Note: Descriptions are shown in the official language in which they were submitted.
~2~3~7~'~7
--1--
"PEPTIDES USEFUL IN VACCINATION AGAINST ENTEROVIR~SES"
This invention relates to peptides having
biological activity, particularly for use in vaccines for
diseases caused by enteroviruses and in particular
polioviruses.
Polioviruses are divisible into three serotypes
on the basis of their neutralization reactions with
specific immune sera. However, they have similar
virological properties and clinical effects and the nucleic
acid and amino-acid sequences of all three serotypes are
strikingly similar (Stanway et al Nucleic Acids Research
_ , 5629 - 5643, 1983; Stanway et al Proc. Natl. Acad. Sci.
USA 81, 1539 - 1543, 1984).
We have previously identified the location oE a
single major antigenic site involved in the neutralization
of poliovirus type 3 (Minor et al, Nature 301, 674 - 679,
1983; Evans et al, Nature 304, 459 - 462, 1983). In GB-A-2
128 621 we have described and claimed a synthetic
polypeptide, suitable for use in vaccination against or
diagnosis of a disease caused by an enterovirus, which
comprises an antigenically effective hexapeptide coded for
by codons 93 to 98 in the RNA sequence coding for the
structural capsid protein VPl of a poliovirus type 3 Sabin
strain or by equivalent codons of another enterovirus. The
codon numbers herein are counted from the 5'-terminus of
the nucleotide sequence coding for the VPl capsid protein.
~2~7~a~7
-- 2
In contrast to this, it has been suggested that
neutralization of poliovirus type`l involves multiple
independent antigenic sites (Emini et al J. Virol. 46,
466-474, 1983). If correct, this di~ference between
closely related viruses would make the satisfactory
prediction of viral peptide sequences for use as vaccines
more difficult than hitherto supposed.
We have now identified a second antigenically
significant peptide coded for by an RNA sequence within the
genome region coding for the structural capsid protein VPl
of an enterovirus.
Accordingly the present invention provides a synthetic
peptide, suitable for use in vaccination against or
diagnosis of a disease caused by an enterovirus, which is
the peptide coded for by codons 286-290 in the RNA sequence
coding for the structural capsid protein VP1 of poliovirus
type 3 Sabin strain or by equivalent codons of another
enterovirus or is an antigenic equivalent of the said
peptide, the antigenic equivalent ~eing:
(i) a said peptide modified by the inclusion therein
of one or more changes to the amino acid
sequence;
(ii) a first longer peptide which incorporates the
sequence of a said peptide or a said modified
peptide and which has up to four extra amino acid
residues attached to the C-terminal end of the
said sequence or up to four extra amino acid
residues attached to the N-terminal e~d of said
seguence or up to four extra amino acid residues
attached to the C-terminal end and up to ~our
extra amino acid residues attached to the
N-terminal end of said sequence;
(iii) a second longer peptide which incorporates the
sequence of a said peptide, a said modified
peptide or a said first longer peptide, to which
12l~7~7
- 2a -
sequence is linked directly, by means of a
further amino acid residue or by means of a
disulphide bridge between Cys residues attached to
each sequence, a sequence of up to eighteen amino
acid residues which comprises a hexapeptide
sequence coded for by codons 93 to 98 in said RNA
sequence or by equivalent codons of another
enterovirus, or
(iv) a third longer peptide which incorporates the
a sequence of a said peptide, a said modified
peptide or a said first longer peptide, to which
sequence is linked directly, by means of a further
amino acid residue or by means of a disulphide
bridge between Cys residues attached ta each
sequence, a sequence comprising residues 58 to 59
of the VP3 capsid protein of an enterovirus of the
same type but starting with a residue numbered no
lower than 53 and ending with a resi~ue numbered
no higher than 68; each said antigenic equivalent
being capable of raising antibodies capable of
neutralizing the same strain and type of
enterovirus as said peptide to ~hich said
antigenic equivalent corresponds and the numbers
of the codons being counted from the ~'-terminus
of the nucleotide saquence for the VP1 capsid
protein.
The peptides of the invention therefore comprise an
antigenically effective peptide unit coded for as
indicated. ~hey are not naturally-occurring peptides, such
as the VPl capsid protein i.tself, which have been recovered
in a suitably pure form after disrupting an enterovirus.
