Plasmid DNA encoding targeted naturally processed peptides generates protective cytotoxic T lymphocyte responses in immunized animals.

Genetic immunization has been widely applied in efforts to find novel and efficient mechanisms of stimulating the immune response. An effective attack against viral pathogens or tumors often requires activation of T cell-mediated immunity and the generation of cytotoxic T cells. Intramuscular immunization with plasmid DNA containing cDNAs that encode proteins results in expression and secretion of the foreign antigen by muscle cells. T cell activation occurs when peptide fragments of the exogenous protein are presented by major histocompatibility complex class I molecules on the surface of professional antigen-presenting cells. Identification of specific peptide epitopes from a protein antigen presented to T cells during an infectious process or tumor situation would provide all of the antigenic information needed to stimulate effective T cell-mediated immunity. Such peptides represent the naturally processed epitopes selected by the processing machinery of antigen presenting cells. Delivery of this information to the appropriate cells in vivo might be sufficient to stimulate T cell immunity and overcome the difficulties associated with overexpression of large protein antigens or those with potentially toxic side effects. This report describes the use of naturally processed T cell epitopes, administered in plasmid DNA vaccines, to stimulate cytotoxic T cell responses to two viral antigens effectively.

The discovery and use of HLA-associated epitopes as drugs.

MHC receptors "display" peptide fragments to T cells. These peptides are predominantly derived from proteins expressed within or ingested by the presenting cell. Since empty MHC molecules are highly unstable, peptide ligands are bound prior to MHC surface expression and the ensuing t1/2 off rates are often on the order of days. It is the remarkable stability of MHC/peptide complexes, which provide us an opportunity to purify MHC molecules from infected, transfected, or antigen pulsed cells and subsequently identify the naturally processed peptides being presented. On the other hand, the stability of MHC/peptide complexes substantially reduces the potency of parenterally administered peptides in vivo. Using serial immuno-affinity chromatography and mass spectrometry, naturally processed peptides can be identified. When these peptides are then encoded into nucleic acid and delivered parenterally, they are highly immunogenic. Application of these techniques to induce vigorous CTL responses will be discussed.

Crystallographic analysis of endogenous peptides associated with HLA-DR1 suggests a common, polyproline II-like conformation for bound peptides.

The structure of the human major histocompatibility complex (MHC) class II molecule HLA-DR1 derived from the human lymphoblastoid cell line LG-2 has been determined in a complex with the Staphylococcus aureus enterotoxin B superantigen. The HLA-DR1 molecule contains a mixture of endogenous peptides derived from cellular or serum proteins bound in the antigen-binding site, which copurify with the class II molecule. Continuous electron density for 13 amino acid residues is observed in the MHC peptide-binding site, suggesting that this is the core length of peptide that forms common interactions with the MHC molecule. Electron density is also observed for side chains of the endogenous peptides. The electron density corresponding to peptide side chains that interact with the DR1-binding site is more clearly defined than the electron density that extends out of the binding site. The regions of the endogenous peptides that interact with DRI are therefore either more restricted in conformation or sequence than the peptide side chains or amino acids that project out of the peptide-binding site. The hydrogen-bond interactions and conformation of a peptide model built into the electron density are similar to other HLA-DR-peptide structures. The bound peptides assume a regular conformation that is similar to a polyproline type II helix. The side-chain pockets and conserved asparagine residues of the DR1 molecule are well-positioned to interact with peptides in the polyproline type II conformation and may restrict the range of acceptable peptide conformations.

Staphylococcal enterotoxin A has two cooperative binding sites on major histocompatibility complex class II.

