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  Pharmaceutical Patents  

 

Title:  Naturally processed measles virus peptides eluted from class II HLA molecules
United States Patent: 
7,579,004
Issued: 
August 25, 2009

Inventors:
 Poland; Gregory A. (Rochester, MN), Ovsyannikova; Inna G. (Rochester, MN), Muddiman; David C. (Raleigh, NC), Johnson; Kenneth L. (Rochester, MN)
Assignee:
  Mayo Foundation for Medical Education (Rochester, MN)
Appl. No.:
 11/266,957
Filed:
 November 4, 2005


 

Executive MBA in Pharmaceutical Management, U. Colorado


Abstract

A preparation of peptides eluted from class II HLA molecules is disclosed. Methods of decreasing measles infections comprising inoculating human patients with a vaccine comprising one or more of the peptides and methods of diagnosing measles infections or immunity comprising analyzing human patients for the presence of one or more of the peptides or antibodies to the peptide(s) are also disclosed.

Description of the Invention

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention is a preparation of an HLA class II binding peptide selected from the group consisting of SEQ ID NOs:1-13 and functional variants thereof. Preferably, the peptide is SEQ ID NO:1 or functional variant thereof or SEQ ID NO:2 or functional variant thereof.

In another embodiment, the present invention is a nucleic acid molecule encoding the peptides described above.

In another embodiment, the present invention is a method of decreasing measles infection comprising inoculating a human patient with a vaccine comprising or encoding a peptide selected from the group consisting of SEQ ID NOs:1-13 or functional variants thereof.

Preferably, the method comprises inoculating a human patient with a vaccine comprising or encoding at least two peptides selected from the group consisting of SEQ ID NOs:1-13 or functional variants thereof.

In another embodiment, the present invention is a method of diagnosing measles infection or immunity comprising analyzing a human patient for the presence of a peptide selected from the group of SEQ ID NOs:1-13 or antibodies to peptides SEQ ID NOs:1-13.

DETAILED DESCRIPTION OF THE INVENTION

In General

We have adopted an approach, developed in the field of proteomics, to resolve the profound biological complexity presented in investigations of peptides bound to HLA class II molecules. The approach is based on two truly orthogonal separation techniques, namely, (1) strong cation exchange (SCX) chromatography, which separates peptides based on their charge, and (2) nano-scale reversed phase liquid chromatography which uses hydrophobicity (Link, et al., Nat. Biotechnol. 17:676-682, 1999; Washburn, et al., Nat. Biotechnol. 19:242-247, 2001). This fully automated, multi-dimensional chromatography-MS approach affords a geometric increase in the overall peak capacity that dramatically increases the effective dynamic range and the number of peptides which can be dissociated (sequenced using data-dependent tandem-MS) for any given sample.

An overview of the methodology we developed for identifying MHC Class II peptides originating from measles virus is shown in FIG. 1 (see Original Patent). This methodology provides a powerful tool for the identification of pathogen-derived HLA class II peptides that in turn can be evaluated as potential subunit vaccine candidates. We describe naturally processed measles phosphoprotein (P)-derived peptide and nucleoprotein (N)-derived peptide which were isolated and sequenced from class II HLA-DR3 molecules of measles virus infected EBV-transformed B (EBV-B) cell lines by mass spectrometry in Examples 1-3. We also describe 11 additional class II HLA-DR3 peptides in Example 4.

Isolated Peptides

In one embodiment, the present invention is a preparation comprising one of the peptides described below and in Table 10 (see Original Patent), SEQ ID NOs:1-13. These peptides are defined by their amino acid composition as follows -- see Original Patent.

By "preparation" we mean any concentration of the peptide that is enhanced or purified relative to its natural occurrence. Preferably, the preparation is substantially pure or is combined with other ingredients into a pharmaceutical preparation. A preparation of the present invention will likely include adjuvants or carriers that might be coupled to the peptide sequence.

