Lymphocytes express Ia antigens of foreign haplotype following treatment with neuraminidase

Evidence is presented which indicates that neuraminidase (NA) treatment of spleen cells both destroys old Ia antigens and reveals new Ia specificities which are not normally expressed by splenocytes. It was found that NA treatment unmasked alien I-A^k-like specificities on A.TH (I ^s ) spleen cells, and I^s-like antigens on A.TL (I ^k ) spleen cells. These conclusions were based on direct testing of NA-treated targets with a range of alloantisera and on cell-absorption experiments. Furthermore, the cellular distribution of NA-exposed antigens resembled that of convential Ia antigens, the new antigens being expressed on more than 90 percent of splenic B cells and a subpopulation of splenic T cells. However, although some of the antigens exposed by NA on A.TH cells appeared to resemble the Ia. 3 and 15 specificities, additional antigens were involved which did not correlate with any previously described Ia antigens.Sugar inhibition experiments demonstrated the NA-exposed antigens to be carbohydrate in nature, D-galactose being an effective inhibitor in these studies. The proportion of- and-linked D-galactose residues associated with the new antigens depended upon the target cell used and the anti-Ia serum tested. Furthermore, glycolipid extracts from lymphoid cells were shown to contain the NA-exposed antigens.Collectively, these results support the existence of carbohydrate-defined Ia antigens. The simplest interpretation of the findings is that NA clips off terminal sialic acid residues from carbohydrate-defined Ia antigens on the cell surface and exposes subterminal sugars which resemble antigens expressed by otherI-region haplotypes.     

The overlooked "nonclassical" functions of major histocompatibility complex (MHC) class II antigens in immune and nonimmune cells

Besides their "classical" antigenic peptide-presenting activity, major histocompatibility complex (MHC) class II antigens can activate different cellular functions in immune and nonimmune cells. However, this "nonclassical" role and its functional consequences are still substantially overlooked. In this review, we will focus on these alternative functional properties of MHC class II antigens, to reawaken attention to their present and foreseeable immunobiologic and pathogenetic implications. The main issues that will be addressed concern 1) the role of MHC class II molecules as basic components of exchangeable oligomeric protein complexes with intracellular signaling ability; 2) the nonclassical functions of MHC class II antigens in immune cells; 3) the pathogenetic role of MHC class II antigens in inflammatory/autoimmune and infectious disease; and 4) the functional role of MHC class II antigens in solid malignancies.     

Genetics of Ia-like alloantigens in chickens and linkage with B major histocompatibility complex

Two sets of backcross matings were performed to test for linkage between genes coding for the Ia-like antigens ("Ia") and the B erythrocyte antigens (Ea-B) of the chicken. Evidence is presented which indicates that the "Ia" antigens are determined by a single codominant locus and that the Ea-B and "Ia" loci are on the same chromosome. Failure to detect a single recombinant between the Ea-B and "Ia" loci out of 208 progeny suggests close linkage of the two genes with a map distance of up to about 2 centimorgans. The "Ia" genes are thus included in the B major histocompatibility complex of the chicken.     

Biochemical and serological characteristics of soluble yeast phase antigens of Histoplasma Capsulatum

Soluble antigens of whole yeast-phase cells were extracted with a 0.1 M phosphate buffer containing 0.1 M sodium chloride and 0.02% iodoacetate. After being separated by differential filtration into fractions less than or greater than 50,000 daltons, these antigens were purified by molecular sieve and chromatographic separations on ionic exchange resins. Two high molecular weight fractions obtained from diethylaminoethyl-cellulose (DEAE) at pH 8.0 and 7.0 with tris (hydroxymethyl) aminomethane (Tris) buffer were M antigens; those obtained at pH 4.0 and 4.0 with salt were H antigens. The four fractions had protein to carbohydrate ratios of 7.3, 14.0, 8.4, and 6.5 respectively, and all had essentially the same amino acid composition with no methionine and tyrosine and little histidine, arginine, phenylalanine and lysine. They had high concentrations of glucose, less mannose and traces of galactose. The low molecular weight fractions had the new complex Y antigen, M antigen, and H antigen with protein to carbohydrate ratios of 1.4, 1.4 and 0.3 respectively. The amino acid and sugar composition of Y antigen strongly resembled the composition of the low molecular weight H and M antigens. Unlike the high molecular weight antigens, these low molecular weight antigens had methionine in relatively high concentrations; they had the same sugars as their respective high molecular weight counterparts. The yeast phase antigens differed from their respective mycelial counterparts in the following ways: glucose was the major sugar in the yeast phase with less amounts of mannose and traces of galactose, whereas in the mycelial antigens, mannose was the major sugar, with lesser amounts of galactose, glucose, and hexosamine. The H and M antigens of the yeast phase had high concentrations of glycine and alanine, whereas in the mycelial phase, these antigens had high concentrations of threonine and proline; the H and M antigens of the yeast phase had 5 to 16 times the protein to carbohydrate ratio observed for the same antigens of histoplasmin.     

