indirect immunofluorescence. An antigenic relationship was detected, however, among the late proteins of K virus, polyoma virus, simian virus 40, and the human papovavirus BKV, when tested with either hyperimmune sera prepared against polyoma virus and simian virus 40 or sera prepared against disrupted virions. The nucleic acids of K and polyoma viruses were compared by agarose gel electrophoresis and restriction endonuclease analysis. No nucleotide sequence homology between the genomes of these two viruses was detectable by DNA-DNA hybridization techniques under stringent conditions. The genome of K virus was found to be slightly smaller than that of polyoma virus, and the cleavage patterns of the viral DNAs with six restriction endonucleases were different. These findings indicate that there is little relationship between these two murine papovaviruses.     

Human Papovavirus, BK Strain: Biological Studies Including Antigenic Relationship to Simian Virus 40

Some of the properties of a new human papovavirus, BK, have been examined. Host range studies of BK virus (BKV) showed human cells to be more sensitive to infection than monkey cells; human fetal brain cells appear to be highly sensitive to BKV, with the production of extensive cytopathology characterized by cytoplasmic vacuolization. The hemagglutinin of BKV is associated with the virion and is resistant to ether or heating at 56 C for 30 min. Fluorescent antibody as well as neutralization tests indicated antigenic similarities between simian virus 40 (SV40) and BKV. Cells undergoing lytic infection with BKV synthesized intranuclear T antigen(s) which reacted with SV40 T antibody demonstrable by immunofluorescence. However, BKV did not appear to induce SV40 transplantation antigens in transplantation-resistance tests. Evidence was obtained that BKV was present in humans prior to the widespread use of polio vaccines, thus ruling out the possibility that BKV is an SV40-related monkey virus, introduced into the human population by accidental contamination of poliovirus vaccines.     

Polyoma Viral DNA Replicated as a Nucleoprotein Complex in Close Association with the Host Cell Chromatin

Polyoma viral DNA is shown to be replicated in close association with the mouse cell chromatin. Two virus-specific nucleoprotein complexes, designated complex A and B, can be dissociated from the isolated chromatin by gentle homogenization in 0.5 M NaCl. Complex A contains only replicating polyoma (Py) DNA whereas complex B contains only mature Py DNA I. The results show, furthermore, that complex A, containing viral DNA in different stages of replication, and complex B are both nucleoproteins with the same buoyant density. The data presently available suggest that newly synthesized stretches of Py DNA are immediately complexed with mouse cell histones and that complex B becomes the "core" of progeny Py virions. These results suggested that Py-induced replication of the mouse cell chromatin may be necessary to provide replicating Py DNA with histones.     

Alpha4beta1 integrin acts as a cell receptor for murine polyomavirus at the postattachment level

The initial interaction of murine polyomavirus (Py) with host cells occurs through direct binding of the major capsid protein VP1 with cell membrane molecules containing terminal sialic acids; however, these Py receptor molecules have not yet been identified. Analysis of the capsid protein primary sequences of all murine strains revealed the presence of integrin ligand motifs in the DE and EF loops of VP1 (LDV and DLXXL, respectively) and at the N terminus of VP2 (DGE). We show that infectivity of the Py A2 strain in mouse Swiss 3T3 fibroblasts is significantly reduced only in the presence of natural integrin ligands carrying an LDV motif or antibodies directed against the alpha4 and beta1 integrin subunits. Furthermore, we demonstrate that expression of the alpha4 subunit in the alpha4-deficient BALB/c 3T3 cells increases viral infectivity. Addition of alpha4 function-blocking antibodies, prior to or after virus adsorption, blocks this increased infectivity without affecting virus binding to cells. Taken together, these data indicate that expression of alpha4 integrin enhances permissivity to Py, probably by acting as one of the postattachment receptors.     

A serologic comparison of Moloney lymphoma cell surface and Moloney oncornavirus antigens.

Moloney lymphomas and Moloney sarcomas share strong tumor antigens. In this report we analyze the cell-surface antigens on a Balb/c Moloney lymphoma, LSTRA, using hyperimmune sarcoma regressor sera (alphaMo) as a primary reagent. We also use heterologous anti-viral p30 and gp70 sera for a direct analysis of virion protein antigens on the LSTRA surface. Using radiolabeled alphaMo-binding assays, we demonstrate that LSTRA tumor antigens detected by these sera are all Moloney viral antigens; approximately 1/3 of these antigenic determinants are expressed on the intact virus, and the other determinants are revealed by detergent lysis of the virus. The major viral antigens expressed on the LSTRA cell surface are viral env gene products, whereas gag gene products are only sparsely represented. We conclude that alphaMo sera detect almost exclusively viral antigens on LSTRA cells, and these antigens are almost exclusively virion env gene products.     

Analysis of the major histocompatibility complex in Syrian hamsters. III. Cellular and humoral immunity to alloantigens.