Each of these peptides was directly eluted out of class II human HLA molecules after natural processing and presentation. The implication of this is that these peptides are exactly what is presented to, and seen by, the human immune system. To our knowledge, this has never before been reported for class II-bound measles peptides or proteins. The immune system then initiates a variety of immune responses to these peptides. In turn, this suggests several useful applications for these peptides.

The present invention also includes functional variants of the peptides disclosed in SEQ ID NOs:1-13. One of skill in the art of molecular biology would understand that the N-1 peptide and the P-1 peptide (SEQ ID NOs:1 and 2) and peptides SEQ ID NOs:3-13 could be modified in trivial or conservative ways and yet still retain their biological activity. For example, while we have demonstrated that N-I and P-1 peptides are indeed immunogenic and initiate long-term memory or "recall" immune responses in human cells previously exposed to measles virus, it is also extremely likely that variations of these peptide sequences are also immunogenic. These measles-derived peptides are bound in HLA allele peptide binding grooves (indeed, we directly eluted these peptides out of the peptide binding grooves). Once in these grooves, the peptide is presented to T cells, which then triggers a cascade of events--ultimately leading to a spectrum of immune responses to the peptide.

It is well known that certain and specific amino acids within the peptide sequence are crucial to proper binding (for both electrical charge and space-filling reasons) within the HLA molecule's peptide binding groove. In turn, such peptide binding conforms to "pockets" or "anchors" within the peptide binding grooves that bind these crucial amino acids which in part compose the peptide(s) of interest. In the case of the class II HLA molecules we are discussing herein, the binding groove is approximately 9 amino acids long. It has been demonstrated that an unusual feature of the class II binding groove is that only 2-3 of the 4-5 possible anchors have to be occupied by the usually proscribed amino acid. In turn, this implies that as long as the crucial amino acids are in place, the remaining amino acids may be more promiscuous--allowing different amino acid combinations to be present or absent.

The importance of the preceding discussion is that it is quite likely that "trimming" these identified peptides by 1-3 or more amino acids on either end of the peptide would not adversely impact the ability of these peptides to be bound within the peptide binding groove and the significance or immunogenicity of these peptides. In fact, peptides as small as 8 amino acids are known to contain functional epitopes.

Conversely, amino acids could be added without ill effect as both ends of the class II peptide binding groove are open, and peptides as long or longer than 24 amino acids have been identified. Nonetheless, the amino acid binding cleft contains only a 9 amino acid length, usually with 2-8 amino acid residues on both ends (so called "ragged ends") to enhance affinity.

Therefore, as noted above, the invention embraces functional variants of the class II binding peptides SEQ ID NOs:1-13. As used herein, a "functional variant" or "variant" of a HLA class II binding peptide is a peptide which contains one or more modifications to the primary amino acid sequence of the HLA class II binding peptide and yet retains the HLA class II and T cell receptor binding properties disclosed herein. One would preferably use the procedures disclosed below in Examples 1-4 to determine whether a peptide retains HLA class II and T cell receptor binding properties. Preferably, the peptide of SEQ ID NOs:1-13 would be modified at 1, 2, 3, 4 or 5 amino acid residues.

For example, in the process of verifying these sequence from the Genbank database, we found that SEQ ID NO:12 from measles nucleoprotein also exists as an additional form (or variant) of the nucleoprotein where the " . . . ATES . . . " portion of SEQ ID NO:12 is " . . . ASES . . . " (substitution of an S for a T). Both sequences exist in the database. In our work, we have identified the form containing " . . . ATES . . . . " The " . . . ASES . . . " form of SEQ ID NO:12 is a suitable functional variant of the present invention.