The superantigen staphylococcal enterotoxin A (SEA) binds to major histocompatibility complex (MHC) class II molecules at two sites on either side of the peptide groove. Two separate but cooperative interactions to the human class II molecule HLA-DR1 were detected. The first high affinity interaction to the DR1 beta chain is mediated by a zinc atom coordinated by H187, H225, and D227 in SEA and H81 in the polymorphic DR1 beta chain. The second low affinity site is to the DR1 alpha chain analogous to SEB binding and is mediated by residue F47 in SEA. Binding of one SEA to the DR1 beta chain enhances the binding of a second SEA molecule to the DR1 alpha chain. The zinc site is on the opposite side of the SEA molecule from residue F47 so that one SEA molecule can readily bind two class II molecules. Both binding sites on SEA are required for maximal activity. Thus, unlike, SEB, SEA requires two separate binding sites for optimal activity, which may allow it to stabilize SEA interaction with T cell receptors, as well as to activate the antigen-presenting cell by cross-linking MHC class II.

Macaques immunized with HLA-DR are protected from challenge with simian immunodeficiency virus.

Macaques immunized with uninfected human cells have been shown to be protected from challenge with simian immunodeficiency virus (SIV) propagated in human cells. To identify the potential antigens involved in this protection, macaques were immunized with uninfected human cells, sucrose density gradient-purified culture fluid from uninfected human cells (mock virus), beta-2 microglobulin (beta 2M), immunoaffinity-purified HLA class I and class II proteins from these human cells, and adjuvant. Although all macaques immunized with beta 2M and HLA class I developed high antibody titers to beta 2M, these animals were not protected from a subsequent challenge with infectious SIV grown in human cells. In contrast, the macaques immunized with class II protein (HLA-DR) and mock virus developed antibodies to class II protein and were protected from the intravenous infectious virus challenge. The class II protein- and mock virus-immunized animals which were protected from challenge were given boosters of the appropriate antigen and challenged with the same SIV propagated in macaque cells. All animals became infected, indicating that the protection seen with human class II protein did not extend to protection from infection with SIV containing macaque class II proteins. Since the virus released from SIV-infected macaque cells would contain macaque class II proteins, our results suggest that the initial SIV infected was completely prevented. In addition, the lack of protection from challenge with SIV propagated in macaque cells provided strong evidence that the protection was due to an immune response to the cellular proteins and not to epitopes cross-reactive between class II proteins and the viral proteins, since the identical virus proteins were present in both challenge stocks. These results are the first demonstration that immunization with a purified cellular protein can protect from virus infection.

Direct binding of the Mtv7 superantigen (Mls-1) to soluble MHC class II molecules.

The superantigen encoded by the mouse mammary tumor virus (MMTV) is a potent stimulator of T cells when bound to MHC class II molecules. Recent data from this laboratory have shown that the Mtv7 superantigen, Mls-1, elicits a strong T cell response when presented by HLA-DR. To expand these observations further, we have produced the 28 kDa extracellular domain and the 18 kDa carboxy-terminal subfragment of the Mls-1 protein in E. coli and studied their interaction with human MHC class II molecules in vitro. In this report, we demonstrate direct binding of these recombinant forms of the Mls-1 protein to soluble HLA-DR1 and HLA-DR4, but not to HLA-A2. Our data imply a unique class II interaction site of retroviral superantigens that is not shared with bacterial superantigens.

Toxic shock syndrome toxin-1 complexed with a class II major histocompatibility molecule HLA-DR1.

The three-dimensional structure of a Staphylococcus aureus superantigen, toxic shock syndrome toxin-1 (TSST-1), complexed with a human class II major histocompatibility molecule (DR1), was determined by x-ray crystallography. The TSST-1 binding site on DR1 overlaps that of the superantigen S. aureus enterotoxin B (SEB), but the two binding modes differ. Whereas SEB binds primarily off one edge of the peptide binding site of DR1, TSST-1 extends over almost one-half of the binding site and contacts both the flanking alpha helices of the histocompatibility antigen and the bound peptide. This difference suggests that the T cell receptor (TCR) would bind to TSST-1:DR1 very differently than to DR1:peptide or SEB:DR1. It also suggests that TSST-1 binding may be dependent on the peptide, though less so than TCR binding, providing a possible explanation for the inability of TSST-1 to competitively block SEB binding to all DR1 molecules on cells (even though the binding sites of TSST-1 and SEB on DR1 overlap almost completely) and suggesting the possibility that T cell activation by superantigen could be directed by peptide antigen.