The "public" determinants HLA-Bw4/Bw6 do not generate cytotoxic T lymphocytes (CTLs)

The human leucocyte antigens (HLA)-Bw4/Bw6 antigens detected serologically are "public" determinants located in the HLA-B molecule. They do not generate cytotoxic T lymphocytes (CTLs) in primary allogeneic cultures (mixed lymphocyte antigens) and secondary (primed lymphocyte typing) cultures indicate that they do not behave like normal HLA "private" cell-mediated lympholysis determinants. Therefore, the contribution of the 79-83 (alpha 1) residues in the generation of the epitopes Bw4/Bw6 does not seem to be critical for the examination by T cell receptor in allogeneic CML. The different overlapping patterns of the serological and CTL examinations are discussed, based on the structure of HLA class I antigens.     

Choice of fixatives for the immunohistochemical study of antigens using immunoadsorption

The effect of fixatives on the rat bone marrow antigens was studied by a method combining immunoadsorption and immunodiffusion. This method allows to estimate rapidly and reliably the effect of fixation on immunochemical properties of the antigen under study, using polyvalence antisera. Acetone and ethanol proved to be the best fixatives for the rat bone marrow antigens since they preserved their immunochemical properties. Aldehydes (formaldehyde and glutaraldehyde) "inactivated" the bone marrow antigens.     

Autoantibodies and immunologic theories]

A hypothesis is put forward to the effect that immunological phenomena represent a particular case of the transport of metabolites, rather than obligatory "defense mechanism". This hypothesis excludes the necessity in additional postulates (forbidden clones, somatic mutations, cells-repressors etc.) to account for the basic immunological phenomena, such as recognition, appearance of autoantibodies and tolerance. It suffices to assume that: 1) autolytic enzymes destroy "their" antigens but cannot destroy completely "foreign" antigens; 2) as a result of decomposition of antigens by enzymes, "tolerogens" may appear which block the receptors in immunocompetent cells thus preventing the appearance of antibodies; 3) cells capable to synthesize autoantibodies exist in the normal organism but not activated due to the absence of "their" antigens. When such antigens appear, they initiate the synthesis of antibodies. The hypothesis advanced may appear too simple as compared with the existing theories, but experiments have to confirm it.     

Soluble Antigens of Vaccinia-infected Mammalian Cells: III. Relation of “Early” and “Late” Proteins to Virus Structure

The structural proteins of vaccinia virus can be divided into two classes on the basis of their times of synthesis in the infected cell. The production of one of these classes of proteins begins prior to the onset of viral deoxyribonucleic acid (DNA) replication. These are referred to as "early" proteins. Synthesis of the second class of structural proteins follows the onset of viral DNA replication; hence, the term "late" proteins for this class. We are able, by immunological procedures, to identify three "early" virus-structural proteins. These materials, when incorporated into virions, appear to be associated with the "core" of the virion and do not elicit production of virus-neutralizing antibody. It would seem, therefore, that those virus-structural proteins synthesized early in the course of infection act as internal components of the virion. The "late" proteins may be subdivided into two groups on the basis of certain physical properties and molecular weight differences. The first of these groups, comprised of at least two proteins, corresponds to the classical LS antigens and elicits production of neutralizing antibodies. These proteins, when incorporated into virions, are found only in the outer ("coat") fraction of the virion. The second group of "late" antigens, also comprised of two proteins, termed the G antigens, do not elicit synthesis of neutralizing antibody. One of these proteins is associated with the virus "core"; the other is found in the "coat" fraction of the virion and appears to occupy an intermediary, subsurface position. Procedures suitable for the isolation of the G antigens are described, in addition to the partial characterization of these antigens.     

