Evidence for two homologous, but nonidentical, Ia molecules determined by the I-EC subregion.

Sequential immunoprecipitation, two-dimensional gel electrophoresis and peptide mapping analyses of B10A(3R), 35S-methionine-labeled, I-EC subregion products were performed. Evidence is presented here for the presence of two structurally homologous, but nonidentical, gene products of the I-EC subregion. These two Ia molecules are independently immunoprecipitable, identical in molecular size and charge, but differ by approximately equal to 20% in their peptides obtained by partial digestion with Staphylococcus aureus protease V8.

Predominant role for directly transfected dendritic cells in antigen presentation to CD8+ T cells after gene gun immunization.

Cutaneous gene (DNA) bombardment results in substantial expression of the encoded antigen in the epidermal layer as well as detectable expression in dendritic cells (DC) in draining lymph nodes (LNs). Under these conditions, two possible modes of DC antigen presentation to naive CD8+ T cells might exist: (a) presentation directly by gene-transfected DC trafficking to local lymph nodes, and (b) cross-presentation by untransfected DC of antigen released from or associated with transfected epidermal cells. The relative contributions of these distinct modes of antigen presentation to priming for cytotoxic T cell (CTL) responses have not been clearly established. Here we show that LN cells directly expressing the DNA-encoded antigen are rare; 24 h after five abdominal skin bombardments, the number of these cells does not exceed 50-100 cells in an individual draining LN. However, over this same time period, the total number of CD11c+ DC increases more than twofold, by an average of 20,000-30,000 DC per major draining node. This augmentation is due to gold bombardment and is independent of the presence of plasmid DNA. Most antigen-bearing cells in the LNs draining the site of DNA delivery appear to be DC and can be depleted by antibodies to an intact surface protein encoded by cotransfected DNA. This finding of predominant antigen presentation by directly transfected cells is also consistent with data from studies on cotransfection with antigen and CD86-encoding DNA, showing that priming of anti-mutant influenza nucleoprotein CTLs with a single immunization is dependent upon coexpression of the DNAs encoding nucleoprotein and B7.2 in the same cells. These observations provide insight into the relative roles of direct gene expression and cross-presentation in CD8+ T cell priming using gene gun immunization, and indicate that augmentation of direct DC gene expression may enhance such priming.

The conformational flexibility of class I H-2 molecules as revealed by anti-peptide antibodies specific for intracytoplasmic determinants: differential reactivity of beta 2-microglobulin "bound" and "free" H-2Kb heavy chains.

Immunoprecipitation experiments using anti-peptide antisera prepared against exon 6, exon 7 and exon 8-encoded intracytoplasmic regions of the H-2Kb gene product indicated that approximately 1/3 of the H-2Kb heavy chains in a cell surface-labelled glycoprotein fraction from EL-4 cells, or H-2b spleen cells, is not associated with beta 2-microglobulin (beta 2-m). This population of "free" H-2Kb heavy chains failed to react with alloantisera or monoclonal antibodies specific for conventional H-2Kb serological determinants, suggesting that significant conformational alterations were induced in the extracellular domains upon dissociation of beta 2-m. In addition, although antibodies to intracytoplasmic peptide 8 were able to react with both "free" and beta 2-m "bound" heavy chains, the determinants seen by anti-peptide 6 and anti-peptide 7 were only recognized in the "free" heavy chain. These data suggest that the conformational perturbation of the extracellular domains induced by beta 2-m dissociation can be "transmitted" to the intracytoplasmic region of the heavy chain. These results indicate the potential for a class I heavy chain-mediated transmembrane signalling event, and suggest that the "free" class I heavy chain might have a role to play in the major histocompatibility complex (MHC)-restricted presentation of T-cell determinants to cytotoxic T-lymphocytes.

Inter- and intramolecular disulfide bonding among lymphocyte plasma membrane proteins and glycoproteins.

The disulfide bonding characteristics of the pig lymph node plasma membrane (PM) proteins and glycoproteins have been examined by 1- and 2-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Reaction of the purified PM vesicles with N-ethyl maleimide (NEM) prior to detergent solubilization was found to markedly reduce the extent of intermolecular disulfide bonding subsequently observed. Thus the blocking of free sulfhydryl groups with NEM prevented the detergent-induced disulfide bonding of numerous components, including PM-bound actin. The extent of intermolecular disulfide bonding among the NEM-pretreated PM glycoproteins purified by lentil lectin affinity chromatography was found to be relatively limited, with only 3% of the total glycoprotein present as intermolecular disulfide-bonded complexes. In contrast, the degree of intramolecular disulfide bonding revealed by a modified 1-dimensional SDS-PAGE technique was quite striking. Among those polypeptides demonstrating a clearly altered mobility upon reduction was the heavy chain of class I and beta-chain of class II major histocompatibility complex (MHC) antigens. The class II alpha-chain, however, was much less affected. These changes have been compared with those observed for proteins containing intramolecular disulfide-bonded domains of known size and number, and considered in the light of recent information on the structure of MHC antigens.