Rather, the hand of man has been involved in the making of
the peptides of the invention. In particular, the peptides
~ -",Y `
12~ 7
--3--
of the invention can be prepared by chemical synthesis rom
single amino acids or s~aller preformed peptides; or by
employing the methods o~ genetic engineering to produce an
organism which makes the peptides in recoverable ~orm.
By 'lequivalent codons" is meant a sequence of
codons in the RNA sequence coding for the structural capsid
protein VPl of another enterovirus, corresponding to the
codon sequence 286-290 in the RNA sequence coding for the
structural capsid protein VPl of the
poliovirus type 3 Sabin strain. The "equivalent codons"
are therefore the counterpart codons in the RNA sequence
coding for ~he capsid protein VPl of another enterovirus to
codons 286-290 for the poliovirus type 3 Sabin strain.
The counterpart codons can readily be determined
by lining up the base sequence coding for the VPl protein
in the RNA sequence of another enterovirus with the
corresponding base sequence of the poliovirus type 3 Sabin
strain. While it is possible that the equivalent codons in
the other enterovirus may also be 286-290, this is not
necessarily the case. In the poliovirus type 3 Leon
strain, which is the virulent progenitor of the attenuated
Sabin strain, the equivalent codons are 286-290. The
equivalent codons are 2a6-290 too or the
poliovirus type 2 Sabin strain. However, in the poliovirus
type 1 Sabin strain, there are si~ equivalent codons,
~287~ ~7
--4--
287-292. Thus, when the base sequences for the Sabin
strains of each of the three types of poliovirus are lined
up (Toyoda et al, ~. Mol. Biol. 174, 561-585, l9a4), the
result is as
follows:
type 3 Sabin:codon 286 287 288 289 290
AGG AAC AAC UUG GAC
type 2 Sabin:codon 286 287 288289 290
AAA GAU GGGCUC ACC
type l Sabin:codon 287 288 289290 291 292
AAG GAU GGU ACG CUU ACA
An "antigenic equivalent" of any particular
"natural" peptide sequence coded for by an existing
enterovirus twhether wild-type or mutant) is a peptide which,
if not itself immunogenic, when linked to material which
renders it immunogenic is capable of inducing the same or a
very similar antibody response as the "natural" peptide, i.e.
the antibody produced, though possibly not precisely
identical, would neutralize the same strain and type of
enterovirus and hence antigenicity is effectively equivalent.
An antigenic equivalent of a "natural" peptide
sequence may be a peptide of the same length which, however,
is not coded for by a wild-type or known mutant enterovirus
but includes one or more changes to the amino acids in the
sequence which does not affect the antigenicity. Thus, one
or more amino acids of a "natural" peptide sequence may be
~Z~74~7
--5--
replaced by, respectively, one or more other amino acids
which preserve the physico-chemical character of the
original, i.e. in terms of charge density,
hydrophilicity/hydrophobicity, size and configuration, and
5 hence preserve the immunological structure. For example, Thr
may be replaced by Ser and vice versa, Asp may be replaced by
Glu and vice versa and Asn may be replaced by Gln and vice
versa.
An antigenic equivalent may also be a longer
peptide which comprises a "natural" peptide sequence but
still has equivalent antigenicity. The "natural" peptide
sequence will thus be exposed in the longer peptide so as to
be available to induce the appropriate immune response and
not "buried" in the interior of the longer peptide and
consequently unable, itself, to provoke an immune response.
Yet ~urther antigenic equivalents may be formed by
modifying reactive groups within a "natural" sequence or
modifying the N-terminal amino and/or C-terminal carboxyl
group. Such equivalents can include salts formed with acids
and/or bases, particular physiologically acceptable inorganic
and organic acids and bases. Other equivalents may include
modified carboxyl groups to produce esters or amides or may
include typical amino acid protecting groups such as
N-t-butoxycarbonyl. Preferred modifications of this type are
those which enable the production of a more stable, active
peptide which will be less prone to enzymic degradation in
vivo.
lZ~7~7
--6--
A combination of two or more of the types of
variations of a "natural" sequence described above may be
used to arrive at an antigenic equivalent peptide of the
invention. For example, a peptide sequence which has been
derived from a "naturall' sequence by changing one or m~re of
the amino acids in the "natural" sequence may be incorporated
in a longer peptide.