Vaccination againstBoophilus microplus: Localization of antigens on tick gut cells and their interaction with the host immune system

Cattle have been vaccinated againstBoophilus microplus with antigens derived from partially fed female ticks. The immune response of the host lyses the gut cells of adult ticks, causing a reduction in the number, weight and reproductive capacity of engorging ticks. This response is different from the immunity that cattle acquire after repeated tick infestation. Evidence is presented that the antigens used in vaccination are located on the plasma membrane of the gut cells and it is unlikely that these antigens are secreted into the host during feeding. Vaccination using such concealed antigens may not encounter the mechanisms of immune evasion that parasites usually demonstrate.In-vitro assays suggest that vaccination immunity is not dependent on the need to stimulate cell-mediated responses. Immunoglobulin G alone, or with the aid of complement, is enough to damage tick gut.The normal function of the one protein antigen isolated so far is unknown but we speculate that it serves some vital function on the cell plasma membrane.     

Immunofluorescent research on the species specificity of the antigens of hematopoietic cells in rodents

The bone marrow cells of mice, rats, guinea pigs, Syrian and dwarf hamsters exhibit a positive immunofluorescence reaction with antisera against insoluble antigens of the bone marrow cells of mice, Syrian and dwarf hamsters and, hence, contain common "cross-reacting" antigens. The use of different methods of antiserum absorption made it possible to reveal, in addition, antigens of "narrow" specificity in (1) mice, Syrian and dwarf hamsters, (2) Syrian and dwarf hamsters, as well as species specific antigens of the bone marrow cells of mice, Syrian and dwarf hamsters.     

The HLA system and its contribution to the genetics of rheumatic diseases

The results of the study of histocompatibility antigens at loci A, B and Dr in patients with RA and SLE, and their first degree relatives are presented. HLA antigens B12. B18, B27, Dr2 and Dr4 were associated with RA. The antigens HLA A11, B7, B35, Dr2 and Dr3 were associated with SLE. The influence of HLA antigens on formation of clinical picture of RA and SLE was determined. Evaluation of interallelic and interloci antigens interaction in a relative risk of disease suggests that, in some cases, there is a "superdominance" effect. Some combinations of HLA antigens at loci B and Dr increase the disease risk for RA and SLE. Analysis of test-marker linkage to genes predisposed to RA and SLE provides no direct confirmation of the hypothesis of their location on the short arm of the sixth chromosome between loci B and Dr, though this possibility cannot be completely excluded.     

Development of basal lamina in synaptic and extrasynaptic portions of embryonic rat muscle

Each vertebrate skeletal muscle fiber is ensheathed by a basal lamina (BL) which passes through the synaptic cleft of the neuromuscular junction. In the adult, the synaptic portion of the BL is both functionally and chemically specialized. We have used an immunofluorescence method to compare the development of synaptic and extrasynaptic portions of BL in embryonic rat intercostal muscles. Immunohistochemical staining of adult muscle fibers with monoclonal and serum antibodies defines "synaptic" antigens (including acetylcholinesterase) that are concentrated in synaptic BL, "extrasynaptic" antigens that are concentrated in extrasynaptic regions, and "shared" antigens (including collagen IV, fibronectin, laminin, and a heparan sulfate proteoglycan) that are present in both synaptic and extrasynaptic BL ( Sanes and Chiu , 1983). Synapses appear on newly formed myotubes on embryonic Day 14 (E14; birth is on E22 ). Patches of BL that contain shared and extrasynaptic antigens are present on myotube surfaces by E15, and BL forms a continuous sheath by E17. Shared antigens are present at but not confined to synaptic areas by E15. Two synaptic antigens appear in synaptic areas a day later, and are not detectable extrasynaptically . At least one extrasynaptic antigen is present at immature synapses, and lost or masked by E19 . Thus synaptic BL is not assembled as a unit; rather, components are added, lost, or modified as synaptogenesis proceeds.     