Fractionation and identification of pig lymph node plasma membrane glycoproteins.

Plasma membrane vesicles purified from pig mesenteric lymph node tissue were solubilized in 1% (w/v) sodium deoxycholate and the fractionation of both membrane proteins and glycoproteins assessed by gel filtration on AcA 34. Milligram quantities of the glycoprotein fraction, consisting of 12 distinct bands on SDS-PAGE ranging in apparent mol. wt from 12,000 to 250,000, were purified by affinity chromatography on lentil lectin-Sepharose. Identified among these by specific immunoprecipitation were the major histocompatibility antigen, SLA (band 9), in association with beta 2-microglobulin (band 12); as well as the alpha (band 10) and beta (band 11) subunits of the Ia-like antigens. A tentative identification of the membrane-bound immunoglobulins (IgM, IgG and IgA) was also proposed on the basis of their known affinities for protein A. Thus, the domestic pig represents and inexpensive, abundant source of defined lymphocyte plasma membrane glycoproteins suitable for further structural analysis.

A structural analysis of the carbohydrate side chains on class I and class II histocompatibility antigens of the swine facilitated by heteroantisera specific for the denatured polypeptides.

Rabbit heteroantisera specific for the denatured glycoprotein subunits of swine class I and class II major histocompatibility complex (MHC) antigens have been prepared and utilized to monitor changes in the mobilities of these polypeptides on SDS-polyacrylamide gel electrophoresis subsequent to various deglycosylation procedures. This information, in combination with lectin reactivity patterns for the glycoproteins bound to nitrocellulose, has made it possible to define specific structural features of the MHC antigen-associated carbohydrate side chains. Both the class I heavy (alpha) chain and the class II light (beta) chain bear a single, N-linked, complex-type oligosaccharide which reacts with lentil lectin (LcH), but not concanavalin A (Con A); a reactivity pattern suggesting the possibility of a special triantennary structure. In contrast, the class II heavy (alpha) chains appear to possess two carbohydrate units, one an N-linked, LcH-reactive, complex-type side chain, and the other, an N-linked, Con A-reactive, high-mannose-type of oligosaccharide. The data suggest considerable homology between the swine and human MHC antigens with respect to the structure of their carbohydrate side chains. The analysis also serves to illustrate how antibodies specific for the denatured polypeptide backbone of individual glycoproteins, along with lectin reactivity patterns, can be used to extract structural information about the attached carbohydrate moieties using minimal amounts of partially purified glycoproteins.

Adjuvant-independent immunization by immunotargeting antigens to MHC and non-MHC determinants in vivo.

Using avidin as a model protein antigen, and biotinylated monoclonal antibodies as a convenient means of forming stable complexes with avidin, we have investigated the adjuvant-independent immunization of three mouse strains, C57BL/6, C3H and (C57BL/6 x C3H)F1, with immunoconjugates targeted to different class II MHC and non-MHC sites. The results confirm the effectiveness of anti-I-Ak and anti-I-Ab immunoconjugates with respect to priming for secondary IgG responses in (H-2b x H-2k)F1 mice, while indicating a lack of response in strains which are homozygous for the targeted allele. In terms of non-MHC targets in the monocyte-macrophage lineage, neither anti-MAC-1 nor anti-MAC-2 immunoconjugates were effective in any of the three strains. However, the 33D1 anti-dendritic cell antibody gave significant responses in all three strains, with the F1 response being more than 10-fold greater than the anti-class II immunoconjugates in either strain. These findings indicate that immunotargeting a protein antigen to a non-MHC determinant on dendritic cells in vivo can be an effective means of inducing an adjuvant-independent serological response, and that this approach can have significant advantages over anti-class II MHC immunotargeting.

Delivery of synthetic peptides by anti-class II MHC monoclonal antibodies induces specific adjuvant-free IgG responses in vivo.