The present invention will now be described with
particular reference to polioviruses, though it will be
appreciated that the concept of the invention is considered
- to apply equally well to other enteroviruses, i.e. viruses
which are found in the intestine, e.g. ECHO (Enteric
Cytopathic Human Orphan) and Coxsackie B viruses. In
accordance with convention, the bases referred to herein are
as follows:
A = Adenine
G = Guanine
C = Cytosine
U = Uracil.
Similarly, in accordance with convention, the
following abbreviations are used for the amino acid radicals:
Alanine = Ala
Arginine = Arg
Asparagine = Asn
25 Aspartic acid = Asp
Cysteine = Cys
Glutamine = Gln
Glutamic Acid = Glu
~Z~7~47
--7--
Glycine = Gly
Histidine = His
Isoleucine = Ile
Leucine = Leu
Lysine = Lys
Methionine = Met
Phenylalanine = Phe
Proline = Pro
Serine = Ser
Threonine = Thr
Tryptophan = Trp
Tyrosine = Tyr
Valine = Val
~herever these amino acids are mentioned, they
lS cover both the D- and L-configurations. However, it is
preferred in accordance with the invention that the amino
acids should take the natural, i.e. the L-, configuration.
The Figure of the accompanying drawing shows the
RNA sequence for the VPl capsid protein in the poliovirus
type 3 Sabin strain. Within this sequence, codons 93-98 and
286-290 are underlined.
It has not yet been unequivocably established
whether the nucleotide sequence coding for the VPl capsid
protein of the poliovirus type 3 Sabin strain actually
commences with the codons GGU AUU . . . as shown in the
Figure or with the codon GGC . . . which is the twelfth codon
~L287~4~
-- 8 --
in the Figure. Nevertheless, herein the codons for the
nucleotide sequence of the VPl capsid protein of poliovirus
type 3 Sabin strain are counted from the first codon in the
Figure, GGU.
In accordance with this notation, the appropriate RNA
sequence coded for by codons 286-290 for Sabin type 3
poliovirus and the corresponding tripeptide are as follows:
286 287 288 289 290
AGG AAC AAC W G GAC
Arg-Asn-Asn-Leu-Asp
Table 1 below sets out the codons and amino acid
residues of the Leon strain and a mutant strain of
poliovirus type 3, of Sabin strain poliovirus type 2 and of
the Sabin and Mahoney strains of poliovirus type 1 in
15 comparison to codons 286-290 for the Sabin strain of
poliovirus type 3.
A preferred peptide according to the invention, suitable
for use in vaccination against or diagnosis of a disease
caused by type 3 poliovirus, comprises the sequence (I):
Ao-Al-A2-Leu-A3 (I)
in which (i) Ao is Arg, each of Al and A2 is independently
Asn or Gln and A3 is Asp or Glu or (ii), with the others of
Ao to A3 being as defined under (i), Ao is Lys or A~ or A2
is Asp or Glu. Preferably, A~ ir Arg, both A1 and A2 are
Asn and A3 is Asp.
A preferred peptide, suitable for use in vaccination
against or diagnosis of a disease caused by type
~Z874 ~7
g
2 poliovirus, comprises the sequence (II):
Lys-Al-Gly-Leu-A3 (II)
in which Al is Asp or Glu and A3 is Thr or Ser. More
preferably, Al is Asp and A3 is Thr.
A preferred peptide, suitable for use in vaccination
against or diagnosis of a disease caused by type 1
poliovirus, comprises the sequence (III):
~ ys-A4-Gly-As-Leu-A6 (II)
in which A4 is Asp or Glu, and each of A5 and A6 is
inde~endently Ser or Thr. More preferably, A4 is Asp and
A5 and A6 are both Thr.
The present invention also includes peptides longer than
the basic pentapeptide, in the case of polioviruses types 2
and 3, or hexapeptide, in the case of poliovirus type 1.