Facts and considerations about sex-specific antigens

Summary The status of the present knowledge on the mammalian sex-specific antigens (H-Y antigens) is reviewed and critically discussed. Special weight is given to problems related to three major topics, i.e., the immunology, genetics, and biological function(s) of these antigens. Current hypotheses as to the function(s) and the genetic control of the sex-specific antigens are individually scrutinized. Finally, some prospects for further H-Y research which seems especially urgent are briefly suggested.     

Production of Reference Enteroviruses

Forty-five human enterovirus reagents of certified purity and quality were prepared for use as seed viruses and as immunizing antigens. One of the reagents was ampouled as "untreated" seed virus, whereas 14 were ampouled as "MgCl(2)-stabilized" reagents. The remaining 30 reagents were ampouled as "untreated" seed viruses and as "MgCl(2)-stabilized" reagents. Thirty of the reagents were propagated on primary African green monkey kidney cells, 3 on primary baboon kidney cells, 3 on primary rhesus monkey kidney cells, and the remaining 9 on human amnion cells. Forty-two of the viral antigens were concentrated for use in the production of high-titered specific antisera in large animals.     

Studies of the antigenicity and immunogenicity of bromelain-pretreated red blood cells

The effects of the proteolytic enzyme bromelain (Br) on the antigenicity and immunogenicity of sheep and mouse red blood cells (RBC) have been investigated. The results presented support the previous claim that there are antigens present on Br RBC that are not present in an exposed form on untreated RBC and that Br RBC have lost some of the antigens present on the surface of normal RBC. The susceptibility of Br RBC to osmotic lysis was very similar to that of normal RBC, implying that the modified RBC were not more fragile than normal RBC. Injection of mice with Br mouse-RBC did not increase the unusually high "background" number of cells producing IgM antibodies against Br mouse-RBC and mice did not mount delayed-type hypersensitivity reactions against Br mouse-RBC, either before or after sensitizing injections of Br mouse-RBC. However, mouse-RBC and Br mouse-RBC elicited similar antibody responses in rabbits and guinea pigs. Although mice appeared unresponsive to Br mouse-RBC injections, delayed-type hypersensitivity responses and antibody production in primary and secondary responses were of similar levels irrespective of whether sheep-RBC or Br sheep-RBC were used as immunogens. From these studies it appears that mice have B-cells producing antibodies against the "new" antigens on Br mouse-RBC, but there are no T-cells that respond to these antigens by way of "helper" activity in antibody production or by way of cell-mediated immune reactions.     

Inefficient serotype knock down leads to stable coexistence of different surface antigens on the outer membrane in Paramecium tetraurelia

Expression of surface antigens is usually mutually exclusive, meaning that only one protein is present on the cell surface. With the RNAi feeding technology we induce serotype shifts in Paramecium tetraurelia which are demonstrated to be incomplete, meaning that the cells remain in a shifting state. The coexpression of "old" and "new" protein on the surface can be detected to be stable for more than 15 divisions over a 5-day feeding procedure, a time period different from that reported for temperature-induced shifts. A characteristic heterogenic distribution of the different surface antigens is demonstrated by double indirect-immunofluorescent-staining and we show antigen transport mechanisms related to the tips of cilia. Therefore, we discuss release mechanisms, potential sorting mechanisms for glycosylphosphatidylinositol-anchored proteins and the localizations of surface antigens, which are important for the reported classical immobilization reaction.     

Suppression of delayed-type hypersensitivity to third party "bystander" alloantigens by antigen-specific suppressor T cells

Delayed-type hypersensitivity (DTH) against alloantigens can be induced by sc immunization with allogeneic cells. The induction of DTH can be suppressed by iv preimmunization of the mice with similar allogeneic spleen cells, provided the cells are irradiated before injection. This suppression is mediated by T cells. The suppressor activity can be induced not only by H-2-and non-H-2-coded antigens, but also by H-2 subregion-coded antigens. Suppression induced by K, I, or D subregion-coded antigens is specific for that particular subregion as well as for its haplotype. I-J-coded alloantigens were found to not be necessary for the induction of antigen-specific suppressor T cells. After restimulation of suppressor T cells by the "specific" alloantigens, the DTH to simultaneously administered third-party alloantigens becomes suppressed as well. This nonspecific suppression of DTH to third party "bystander" alloantigens also occurs when the specific and the third-party antigens are presented on separate cells, provided that both cell types are administered together at the same site. The simultaneous presentation of both sets of alloantigens during the induction phase of DTH only is sufficient to prevent the normal development of DTH to the third-party antigens.     