Two synthetic peptides from different regions of the glycoprotein D of herpes simplex virus (HSV) have been used to determine whether or not anti-peptide IgG responses can be generated in the absence of adjuvant. Based on an earlier demonstration (Carayanniotis and Barber, 1987) that protein antigens could be made immunogenic in the absence of adjuvant, if coupled to MAbs specific for the recipient's class II major histocompatibility complex (MHC) antigens, the HSV synthetic peptides were photocoupled to avidin and mixed with biotinylated anti-class II or control antibodies. Peptide-specific IgG responses were obtained when avidin-peptide conjugates coupled to an anti-I-Ak MAb were injected into (H-2k x H-2b) F1 mice, but not when injected into H-2b mice. The same avidin-peptide conjugates were non-immunogenic when coupled to a control anti-influenza virus nucleoprotein antibody or biotinylated bovine serum albumin. These data indicate that targeting synthetic peptides to class II bearing cells in vivo can elicit peptide-specific IgG responses without the need for adjuvant. These findings offer a new approach to the design and delivery of adjuvant-independent vaccine agents based on synthetic peptide antigens.

The preparation and characterization of anti-peptide heteroantisera recognizing subregions of the intracytoplasmic domain of class I H-2 antigens.

Peptides corresponding to each of the three intracytoplasmic exons (i.e. exons 6, 7 and 8) of the murine class I H-2Kb gene were synthesized, coupled to bovine serum albumin and used as immunogens in rabbits. In each case the antisera were found to react with the immunizing peptide coupled to a heterologous carrier, and recognized class I heavy chains electrophoretically transferred from SDS-polyacrylamide gels to nitrocellulose. Immunoprecipitation of class I antigens from Nonidet P-40 (NP-40) solubilized EL-4 (H-2b) tumour cells by each of the antisera reflected their ability to recognize the corresponding determinants in non-denatured class I molecules. The same sera were also able to immunoprecipitate class I molecules from NP-40 solubilized RDM-4 (H-2k) and P815 (H-2d) tumour cells, indicating the cross-reactive nature of these antisera for different class I alleles. In addition to reacting with the class I heavy chain in its conventional form as a dimer with beta 2-microglobulin, the antiserum specific for the exon 8 peptide was able to react with "free" (i.e. non-beta 2-microglobulin-associated) class I heavy chains. Thus, a unique set of immunological reagents has been prepared which offer a new approach to studying the structural and functional features of the cytoplasmic domain of class I H-2 antigens.

Enhanced immunogenicity of B cell lymphoma genetically engineered to express both B7-1 and interleukin-12.

The A20 murine B cell lymphoma was transfected with B7-1 and subsequently these variants and vector control variants were retrovirally infected to express murine interleukin-12 (mIL-12). In vitro data showed that the B7-1 variants enhanced secretion of IL-2 and IL-4 by allogeneic T cells in mixed lymphocyte tumor cultures. While IL-12 variants stimulated IFN-gamma, variants expressing both B7-1 and IL-12 stimulated IFN-gamma, IL-2, and IL-4 secretion. Tumorigenicity experiments showed that whereas B7-1 delayed tumor onset, only the mIL-12 variants with or without B7-1 were completely rejected in syngeneic hosts. In addition, tumor-free mice were protected against subsequent challenge with the parental unmodified cells and had enhanced cytotoxic T lymphocyte (CTL) lysis activity. Results from minimal disease mixing experiments demonstrated that only the A20/B7-1/mIL-12 variant was able to reject A20 unmodified cells inoculated at the same site, whereas prolonged survival was observed when the A20 parental cells were inoculated at different sites. Depletion studies and injections into nu-/nu- mice demonstrated that both CD4+ and CD8+ T cells may mediate immunity. These data suggest that vaccinations with tumor cells genetically modified to express both B7-1 and IL-12 may alter cytokine profiles and generate CTL activity and, thus, the mechanisms of enhanced antitumor immunity may be multifactorial.

A cautionary note on the use of lentil lectin affinity chromatography in the presence of sodium deoxycholate.

When affinity chromatography with lentil lectin (LcH)-Sepharose was carried out in 0.5% (w/v) sodium deoxycholate (DOC), 10 mm Tris, pH 8.2, LcH consistently appeared in the 0.1 M alpha-methyl-mannoside (alpha-MeMan)-eluted fraction. Eluted LcH was recovered in both the control and specific immunoprecipitates with antisera directed against components of the glycoprotein fraction, suggesting that at least a portion of the LcH retained the capacity to interact with protein-bound carbohydrate. We suggest that the presence of functional LcH in the purified glycoprotein fraction is a potential source of artifact which may not be generally appreciated. When the non-ionic detergent. Nonidet P-40 was substituted for DOC in the same type of LcH-Sepharose fractionation, LcH did not appear in the 0.1 M alpha-MeMan-eluted fraction.