Modifications which create an HLA class II binding peptide functional variant can be made (1) to enhance a property of a HLA class II binding peptide, such as peptide stability in an expression system or the stability of protein-protein binding such as HLA-peptide binding; (2) to provide a novel activity or property to a HLA class II binding peptide, such as addition of an antigenic epitope or addition of a detectable moiety; or (3) to provide a different amino acid sequence that produces the same or similar T cell stimulatory properties. Modifications to the HLA class II binding peptides of SEQ ID NOs:1-13 can be made to nucleic acids which encode the peptide and can include deletions, point mutations, truncations, amino acid substitutions and additions of amino acids. Alternatively, modifications can be made directly to the polypeptide, such as by cleavage, addition of a linker molecule, addition of a detectable moiety, such as biotin, addition of a fatty acid, substitution of one amino acid for another and the like. Preferably the substitutions are not made at anchor residues of a HLA binding epitope. Lipids may be attached as possible modifiers, (see Jackson, et al.'s report of a synthetic vaccine of generic structure that targets Toll-like receptor 2 on dendritic cells and promotes antibody or cytotoxic T cell responses. Jackson, et al., Proc. Natl. Acad. Sci. USA 101:15440-15445, 2004).

Variants also can be selected from libraries of peptides, which can be random peptides or peptides based on the sequence of peptides SEQ ID NO:1-13 including substitutions at one or more positions (preferably 1-5). For example, a peptide library can be used in competition assays with complexes of peptides bound to HLA class II molecules (e.g. dendritic cells loaded with the peptides). Peptides which compete for binding of the peptide to the HLA class I molecule can be sequenced and used in other assays (e.g. CD4 lymphocyte proliferation) to determine suitability as a peptide functional variant.

In another embodiment, the present invention is a peptide or use of a peptide comprising SEQ ID NO:1, 3 or 6. These peptides are shorter versions of other peptides disclosed in Examples 1-4 and represent "core" sequences. For example, peptide SEQ ID NO:3 is shorter at either end than SEQ ID NO:2 or SEQ ID NOs:4 or 5. One of skill in the art would understand that SEQ ID NO:3, for example, could have additional residues (preferably 1-5) added at either end and still be functional as a class II HLA molecule.

To obtain the peptides of the present invention, one would most easily chemically synthesize the peptides. Of course, other methods in the art would be appropriate. A variety of methods are available now and in the future to obtain the peptides of interest. The easiest and most obvious is simple chemical synthesis of each peptide. The next would be inserting the genetic code for the amino acids of interest (which then compose the peptide of interest) into a plasmid and inserting it into a vector (or delivery vehicle) which is then delivered to the host and induced to transcribe the genetic code into the peptide of interest--this can be accomplished by naked DNA immunization, or infection by a vector organism. It is also possible to insert the coding sequence for a larger protein into the host organism if it were certain that the protein would then be processed into smaller peptide components that would result in the identified peptides of interest or a functionally equivalent variant.

In another embodiment, the present invention is a nucleic acid sequence which codes for the class II binding peptides or variants thereof and other nucleic acid sequence which hybridize to a nucleic molecule consisting of the above-described nucleotide sequences under high stringency conditions. The term "stringent conditions" as used herein refers to parameters with which the art is familiar. For example, nucleic acid hybridization parameters may be found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. More specifically, high stringency conditions as used herein, refers to hybridization at 65.degree. C. in hybridization buffer (3.5.times.SSC, 0.02% Ficoll, 0.02% Polyvinyl pyrolidone, 0.02% Bovine Serum Albumin, 25 mM NaH.sub.2PO.sub.4 (pH 7), 0.5% SDS, 2 mM EDTA). SSC is 0.15M Sodium Chloride/0.015M Sodium Citrate, pH 7; SDS is Sodium Dodecyl Sulphate; and EDTA is Ethylene diaminetetraacetic acid. After hybridization, the membrane upon which the DNA is transferred is washed at 2.times.SSC at room temperature and then at 0.1-0.5.times.SSC/0.1.times.SDS at temperatures up to 68.degree. C., e.g., 55.degree. C., 60.degree. C., 65.degree. C. or 68.degree. C. Alternatively, high stringency hybridization may be performed using a commercially available hybridization buffer, such as ExpressHyb.TM. buffer (Clontech) using hybridization and washing conditions described by the manufacturer.