Isolation of a cDNA clone from the B-G subregion of the chicken histocompatibility (B) complex

The B-G antigens are highly polymorphic antigens encoded by genes located within the major histocompatibility complex (MHC) of the chicken, the B system. The B-G antigens of the chicken MHC are found only on erythrocytes and correspond to neither MHC class I nor class II antigens. Several clones were selected from a gt11 erythroid cell expression library by means of rabbit antisera prepared against a purified, denatured B-G antigen. One clone chosen for further study, bg28, was confirmed as a B-G subregion cDNA clone by the results obtained through using it as a nucleic acid hybridization probe. In Northern hybridizations bg28 anneals specifically with erythroid cell mRNA. In Southern blot analyses the bg28 clone could be assigned to the B system-bearing microchromosome of the chicken karyotype on the basis of its hybridization to DNA from birds disomic, trisomic, and tetrasomic for this microchromosome. The cDNA clone was further mapped to the B-G subregion on the basis of its pattern of hybridization with DNA from birds of known B region recombinant haplotypes. Southern blot analyses of the hybridization of bg28 with genomic DNA from birds of known haplotypes strongly suggest that the B-G antigens are encoded by a highly polymorphic multigene family.     

Protectome Analysis: A New Selective Bioinformatics Tool for Bacterial Vaccine Candidate Discovery