Assembly and peptide binding of major histocompatibility complex class II heterodimers in an in vitro translation system.

In vitro transcription/translation of HLA-DR1 cDNAs in the presence of microsomal membranes was used to study the association of major histocompatibility complex class II molecules with peptide and invariant chain (Ii) in the endoplasmic reticulum (ER). HLA-DR alpha and HLA-DR beta subunits assembled into SDS-unstable heterodimers in the absence of exogenous peptide. The inclusion of synthetic peptides during the alpha/beta assembly process promoted their conversion to SDS-resistant heterodimers. Addition of Ii RNA during the translation of HLA-DR alpha and HLA-DR beta RNAs resulted in the formation of alpha/beta/Ii complexes. Peptide binding by class II molecules was detected even when excess Ii was present during alpha/beta assembly. These findings indicate that peptides can bind alpha/beta heterodimers in the ER microenvironment and suggest that peptides derived from cytosolic proteins that are presented by class II molecules at the cell surface may have bound to HLA-DR in the ER.

Selective release of some invariant chain-derived peptides from HLA-DR1 molecules at endosomal pH.

The predominant peptides bound to major histocompatibility complex class II molecules expressed on human B cells are derived from a relatively limited number of self proteins. To determine whether any of the prebound self peptides might be released in endosomes during recycling, water-soluble HLA-DR1 molecules were incubated with a high affinity synthetic peptide at pH 4.0 and 7.0 at 37 degrees C. The resulting bound peptide repertoire was then acid extracted, and separated by reversed-phase high performance liquid chromatography. Using a combination of mass spectrometry and ultraviolet spectroscopy, prebound self peptides and newly bound synthetic peptide were characterized. Most self peptides bound to HLA-DR1 were not appreciably released during extended exposure to pH 4.0. However, some invariant chain-derived peptides were uniquely released at this pH.

Analysis of MHC-presented peptides: applications in autoimmunity and vaccine development.

T cells recognize antigenic peptides presented on the cell surface by major histocompatibility complex (MHC) molecules. Here, Roman Chicz and Robert Urban describe the features of peptides bound to MHC molecules and the mechanism by which these surface proteins bind diverse peptide ligands with high affinity. In addition, they discuss the application of new technologies to the identification of MHC-associated peptides.

Three-dimensional structure of a human class II histocompatibility molecule complexed with superantigen.

The structure of a bacterial superantigen, Staphylococcus aureus enterotoxin B, bound to a human class II histocompatibility complex molecule (HLA-DR1) has been determined by X-ray crystallography. The superantigen binds as an intact protein outside the conventional peptide antigen-binding site of the class II major histocompatibility complex (MHC) molecule. No large conformational changes occur upon complex formation in either the DR1 or the enterotoxin B molecules. The structure of the complex helps explain how different class II molecules and superantigens associate and suggests a model for ternary complex formation with the T-cell antigen receptor (TCR), in which unconventional TCR-MHC contacts are possible.

Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide.

An influenza virus peptide binds to HLA-DR1 in an extended conformation with a pronounced twist. Thirty-five per cent of the peptide surface is accessible to solvent and potentially available for interaction with the antigen receptor on T cells. Pockets in the peptide-binding site accommodate five of the thirteen side chains of the bound peptide, and explain the peptide specificity of HLA-DR1. Twelve hydrogen bonds between conserved HLA-DR1 residues and the main chain of the peptide provide a universal mode of peptide binding, distinct from the strategy used by class I histocompatibility proteins.

A subset of HLA-B27 molecules contains peptides much longer than nonamers.