There are other conditions, reagents, and so forth which can be used, which result in a similar degree of stringency familiar to one of skill in the art. It will be understood, however, that the skilled artisan will be able to manipulate the conditions in a manner to permit the clear identification of homologs and alleles of nucleic acids encoding the HLA class II binding peptides of the invention. The skilled artisan also is familiar with the methodology for screening cells and libraries for expression of such molecules which then are routinely isolated, followed by isolation of the pertinent nucleic acid molecule and sequencing.

It will also be understood that the invention embraces the use of the sequences in expression vectors, as well as to transfect host cells and cell lines, be these prokaryotic (e.g. E. coli), or eukaryotic (e.g., dendritic cells, CHO cells, COS cells, yeast expression systems and recombinant baculovirus expression in insect cells). The expression vectors require that the pertinent sequence, i.e., those described supra, be operably linked to a promoter.

In another embodiment, the present invention is an antibody, either monoclonal or polyclonal, that specifically binds to a peptide selected from the group consisting of SEQ ID NOs:1-13 or functional variants thereof. One of skill in the art would understand that there are numerous ways to create antibodies specific to the peptides described above.

Peptide-Based Vaccine

The peptides of the present invention, either alone or in combination with other measles peptides, could be used in a peptide-based vaccine to protect against measles. These identified measles-derived peptides, potentially in combination with other yet to be identified peptides, logically could and will be used in the directed design of newer measles vaccines. The major advantage of such an approach includes avoidance of the safety problems and contraindications present for any live viral vaccine (i.e. there are persons who cannot safely receive a live viral vaccine, such as a highly immunocompromised person), and the ease and lower cost of manufacturing such a vaccine.

In one embodiment, the present invention is a peptide vaccine comprising or encoding at least one of the peptides disclosed at SEQ ID NOs:1-13 or functional variants. Applicants specifically envision that one may wish to use the peptides of SEQ ID NOs:1-13 wherein the sequences have been modified by "trimming" or deleting 1-5 amino acids from each end. These amino acids may be replaced with conservative or inert substitutions, may be deleted or may be replaced with amino acid residues designed to supply the vaccine with an additional feature, preferably as described above. The vaccine preferably comprises or encodes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or all of the peptides.

Multiple options for use of the peptides as a vaccine are possible. A promising technique is the so-called "chain of beads" approach. Here each peptide is chemically linked to the next peptide either with or without an immunologic adjuvant, directly administered as a vaccine. Such an approach has, in essence, been used in the design of the currently licensed pneumococcal and Haemophilus influenzae type b vaccines. Another approach, as discussed above, is simply to immunize with the genetic code for the peptides or proteins of interest. Yet another approach would be to encapsulate one or several peptides into viral-like particles (VLP) that again could be directly administered as a vaccine. Other possible and successful delivery methodologies of these peptides may include transcutaneous or mucosal delivery (such as by direct application to the skin via a "patch", nasal spray, eyedrops, or inhaled formulations), with or without an accompanying adjuvant. At the current time there is no one directly preferred vector system or delivery methodology.

A particularly good example for development of peptide-based vaccines will be the approach that was taking for development of Haemophilus influenza type b (Hib) capsular polysaccharide conjugate and pneumococcal conjugate vaccines. Usually "the bacterial capsular polysaccharides are poorly immunogenic in young children. The coupling of these polysaccharides with protein carriers renders the polysaccharides visible to the T cells, which then provide help for antibody responses. The big successes of Hib conjugate and pneumococcal conjugate vaccines testify to the power of this strategy. Not only are antibody responses induced in the young children, but the average titers were higher in comparison with those achieved after unconjugated polysaccharide." (Plotkin, Pediatr. Infect. Dis. J. 20:63-75, 2001).

One of skill in the art should review the following references (all incorporated by reference) for exemplary models of peptide or peptide-like vaccines.