Further amino acids and/or peptides can be linked to one or
both ends of the basic peptide chain. 1, 2, 3 or 4 extra
amino acid residues may be attached to the C-terminal of
the basic peptide and/or 1, 2, 3 or 4 extra amino acid
residues can be attached at the N-terminal of the hasic
peptide. Preferably, these additional amino acids
correspond to those in a "natural" sequence. Thus, a
longer peptide according to the invention may correspond to
codons 282 to 292 in the RNA sequence coding for the VPl
capsid protein of poliovirus type 3 Sabin strain or
equivalent codons of another poliovirus such as poliovirus
type 1 or 2 Sabin strain. Such peptides
~Z~ 7
--10--
in relation to the Sabin strains are:
(type 1) Gly-Val-Asp-Tyr-Lys-Asp-Gly-~hr-Leu-Thr-Pro-Leu
(type 2) Gly-Val-Asp-Tyr-Lys-Asp-Gly-~eu-Thr-Pro-Leu
(type 3) Gly-Val-Asp-Tyr-Arg-Asn-Asn-Leu-Asp-Pro-Leu
The longer polypeptides may terminate in a Cys
residue at one or both ends. Alternatively, the basic
peptide chain itself or longer peptides containing this chain
may be linked at one or both ends to a protein and/or some
other carrier.
When, for example, the basic amino acid sequence o
a "natural" peptide or of an antigenic equivalent thereof is
included in a longer peptide, the additional amino acids
attached to the basic peptide preferably correspond to the
amino acids linked to the "natural" peptide in the
corresponding natural VPl capsid protein. In type 3 Sabin
poliovirus, the first N-terminal amino acid which may be
added to the basic pentapeptide is Tyr (coded for by U~U as
can be seen from the Figure of the accompanying drawing).
The first C-terminus amino acid which may be added in this
instance is Pro (coded for by CCC). Appropriate further
amino acids in this case can be determined from the Figure.
La~ger compounds are such that the basic peptide
sequence is positioned so as to be readily available to
lZ~7~7
--11--
induce the appropriate immune response and, in particular, so
that it is not "buried" in the interior of the molecule.
Thus, for example, repeats of basic peptide may be linked
together either by non-covalent or, preferably, covalent
bonds. Where no appropriate amino acid is contained in the
peptide sequence of the present invention, additional acids
can be attached at either terminus for this purpose, in
particular Cys which will enable covalent bonding through
formation of a disulphide linkage.
Alternatively, a longer peptide may be formed into
a loop by including groups which can link together at each
terminus of the chain. A loop can of course be created by
formation of an amide link between the N-terminus and
C-terminus which can occur irrespective oE the amino acids at
those termini. Also, a longer peptide may comprise sequences
for different types of poliovirus. Such a peptide would be
useful as a vaccine for or in the diagnosis o~ two or all
three types of poliovirus.
A peptide of the invention may comprise the basic
peptide sequence linked to the sequence o a polypeptide
according to GB-A-2 128 621. The polypeptides of GB-A-2 128
621 are hexapeptides coded for by codons 93-9a in the RNA
sequence coding for the structural capsid protein VPl of
poliovirus type 3 Sabin strain or by equivalent codons of
another enterovirus or are antigenic equivalents of such a
hexapeptide. Preferably, the basic peptide sequence
acco}ding to the present invention and the polypeptide
according to GB-A-2 128 621 which are linked together are in
12~7~47
-12-
respect of the same type, more preferably the same strain, of
enterovirus.
A preferred hexapeptide according to GB-A-2 128
621, suitable for use as a vaccine for or in the diagnosis of
type 3 poliovirus, has the formula (IV):
11 12 13 14 15 (IV-)
in which (A) Alo is Glu, All is Gln, A12 is Pro, A13 is Thr,
A14 is Thr and A15 is Arg, or
(B) with the remainder of Alo to A15 being as
defined under (A), (a) Alo is Gly or (b) A13 is ~le, Ser,
Ala or Asn or (c) A14 is Asn, Ser or Ile or (d) A15 is Gln,
Trp or Gly or (e) A13 is Ile and A14 is Asn or Ala This
hexapeptide can therefore be linked to the preferred type 3
poliovirus tripeptide of formula (I) above.
A preferred hexapeptide according to Ga-A-2 128
621, suitable for use as a vaccine for or in the diagnosis of
type 2 poliovirus, has the formula (IV) in which Alo is Asp,
All is Ala, A12 is Pro, A13 is Thr, A14 is Lys and A15 is
Arg. This hexapeptide may therefore be linked to the
preferred type 2 poliovirus tripeptide of forMula (II)above.