New generation vaccines are in demand to include only the key antigens sufficient to confer protective immunity among the plethora of pathogen molecules. In the last decade, large-scale genomics-based technologies have emerged. Among them, the Reverse Vaccinology approach was successfully applied to the development of an innovative vaccine against Neisseria meningitidis serogroup B, now available on the market with the commercial name BEXSERO® (Novartis Vaccines). The limiting step of such approaches is the number of antigens to be tested in in vivo models. Several laboratories have been trying to refine the original approach in order to get to the identification of the relevant antigens straight from the genome. Here we report a new bioinformatics tool that moves a first step in this direction. The tool has been developed by identifying structural/functional features recurring in known bacterial protective antigens, the so called “Protectome space,” and using such “protective signatures” for protective antigen discovery. In particular, we applied this new approach to Staphylococcus aureus and Group B Streptococcus and we show that not only already known protective antigens were re-discovered, but also two new protective antigens were identified.Although vaccines based on attenuated pathogens as pioneered by Luis Pasteur have been shown to be extremely effective, safety and technical reasons recommend that new generation vaccines include few selected pathogen components which, in combination with immunostimulatory molecules, can induce long lasting protective responses. Such approach implies that the key antigens sufficient to confer protective immunity are singled out among the plethora of pathogen molecules. As it turns out, the search for such protective antigens can be extremely complicated.Genomic technologies have opened the way to new strategies in vaccine antigen discovery (1, 2, 3). Among them, Reverse Vaccinology (RV)¹ has proved to be highly effective, as demonstrated by the fact that a new Serogroup B Neisseria meningitidis (MenB) vaccine, incorporating antigens selected by RV, is now available to defeat meningococcal meningitis (4, 5). In essence, RV is based on the simple assumption that cloning all annotated proteins/genes and screening them against a robust and reliable surrogate-of-protection assay must lead to the identification of all protective antigens. Because most of the assays available for protective antigen selection involve animal immunization and challenge, the number of antigens to be tested represents a severe bottleneck of the entire process. For this reason, despite the fact that RV is a brute force, inclusive approach (“test-all-to-lose-nothing” type of approach) in their pioneered work of MenB vaccine discovery, Pizza and co-workers did not test the entire collection of MenB proteins but rather restricted their analysis to the ones predicted to be surface-localized. This was based on the evidence that for an anti-MenB vaccine to be protective bactericidal antibodies must be induced, a property that only surface-exposed antigens have. For the selection of surface antigens Pizza and co-workers mainly used PSORT and other available tools like MOTIFS and FINDPATTERNS to find proteins carrying localization-associated features such as transmembrane domains, leader peptides, and lipobox and outer membrane anchoring motifs. At the end, 570 proteins were selected and entered the still very labor intensive screening phase. Over the last few years, our laboratories have been trying to move to more selective strategies. Our ultimate goal, we like to refer to as the “Holy Grail of Vaccinology,” is to identify protective antigens by “simply” scanning the genome sequence of any given pathogen, thus avoiding time consuming “wet science” and “move straight from genome to the clinic” (6).With this objective in mind, we have developed a series of proteomics-based protocols that, in combination with bioinformatics tools, have substantially reduced the number of antigens to be tested in the surrogate-of-protection assays (7, 8). In particular, we have recently described a three-technology strategy that allows to narrow the number of antigens to be tested in the animal models down to less than ten (9). However, this strategy still requires high throughput experimental activities. Therefore, the availability of in silico tools that selectively and accurately single out relevant categories of antigens among the complexity of pathogen components would greatly facilitate the vaccine discovery process.In the present work, we describe a new bioinformatics approach that brings an additional contribution to our “from genome to clinic” goal. The approach has been developed on the basis of the assumption that protective antigens are protective in that they have specific structural/functional features (“protective signatures”) that distinguish them from immunologically irrelevant pathogen components. These features have been identified by using existing databases and prediction tools, such as PFam and SMART. Our approach focuses on protein biological role rather than its localization: it is completely protein localization unbiased, and lead to the identification of both surface-exposed and secreted antigens (which are the majority in extracellular bacteria) as well as cytoplasmic protective antigens (for instance, antigens that elicit interferon γ producing CD4+ T cells, thus potentiating the killing activity of phagocytic cells toward intracellular pathogens). Should these assumptions be valid, PS could be identified if: (1) all known protective antigens are compiled to create what we refer to as “the Protectome space,” and (2) Protectome is subjected to computer-assisted scrutiny using selected tools. Once signatures are identified, novel protective antigens of a pathogen of interest should be identifiable by scanning its genome sequence in search for proteins that carry one or more protective signatures. A similar attempt has been reported (10), where the discrimination of protective antigens versus nonprotective antigens was tried using statistical methods based on amino acid compositional analysis and auto cross-covariance. This model was implemented in a server for the prediction of vaccine candidates, that is, Vaxijen (www.darrenflower.info/Vaxijen); however, the selection criteria applied are still too general leading to a list of candidates that include ca. 30% of the total genome ORFs very similarly to the number of antigens predicted by classical RV based on the presence of localization signals.Here we show that Protectome analysis unravels specific signatures embedded in protective antigens, most of them related to the biological role/function of the proteins. These signatures narrow down the candidate list to ca. 3% of the total ORFs content and can be exploited for protective antigen discovery. Indeed, the strategy was validated by demonstrating that well characterized vaccine components could be identified by scanning the genome sequence of the corresponding pathogens for the presence of the PS. Furthermore, when the approach was applied to Staphylococcus aureus and Streptococcus agalactiae (Group B Streptococcus, GBS) not only already known protective antigens were rediscovered, but also two new protective antigens were identified.     

Identification of a family of human centromere proteins using autoimmune sera from patients with scleroderma

We have examined preimmune serum samples from a patient who progressively developed the symptoms of scleroderma CREST over a period of several years. During this period, anti-centromere antibodies (recognized by indirect immunofluorescence) appeared in the serum. Concomitant with the appearance of the anti-centromere antibodies, antibody species recognizing three chromosomal antigens in immunoblots of SDS polyacrylamide gels appeared in the patient's serum. These antigens migrate with electrophoretic mobilities corresponding to M_r=17, 80, and 140 kilodaltons (kd). Affinity-eluted antibody fractions recognizing the antigens have been prepared from sera of three other patients. Indirect immunofluorescence labeling of mitotic cells using these antibody fractions demonstrates that the antigens are centromere components. We designate them CENP (CENtromere Protein) — A (17kd), CENP-B (80kd), and CENP-C (140kd). The three CENP antigens share antigenic determinants. Immunoblotting experiments show that these patients make antibody species recognizing at least three distinct epitopes on CENP-B and two on CENP-C. Sera from different patients contain different mixtures of the antibody species.