Recombinant antibodies with conformationally constrained HIV type 1 epitope inserts elicit glycoprotein 160-specific antibody responses in vivo.

Although neutralizing epitopes have been identified on the HIV-1 gp120/gp41 envelope complex, efforts to exploit this information through the construction of synthetic peptide vaccines have been largely unsuccessful. Unfortunately, synthetic peptides tend to be poorly immunogenic, and most often lack the conformational characteristics of the corresponding epitope in the native protein. In an effort to circumvent these difficulties, we have utilized an anti-class II MHC antibody as a molecular scaffold for the construction of two conformationally constrained neutralizing HIV-1 epitopes. Previously we demonstrated that anti-class II MHC antibodies can function as vectors for the induction of adjuvant-independent antibody responses to incorporated epitopes. In this instance, one epitope, IHIGPGRAFYT, is the crown of the V3 loop from gp120, and the other, ELDKWAS, is a neutralizing epitope from gp41. The insertion of these epitopes into a specific loop region of the immunoglobulin heavy chain FR3 was found to preserve the anti-class II MHC-binding activity of these recombinant antibodies, and the inserts were recognized by epitope specific monoclonal antibodies. When utilized as immunogens, each of these epitope insertion antibodies was able to induce high-titer anti-HIV-1 gp160 responses in guinea pigs. These responses were conformation specific in that the anti-gp160 binding was not inhibited by the synthetic peptide corresponding to the epitope in question. These data demonstrate the potential to construct conformationally constrained HIV-1 epitope immunogens, and thus establish an alternative approach to the design of an effective HIV-1 subunit vaccine.

Anti-HLA alloantibody is found in children but does not correlate with a lack of HIV type 1 transmission from infected mothers.

Searching for mechanisms of natural resistance to HIV infection with which to guide HIV vaccine design, we have examined antibody responses to HLA class I antigens in children of HIV-1-infected mothers. Anti-HLA antibodies are known to block HIV infectivity in vitro and can be protective against SIV challenge in macaques immunized with purified class I HLA. It was hypothesized that alloantibody to maternal HLA in children might contribute to the prevention of mother-to-child transmission of HIV-1. In fact, a surprisingly high proportion of the children examined, 22%, were found to have antibody against class I alloantigens. This alloantibody, however, did not correlate with the HIV status of the children and was found in a similar proportion of children of HIV-negative mothers. The HLA specificity of the antibody was not correlated with noninherited maternal HLA alleles and occurred with a higher frequency in older children. This result suggests environmental factors, rather than exposure to maternal cells, are involved in the formation of the alloantibody. The finding that anti-allo-class I HLA antibodies are not associated with a decreased risk of mother-to-child transmission indicates that this humoral immune response is unlikely to be the natural mechanism that accounts for the lack of transmission observed in many births. This result, however, does not preclude the further investigation of cellular alloimmune responses, or the use of alloimmunization as an artificial HIV immunization strategy.

Naturally occurring IgG anti-HLA alloantibody does not correlate with HIV type 1 resistance in Nairobi prostitutes.

In an effort to identify an immunological basis for natural resistance to HIV-1 infection, we have examined serum antibody responses to HLA class I antigens in female prostitutes of the Nairobi Sex Workers Study. Anti-HLA antibodies are known to block HIV infectivity in vitro and can be protective against SIV challenge in macaques immunized with purified class I HLA. Thus, it was postulated that broadly cross-reactive alloantibodies recognizing common HLA alleles in the client population might contribute to the prevention of heterosexual transmission of HIV. In fact, 12% of the women were found to have serum IgG antibodies against class I alloantigens. However, this alloantibody did not correlate with the HIV status of the women and was found in a similar proportion of HIV-positive and HIV-resistant women. The observed levels of alloantibody did not increase with HIV infection in susceptible individuals, suggesting that potential antigenic mimicry between HIV and host HLA class I antigens does not significantly increase levels of anti-class I antibodies. The lack of correlation between serum anti-allo-class I HLA antibodies and the risk of sexual transmission indicates that this humoral immune response is unlikely to be the natural mechanism behind the HIV-resistance phenotype of persistently HIV-seronegative women. This result, however, does not preclude the further investigation of alloimmunization as an artificial HIV immunization strategy.