A preferred hexapeptide according to GB-A 2 128
621, suitable for use as a vaccine for or in the diagnosis of
type 1 poliovirus, has the formula (IV) in which All is Ala,
A12 is Ser, A13 is Thr, A15 is Asn and either (A) Alo is Ser
and A14 is Lys or (B) Alo is Pro and A14 is Thr. This
hexapeptide may therefore be linked to the preferred type 1
poliovirus tripeptide of formula (III)above-
~8~
--13--
The hexapeptides of GB-A-2 128 621 can be built up
into longer polypeptides, and these longer polypeptides may
too be linked to peptides according to the present invention.
For example a preferred type 3 poliovirus octapeptide
according to GB-A-2 128 621 has the formula (V):
Alo-All-A12-A13-A14-A15-A16-A17 tV)
in which Alo is Glu, Al~ is Thr or Ser and A15 is Arg. More
preferably, (A) Alo is Glu, All is Gln, A12 is Pror A13 is
Thr, A14 is Thr, A15 is Arg, A16 is Ala and A17 is Gln, or
(B) with the others of Alo to A17 being as defined in (A)
(a) Alo is Gly, or
(b) A13 is Ile, Ala or Asn, or
(c) A14 is Asn, Ser or Ile, or
(d) A15 is Gln or Trp, or
(e) A16 is Thr or Val, or
(f) A17 is Leu, Pro, Arg or His; or further
(g) A13 is Ser, Ile or Asn and A16 is Thr, or
(h) A13 is Ile, A14 is Asn or Ala and A16 is Thr.
A preferred type 2 poliovirus octopeptide has the
formula (V) in which: Alo is ~sP~ All iS Ala~ A12 is Pro~
A13 is Thr, A14 is ~ys, A15 is Arg, A16 is Ala and A17 is
Ser.
A preferred type 1 poliovirus octapeptide has the
--3
12~4'~7
- 14 -
formula (V) in which: All is Ala, A12 is Ser, A13 is Thr,
A15 is Asn, A16 is Lys, A17 is Asp and either tA) Alo is
Ser and A14 is Lys or (B) Alo is Pro and A14 is Thr.
Further amino acids and/or peptides can be linked to one
or both ends of these eight amino acid polypeptide chains
according to GB-A-2 128 621. For example, a dodecapeptide
or an octadecapeptide may be formed respectively by
bonding:
(poliovirus type 3) Glu-Val-Asp-Asn-,
(poliovirus type 2) Glu-Val-Asp-Asn-,
(poliovirus type 1) Thr-Val-Asp-Asn, to residue Alo of
formula (IV), or
(poliovirus type 3) Ala-Ile-Ile-Glu-Val-Asp-Asn and
-Lys-Leu-Phe,
(poliovirus type 2) Ala-Ile-Ile-Glu-Val-Asp-Asn- and
-Arg-Leu-Phe, or
(poliovirus type 1) Ala-Ile-Ile-Thr-Val-Asp-Asn- (when A1o
is Ser and A14 is Lys) or
Thr-Thr-Met-Thr-Val-Asp-Asn- (when Alo is
Pro and A14 is Thr) and -Lys-Leu-Phe, to
residues Alo and A17 of formula (IV).
The basic peptide sequence according to the present
invention may be linked to a polypeptide sequence according
to GB-A-2 128 621 directly or by one or more amino acid
residues or by a disulphide bridge between Cys residues
attached to each sequence. Not only may peptides of the
same poliovirus type be lin~ed
~2~37~'~7
-15-
together but also peptides of different types may be linked
so as to form a single peptide useful as a vaccine for or in
the diagnosis of two or all three types of poliovirus.
A peptide of the invention may also comprise the
basic peptide sequence linked to another peptide comprising
residues 58 and 59, for example residues 58 to 61, of the VP3
capsid protein of the same type, preferably of the same
strain, of enterovirus, in particular poliovirus, as that to
which the basic sequence corresponds. ~or poliovirus type 3,
the other peptide may also comprise VP3 residues 77 and 79.
