Naturally processed measles virus peptides eluted from class II HLA
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
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
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
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.
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
-- 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
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
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.
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,
One of skill in the art should review the following references (all
incorporated by reference) for exemplary models of peptide or peptide-like
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.
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
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.
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