The use of bone marrow-chimeric mice in determining the MHC restriction of epitope-specific cytotoxic T lymphocytes.

Plasmid DNA immunization has emerged as a promising vaccine strategy against infectious agents, as well as a potential intervention for the treatment of cancer, autoimmunity, and allergy (1). Until recently, however, the cellular events by which injected plasmid DNA elicits potent antibody and cytotoxic T-lymphocyte (CTL) responses were largely unknown. Upon intramuscular (i.m.) injection of naked DNA, predominant expression of transfected DNA occurs in the myofibers (2), but no direct transfection of antigen presenting cells (APC) has been reported. There are essentially three different mechanisms by which CTLs can be primed by the injected DNA (3). The first possibility is that the transfected muscle cells directly activate CTLs by presenting the antigenic peptide on their MHC class I molecules. Alternatively, the priming of CTLs may be mediated by professional APC taking up antigen released from muscle cells. Finally, CTL priming may involve direct transfection of APC occurring, albeit at low level, and that the CTLs are activated by the transfected APC.

The immunotargeting approach to adjuvant-independent subunit vaccine design.

Recently there has been considerable interest in exploring adjuvant-independent approaches to the enhancement of immunogenicity. One strategy which has emerged in response to this need is the immunotargeting approach to subunit vaccine design. Immunotargeting involves the conjugation of non-self antigens to monoclonal antibodies specific for cell surface determinants (e.g. class II MHC) on antigen presenting cells in vivo. Saline injections of these immunoconjugates have been shown to promote significant IgG responses to the delivered antigens in a number of different animal model studies. Oral and nasal immunizations in mice with anti-class II MHC immunoconjugates induce both secretory IgA and systemic IgG responses. Recombinant anticlass II MHC antibodies with defined B-cell and T-cell epitopes incorporated into their primary sequence have recently been used to induce epitope specific antibody responses in macaques. Thus the potential now exists for the rational design of adjuvant-independent human vaccine candidates based on a recombinant immunotargeting anti-body framework.

A recommendation for visualizing disulfide bonding by one-dimensional sodium dodecyl sulfate--polyacrylamide gel electrophoresis.

A simple modification of the conventional one-dimensional sodium dodecyl sulfate--polyacrylamide gel electrophoresis technique has been used to visualize inter- and intramolecular disulfide bonding in proteins. The gradient of reducing agent established between adjacent slab gel tracks by electrophoresing identical protein samples next to one another, one containing and the other not containing 2-mercaptoethanol, has been used to visualize the change in mobility of disulfide bond-containing proteins throughout the transition from a reducing to a nonreducing environment. As illustrated by an analysis of immunoglobulin heavy and light chains, the method particularly facilitates the positive identification of proteins containing intrachain disulfide bonds.

Magnetic resonance studies of concanavalin A:CONFORMATIONAL CHANGES INDUCED BY Ca2+ and alpha-methyl-D-mannopyranoside.

Three independent solution spectroscopic techniques (solvent proton relaxation enhancement, circular dichroism, and high resolution 220 MHz proton magnetic resonance spectroscopy) have been utilized to demonstrate mental ion- and monosaccharide inhibitor-induced structural perturbations for the dimeric form of the plant lectin concanavalin A (Con A). The results indicate that (i) the occupation of the transition metal ion site S1 by Mn-2+ or Zn-2+ does not detectably perturn the demetallized protein conformation, (ii) the binding of Ca=2+ to the Con A-Mn-2+ or Con A-Zn-2+ complexes perturbs the protein structure in the vicinity of the S1 site as well as at points remote from the S1-S2 double ion site, and (iii) the binding of the monosaccharide inhibitor alpha-methyl-D-mannopyranoside to the fully metallized Con A complex also significantly perturbs the structural features of the protein. A detailed radio frequency dependence analysis of the Ca-2+ effect on the solvent proton relaxation enhancement properties of the Con A-Mn-2+ complex indicates that the considerable reduction in the observed enhancement upon Ca-2+ binding principally results from an approximate 120-fold decrease in the single Mn-2+ water of hydratio- exchange rate; The 220 MHz proton magnetic resonance spectra for Con A indicate that this form of spectroscopy is the most useful of those utilized in detailing the solution structural features of this lectin, and a tentative assignment for the C-2-H proton of histidine residue 24 (the S1 site ligand) has been proposed.