It is believed that for poliovirus type 3 the pentapeptide
coded for by codons 286-290 in the RN~ sequence coding for
VPl and VP3 residues 58,59,77 and 79, which are thought to
constitute a subsidiary antiqenic site, form a single
operationally distinct antigenic site~
Thus, the invention provides a peptide suitable for
use in vaccination against or diagnosis of a disease caused
by type 3 poliovirus, which com?rises a type 3 pentapeptide
of formula (I) above linked to a VP3 peptide of formula (~I)
ortVIII):
A7-A~-Ag~LYS (VI) 7 A8 Ag Lys Alo~ All (VIII)
in which ~i) each of A7 and Alo is independently Glu or Asp,
each of A8, Ag and All is independently Thr or Ser and Alo
and All are linked to the Lys residue and Alo respectively
either dir~ctly or through intervenin~ amino acid residues or
7~
-16-
(ii), with the others of A7 to All being as defined under
(i), A7 is Asn or Gln or A8 is Arg, Asn or Gln. The peptide
of formula (~TII)may therefore correspond to VP3 amino acid
residues 58 through to 79 of, for example, Sabin or Leon
strain type 3 poliovirus. Preferably, A7 is Glu, A8 and A
are both Ser, Ag is Thr and Alo is Asp.
The invention also provides a peptide suitable for
use in vaccination against or diagnosis of a disease oaused
by type 2 poliovirus, which comprises a type 2 pentapeptide
of formula (I) above linked to a VP3 peptide of formula (V)
or (VI):
A12-A13-A14-Arg ( ) A12 A13~A14~Ar9...His , A15 (VI)
in which each of A12 and A13 is independently Thr or Ser, A14
is Gln or Asn, A15 is Asp or Glu and the His residue and A15
are linked to the Arg residue and the His residue
respectively either directly or through intervening amino
acid residues. The peptide of formula (VI) may therefore
correspond to VP3 amino acid residues 58 through to 79 of,
for example, Sabin strain type 2 poliovirus. Preferably, A12
is Thr, A13 is Ser, A14 is Gln and A15 is Asp.
The invention further provides a peptide suitable
for use in vaccination against or diagnosis of a disease
caused by type 1 poliovirus, which comprise a type 1
hexapeptide of formula (II) above linked to a VP3 peptide of
formula (VII) or (VIII):
3~2~
-17-
A -Ala-Lys-Lys (VII) Al6-Ala-Lys-~ys.~His~Al7 (VIII)
in which A16 is Thr or Ser, A17 is Asp or Glu and the His
residue and A17 are linked to the Lys residue shown adjacent
to the His residue in formula (VIII) and the His residue
respectively either directly or through intervening amino
acid residues. The peptide of formula (VIII) may therefore
correspond to VP3 amino acid residues 58 through to 79 of,
for example, Sabin or Mahoney strain type 1 poliovirus.
Preferably, A16 is Ser and A17 is Asp. The hexapeptide of
formula (II) may be linked to the peptide of formula (VII) or
(VIII), and the type 3 and 2 pentapeptides of formula (I~ may
be linked to the peptide of formula (III) or (IV) and the
peptide of formula (V) or (VI) respectively in the same
manner as the basic peptide sequence according to the
invention may be linked to a polypeptide according to GB-A-2
128 621.
A peptide of the present invention, if not itself
immunogenically active, may be linked to a carrier in order
to create a conjugate which will be immunogenically active.
The carrier in that case may be a protein such as bovine
serum albumin, thyroglobulin, ovalbumin or keyhole limpet
hemocyanin, or palmitic acid. For immunization oE humans,
the carrier must be a physiologically acceptable carier
acceptable to humans and safe. Preferably however, the
peptide is linked to tetanus toxoid and/or diptheria toxoid
thus providing both an immunogen and a multivalent vaccine at
~2~7~4~7
-18-
the same time. Alternatively, the peptide may be chemically
bonded to inert carriers where they can be used to assay
and/or isolate by affinity chromatography antibodies to the
appropriate virus. Examples of such inert carriers are
dextrans e.g. sepharose.
The present invention also provides a process for
the preparation of a peptide of the invention, which process
comprises identifying either (a) the codons in the RNA
sequence coding for the structural capsid protein VPl oE an
enterovirus which are or which are equivalent to codons 286
to 290 for a poliovirus type 3 Sabin strain or (b) the
corresponding codons in a D~A sequence corresponding to said
RNA sequence; and producing a synthetic peptide comprising
the peptide sequence corresponding to the codons thus
identified .