The effectiveness of such a peptide vaccine is easily measured in non-human primate and smaller animal studies. After optimization of the route and dose of peptide vaccine, animals that have and have not been immunized are exposed to sub-lethal and lethal doses of wild measles virus. Efficacy can be defined in multiple ways including lack of evidence of infection, lack of serious consequences to infection, and survival after wild virus infection.

Diagnostic Assays

The peptides of the present invention, either alone or in combination with other measles peptides, could be used in diagnostic assays designed to determine whether the measles virus is present. One would preferably wish to analyze a biological sample taken from a human patient and detect the presence or absence of the peptides by means of specific antibodies or other probes. These peptides, perhaps in conjunction with other yet-to-be-identified measles-derived peptides, could also be used in diagnostic assays. Direct detection of these peptides is possible as a diagnostic modality proving the presence of recent measles virus either within a blood sample, or potentially directly within a tissue specimen (for example in trying to make the diagnosis of SSPE from a slice or biopsy of brain or other tissue).

In addition, because these peptides derive directly from the measles virus, antibody to these peptides unambiguously reflects the presence and/or previous exposure to either wild measles virus or measles vaccine virus. In the case of someone not previously immunized, the presence of IgM and/or IgG antibody to these peptides can only be attributed to infection with the measles virus. In the case of someone previously immunized against measles, an anamnestic (or long-term memory) immune response, measured by detection of IgM antibody to these peptides, is attributed to re-exposure and re-infection by wild measles virus.

These peptides could be directly incorporated onto plastic wells for use in an ELISA antibody assay, into microparticles in an ELISA or luminex technology assay, or even more generically into an ELISPOT assay. Conversely, it would also be possible to make monoclonal antibodies against these peptides, then make animal anti-human anti-measles peptides to these antibodies and use this in a biologic assay for the presence of antibody to these peptides. Thus, these peptides will ultimately also be utilized in the design of subunit antibody assays to these specific measles-derived peptides. It is important to note that the value of this approach may be the fine dissection of the immune response to an otherwise large virus. This assumes particular relevance when one considers that these peptides are in fact, the most prevalent measles-derived peptides as evidenced by the fact that these were found in high abundance on antigen-presenting cells by our methodology.

Other Immunostimulating or Immunotherapeutic Potential

The peptides of the present invention may have other immunostimulating or immunotherapeutic potential in applications. The peptides could be used to stimulate in combination with a variety of other antigens and immune response.

Since both N-1 and P-1 peptides have demonstrable immunostimulatory and immune memory recall properties in humans, it is also possible that these peptides along with SEQ ID NOs: 3-13 could be adapted to stimulate non-specific immune responses against other antigens such as other pathogens, tumors or malignant cells. For example, direct injection or transcutaneous application of these peptides, alone or in conjunction with one another and/or an adjuvant, into warts caused by human papillomavirus infection, perhaps in the setting of a measles immune host, could lead to the "by-stander" effect of destruction of the HPV-infected cells (wart). Similarly, we have taken measles virus, injected directly into malignant lymphoma cells, and demonstrated in animal models, significant clearing of the malignant cells (Grote, et al., Blood 97:3746-3754, 2001). It may therefore be possible that in the setting of a person immune to measles, that injection or delivery of these measles-derived peptides into a tumor, would lead to an immune response that could destroy the surrounding malignant tissue. We are currently conducting human clinical trials to examine the effect of measles virus delivery into women with metastatic peritoneal ovarian cancer and into persons with malignant glioblastoma (brain tumors) to observe the extent of malignant tissue destruction elicited by the anti-measles immune responses.

Similarly, it is also possible that these peptides, combined with vaccines against other pathogens, could "boost" the immune responses to the pathogen of interest, by acting themselves as vaccine adjuvants.
 

Claim 1 of 8 Claims

1. A preparation of an isolated HLA class II binding peptide consisting of SEQ ID NO:13.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.
 

 

     
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