Among the genetic loci incorporated into the major histocompatibility complex in every species studied to date have been prominent genes encoding for strong histocompatibility determinants that elicit detectable alloantibody responses and which are the chief antigenic targets of cell-mediated cytotoxicity reactions. The K and D regions of the H-2 complex in the mouse and the A, B, and C regions of the HLA complex in man are representative examples. Syrian hamsters, as described in this report, do not make alloantibodies to antigens of this type and only very poorly do they carry out in vitro cell-mediated cytotoxicity to target cells putatively bearing these antigens. Since hamsters are quite capable of discriminating analogous antigenic differences in xenogeneic species, and xenogeneic sources cannot distinguish immunologically between the antigens encoded by the two hamster major histocompatibility alleles. Hm-1a and Hm-1b, we conclude that the hamster strains we work with are serologically indistinguishable by the methods used here. However, they obviously differ for determinants which elicit T cell-mediated responses, as evidenced by their ability to express acute skin graft rejection, mixed lymphocyte reactivity, graft-vs-host reactions, and cell-mediated cytotoxicity reactions. Such alloreactivity may reflect a mutation at an SD locus, affecting antigenic sites recognized only by T cells, or that the available hamster strains are SD identical, but differ at loci similar to the I region loci in mice. Alternatively, we cannot exclude the possibility that Syrian hamsters somehow fail to express properly the genes coding for SD determinants.     

55,000-dalton, retrovirus-associated, cell membrane glycoprotein: purification and quantitative measurements of expression in viruses, cells, and tissues.

We have purified to homogeneity and characterized a 55,000-dalton rat cell membrane glycoprotein, gp55. This protein was originally identified in preparations of a defective pseudotype of the Kirsten sarcoma virus and shown to be present in several rodent retrovirus particles. The gp55 was purified from this defective virus by concanavalin A and heparin affinity chromatography, as well as by preparative sodium dodecyl sulfate-gel electrophoresis. Both preparations displayed similar purity and antigenic characteristics. The 125I-labeled gp55 was precipitated by antisera against rodent retroviruses, but not by monospecific antisera against purified type C virus structural proteins, thus indicating that gp55 was retrovirus associated, but unrelated to known retrovirus structural proteins. Competition radioimmunoassay with an anti-rat virus serum which recognized rodent group-specific antigens on gp55 indicated: the presence of gp55 antigens in 15 rodent cell lines, but not 10 nonrodent cell lines; no effect of viral infection or cell transformation on the amount of gp55 expressed; up to 100-fold increases in the concentration of the gp55 antigens in nine rodent retroviruses, but not in five nonrodent viruses, as compared to cells; the presence of gp55 in rodent sera, especially of the NZB mouse, where anti-gp55 antibody was also detected; a lymphoid and epithelial tissue distribution of gp55 in rats and mice. Additional competition radioimmunoassays with a broad-reacting antivirus serum also detected the presence of gp55 in nonrodent, mink, and human cells and thus distinguished rat type, rodent group, and interspecies antigenic determinants on gp55. In conclusion, gp55 is a cell membrane glycoprotein associated in high concentration with retroviruses.     

Potency of adenovirus vaccines: comparison of unconcentrated, concentrated, and soluble antigen preparations

Five adenoviruses were effectively concentrated and partially purified by methanol precipitation. The soluble antigens from one adenovirus concentrate were obtained by adsorption to and elution from calcium phosphate. Formaldehyde-inactivated vaccines prepared from the virus concentrates were more antigenic in guinea pigs than a National Institutes of Health reference vaccine or one prepared from unconcentrated adenovirus. The soluble antigens also proved to be antigenic. The data indicate that adenovirus vaccines of superior potency can be prepared from concentrated virus preparations and that the extracted soluble antigens are immunogenic.     

Unbiased identification of target antigens of CD8+ T cells with combinatorial libraries coding for short peptides

Cytotoxic CD8(+) T cells recognize the antigenic peptides presented by class I major histocompatibility complex (MHC) molecules. These T cells have key roles in infectious diseases, autoimmunity and tumor immunology, but there is currently no unbiased method for the reliable identification of their target antigens. This is because of the low affinities of antigen-specific T cell receptors (TCR) to their target MHC-peptide complexes, the polyspecificity of these TCRs and the requirement that these TCRs recognize protein antigens that have been processed by antigen-presenting cells (APCs). Here we describe a technology for the unbiased identification of the antigenic peptides presented by MHC class I molecules. The technology uses plasmid-encoded combinatorial peptide libraries and a single-cell detection system. We validated this approach using a well-characterized influenza-virus–specific TCR, MHC and peptide combination. Single APCs carrying antigenic peptides can be detected among several million APCs that carry irrelevant peptides. The identified peptide sequences showed a converging pattern of mimotopes that revealed the parent influenza antigen. This technique should be generally applicable to the identification of disease-relevant T cell antigens.