A peptide of the invention may be produced by
chemical synthesis, for example by one of the generally known
methods. In these methods, the peptide is usually built up
either from the N-terminus or, more usually, the C-terminus
using either single amino acids or preformed peptides
containing two or mo~e amino acid residues. Particular
techniques for synthesising peptides include the classical
methods where the peptides of increasing size are usually
isolated before each amino acid or preformed peptide
addition. Alternatively, solid phase peptide synthesis may
be employed where the peptide is built up attached usually to
' '
~2~ 7
--19--
a resin e.g. ~ Merrifield resin. In these syntheses, groups
on the amino acids will generally be in protected form using
standard protecting groups such as t-butoxycarbonyl. If
necessary, these protecting groups are conveniently cleaved
once the synthesis is complete, though they may be retained
where they do not affect the ability of the compound
including the peptide to provoke an appropriate immune
response. Other modifications of the peptide may either be
introduced during ~he synthesis or at the end of it.
A still further possible method for producing the
peptides of the invention is by employing the techniques of
genetic engineering whereby a DNA sequence coding for the
peptide is introduced into a plasmid which itself is
introduced into an oeganism e.g. a bacterium, which can be
lS induced to make the peptide in recoverable ~orm. The present
invention thus not only covers the peptide, but also a DNA or
RNA sequence coding for the peptide which can be used in such
a synthesis. However, in view of the small number of amino
acids in the pepetide chain of the invention, the most
appropriate methods of production are the synthetic methods
for building up the chains described above~
The peptides of the present invention have a
particular application in vaccinating patients against
diseases caused by enteroviruses, in particular polioviruses.
Vaccination is achieved by administering to a patient an
effective amount of a peptide of the invention, either as
~2~ 7
-20-
such or linked to a carrier. Typically, from 100 ug to 1 mg
of the peptide is administered intramuscularly to a human.
When used for this purpose, the material must be
such, particularly of such a size, that it produces an immune
reaction. The peptide is usually therefore coupled to an
immunogenically active carrier such as the proteins mentioned
hereinbefore or be in the form of a longer peptide including
the peptide sequence, which may be achievea by linking the
peptide to a synthetic polypeptide such as poly-lys.
The vaccines may include not just one peptide in
accordance with the present invention, but two or more. By
including several different peptides, for example one for
each of the three different types of poliovirus, a patient
may be vaccinated against all three types of poliovirus and
the vaccine can also take account o variations in the
peptide between different viruses of the same type.
Further, a peptide according to the present
invention may therefore be administered with a polypeptide
according to GB-A-2 128 621 and/or a VP3 peptide as described
above. The peptides may be mixed together or administered
separately over a period of time in any order. Preferably,
peptides in respect oE the same type, more preEerably the
same strain, of enterovirus are administered. Preferred
polypeptides according to GB-A-2 128 621 and preferred VP3
peptides which may be administered simultaneously with or
separately from a peptide according to the present invention
-21-
are those mentioned above. Alternatively, a polypeptide
according to GB-A-2 128 621 and/or a VP3 peptide may be
linked to the same carrier as a polypeptide o~ the present
invention.
It is also preferred to formulate vaccine
compositions as physical mixtures which include other
antigens particularly those commonly used in infant vaccines,
such as tetanus, diphtheria and whooping cough. However, as
indicated before, such antigens may, if desired, be linked
chemically to the peptide of the invention in order to render
it immunogenic.
Although the peptides of the present invention,
when in immunogenic form, can act as vaccines to protect a
patient by inducing the production of the appropriate
antibodies, it is possible that, in addition, the peptide may
have a chemotherapeutic effect. Thus it is believed that the
same peptide sequence which can evoke the production of
antibodies may be the se~uence in the viral capsid protein
which enables the virus to attach itself to a cell within a
patient and thereby cause the infection. Thus the peptide of
the present invention may have a competitive effect and, by
occupying the appropriate cell receptor sites, prevent the
virus itself from infecting the patient. Generally,
immunogens comprising the peptides of the present invention
will be administered by injection which will usually be
intramuscular but can be by routes, such as intraperitoneally
or subcutaneously.
1~87~7
- 22 -
The peptides of the present invention can also be used
to prime the immune system of a patient to exhibit an
enhanced response to vaccination against diseases caused by
enteroviruses. An effective amount, typically 100 ug to
1 mg, of a peptide of the invention can be administered to
a patient and, after a suitable amount of time has elapsed,
the patient can be vaccinated against a disease caused by a
corresponding enterovirus in the conventional manner. Less
material, both of the peptide of the invention and of that
required for the conventional vaccination, may be needed
and fewer challenges may be required to achieve ef~ective
vaccination.