An unusual monoclonal antibody (MARB4) directed against HLA-B27 that reacts with only approximately 5-20% of the cell surface HLA-B27 was used for large-scale purification of these molecules. Subsequent mass spectrometry of HLA-B27-bound peptides showed that the minor MARB4-reactive population contained peptides primarily from 900 to 4000 Da in size (approximately 8-33 amino acid residues), whereas the major HLA-B27 population contained peptides in the mass range of 900-1400 Da (approximately 8-12 amino acid residues). Thus, a subset of HLA-B27 molecules binds to peptides much longer than nonamers. Typical HLA-B27-binding peptides contain arginine in position 2. Further analysis by Edman sequencing of the pooled bound peptides revealed that the major population contained substantial amounts of arginine at positions 1 and 9 (40-50%) and exclusively arginine at position 2, as expected. The minor population of peptides also contained detectable amounts of arginine at these positions, but at the level of only approximately 10%; no marked enrichment at any position was observed. These long HLA-B27-bound peptides could represent either intermediates in the formation of nonamers or adventitiously bound peptides. Lastly, in the TAP2 mutant cell line BM36.1 transfected with HLA-B*2705, MARB4-reactive HLA-B27 molecules were absent from the cell surface, indicating that the peptide transporter was required for delivery of the long peptides. Thus, during the folding of class I heavy chains, peptides of diverse lengths are available and participating.

Specificity and promiscuity among naturally processed peptides bound to HLA-DR alleles.

Naturally processed peptides were acid extracted from immunoaffinity-purified HLA-DR2, DR3, DR4, DR7, and DR8. Using the complementary techniques of mass spectrometry and Edman microsequencing, > 200 unique peptide masses were identified from each allele, ranging from 1,200 to 4,000 daltons (10-34 residues in length), and a total of 201 peptide sequences were obtained. These peptides were derived from 66 different source proteins and represented sets nested at both the amino- and carboxy-terminal ends with an average length of 15-18 amino acids. Strikingly, most of the peptides (> 85%) were derived from endogenous proteins that intersect the endocytic/class II pathway, even though class II molecules are thought to function mainly in the presentation of exogenous foreign peptide antigens. The predominant endogenous peptides were derived from major histocompatibility complex-related molecules. A few peptides derived from exogenous bovine serum proteins were also bound to every allele. Four prominent promiscuous self-peptide sets (capable of binding to multiple HLA-DR alleles) as well as 84 allele-specific peptide sets were identified. Binding experiments confirmed that the promiscuous peptides have high affinity for the binding groove of all HLA-DR alleles examined. A potential physiologic role for these endogenous self-peptides as immunomodulators of the cellular immune response is discussed.

Three-dimensional structure of the human class II histocompatibility antigen HLA-DR1.

The three-dimensional structure of the class II histocompatibility glycoprotein HLA-DR1 from human B-cell membranes has been determined by X-ray crystallography and is similar to that of class I HLA. Peptides are bound in an extended conformation that projects from both ends of an 'open-ended' antigen-binding groove. A prominent non-polar pocket into which an 'anchoring' peptide side chain fits is near one end of the binding groove. A dimer of the class II alpha beta heterodimers is seen in the crystal forms of HLA-DR1, suggesting class II HLA dimerization as a mechanism for initiating the cytoplasmic signalling events in T-cell activation.

Minute quantities of a single immunodominant foreign epitope are presented as large nested sets by major histocompatibility complex class II molecules.

The processing and presentation of immunogenetic peptides is an obligate event in the generation of an immune response. However, the degree of complexity with which an immunogenic foreign epitope is presented is still unclear. This question was addressed by analyzing the naturally processed peptides generated from exogenously-derived hen egg white lysozyme (HEL) bound to the murine major histocompatibility complex (MHC) class II molecule, H-2Ak. Using reversed-phase chromatography (RPC), T cell hybridomas and mass spectrometry, 16 peptides were identified that contain the minimal MHC binding epitope 52-61. These peptides exhibited substantial N- and C-terminal extensions and ranged from 13-28 amino acids in length. In contrast, MHC class I molecules present peptides of 8-11 residues and each foreign epitope appears to be represented by only a single peptide. The data here also show that only approximately 0.8% of the total bound peptide was derived from this single HEL epitope. These findings provide direct evidence that relatively small amounts of processed peptide are required to stimulate an effective T cell response.

Purification of the STB enterotoxin of Escherichia coli and the role of selected amino acids on its secretion, stability and toxicity.

The methanol-insoluble heat-stable enterotoxin of Escherichia coli (STB) was purified and characterized by automated Edman degradation and tryptic peptide analysis. The amino-terminal residue, Ser-24, confirmed that the first 23 amino acids inferred from the gene sequence were removed during translocation through the E. coli inner membrane. Tryptic peptide analysis coupled with automated Edman degradation revealed that disulphide bonds are formed between residues Cys-33 and Cys-71 and between Cys-44 and Cys-59. Oligonucleotide-directed mutagenesis performed on the STB gene demonstrated that disulphide bond formation does not precede translocation of the polypeptide through the inner membrane and that disulphide bridge formation is a periplasmic event; apparently, elimination of either of two disulphides of STB renders the molecule susceptible to periplasmic proteolysis. In addition, a loop defined by the Cys-44-Cys-59 bond contains at least two amino acids (Arg-52 and Asp-53) required for STB toxic activity.

Predominant naturally processed peptides bound to HLA-DR1 are derived from MHC-related molecules and are heterogeneous in size.

Peptides bound to class I molecules are 8-10 amino acids long, and possess a binding motif representative of peptides that bind to a given class I allele. In the only published study of naturally processed peptides bound to class II molecules (mouse I-Ab and I-Eb), these peptides were longer (13-17 amino acids) and had heterogenous carboxy terminals but precise amino-terminal truncations. Here we report the characterization of acid-eluted peptides bound to HLA-DR1 by high-performance liquid chromatography, mass spectrometry and microsequencing analyses. The relative molecular masses of the peptides varied between 1,602 and 2,996 (13-25 residues), the most abundant individual M(r) values being between 1,700 and 1,800, corresponding to an average peptide length of 15 residues. Complete sequence data were obtained for twenty peptides derived from five epitopes, of which all but one were from self proteins. These peptides represented sets nested at both the N- and C-terminal ends. Binding experiments confirmed that all of the isolated peptides had high affinity for the groove of DR1. Alignment of the peptides bound to HLA-DR1 and the sequences of 35 known HLA-DR1-binding peptides revealed a putative motif. Although peptides bound to class II molecules may have some related features (due to the nonpolymorphic HLA-DR alpha-chain), accounting for degenerate binding to different alleles, particular amino acids in the HLA-DR beta-chains presumably define allelic specificity of peptide binding.

Zinc regulates the function of two superantigens.

Staphylococcal enterotoxins bind with high affinity to class II major histocompatibility complex proteins and subsequently stimulate large numbers of T cells via the V beta portion of the T-cell receptor. Binding of enterotoxin A and enterotoxin E to HLA-DR was completely abolished by low levels of EDTA, whereas binding of toxic shock toxin was unaffected. Addition of Zn2+ to as little as 2 microM excess over EDTA completely reconstituted binding, but Ca2+, Mg2+, Cu2+, Fe2+, and Mn2+ had no effect. The dissociation constant (Kd) of 65Zn2+ binding to a single site on purified enterotoxin A was 2 microM, and addition of purified HLA-DR1 did not alter the Kd, indicating that the binding site was exclusive to enterotoxin A. In the presence of saturating levels of zinc the Kd for enterotoxin A binding to purified HLA-DR1 was 25 nM. Thus, zinc binding is an essential first step in the formation of the major histocompatibility complex binding domain of at least two bacterial superantigens. Given the measured Kd of zinc binding to enterotoxin A, serum levels of free zinc (0.2-1.0 microM) may well regulate the toxic sequelae by these two superantigens.