The present invention also provides a pharmaceutical
composition useful as a vaccine against a disease caused by
an enterovirus which composition comprises a
pharmaceutically acceptable carrier or diluent and, as
active ingredient, a peptide of the present invention. The
actual form of the peptide in this composition, i.e.
whether it is lin~ed to another compound or not, will
depend upon the use to which the composition is to be put.
The composition may, for example, comprise an effective
amount of the peptide in a suitable diluent such as Freunds
Complete Adjuvant (FCA) or physiologically acceptable
saline.
An alternative use for the peptides of the present
invention is in the diagnosis of infection by
enteroviruses. This diagnosis may be carried out by the
detection of the
12~3~7~7
-23-
presence or absence of antibody to the approriate virus in
the patient. For this purpose, the peptides are usually
bonded to inert carriers as mentioned hereinbefore and, in
such form, they can also be used as an affinity
chromatography medium in the isolation of antibodies to the
virus. The peptide of the invention may therefore form a
component of a test kit, suitable for use in determining
antibody against an enterovirus, which kit also includes
means for determining antibody bound to the peptide. Any
suitable immunoassay system, for example radioimmunoassay
system, may be used to determine the antibody.
The following Examples illustrate the invention.
Example 1: Identification of the antigenic
site coded for by codons 286-290 in the RNA sequence coding
for the VPl capsid protein, and a subsidiacy antigenic site
coded for by codons including 58,59,77 and 79 in the RNA
se~uence coding for the VP3 capsid protein, of poliovirus
type 3 Sabin strain.
1 Identification of an antigenic site coded for
.
by VPl codons 286-288.
Mutants which were resistant to neutr~lization by
specific monoclonal antibodies were isolated by plaque
formation by antibody-treated virus under an agar overlay
containin~ antibody (Minor et al 1983). They were isolated
from the type 3 poliovirus strain P3 Leon/USA/1937 (Minor et
al 1983, Evans et al 1983) and its attenuated derivative the
- ~287~'~7
-24-
Sabin vaccine strain. The mutants were characterized by
their susceptibility to neutralization by a panel of twelve
monoclonal antibodies as shown in Table 2 below. Mutants
obtained from a total of 213 plaques derived from P3/Leon/37
virus could be classified into 16 distinct groups on the
baiss of the pattern of their neutralization. Similarly
fifteen distinct groups of mutants were selected from the
Sabin vaccine strain from a total of 129 plaques.
Evidence for a second, independent antigenic site
was obtained using a highly strain-specific monoclonal
antibody, 138, which neutralizes the Sabin type 3 vaccine
virus or most strains derived from it, but not P3/Leon/37 or
other strains (Ferguson _ al 1982). All mutants of the
Sabin strain which were resistant to the twelve antibodies
which selected mutants with substitutions in the first site
tsee GB-A-2 128 621), were found to be still sensitive to
antibody 138, while all mutants selected for resistance to
antibody 138 were fully sensitive to these site 1 antibodies.
Antibody 138 was therefore thought to be directed against an
independently mutable site, distinct from the first site.
This second site was identified as follows.
Nucleotide sequencing studies (Stanway et al 1983,
1984) indicate that there are only two predicted amino acid
differences between Leon 12alb tthe Sabin strain) and P3/Leon
USA 1937 in the structural portion of the genome, one in the
region coding for protein VP3, the other at codon 286 from
12l~7~47
-25-
the 5' end of the region coding for VPl r which is a lysine in
P3/Leon/37 and an arginine in the Sabin vaccine strain. A
recombinant plasmid was prepared from whole cloned cDNA
copies of the genomes of Sabin type 3 vaccine virus and
P3/Leon/1937 such that the recombinant genome contained the
VP3 region of the Sabin strain and the VPl of Leon (G
Westrop, unpublished). Virus was recovered by transfection
of cells with this plasmid (Racaniello and Baltimore 1981)
and characterized by partial sequencing of the genome in the
regions coding for VPl and VP3. This recombinant virus
failed to react with antibody 138, suggesting that the
specific site did not involve VP3 of the Sabin strain, but
included the amino acid at position 286 from the N terminus
of VPl.
Mutants resistant to 138 isolated from Sabin virus
proved to have a base substitution in the adjacent codon
(287) leading to the substitution of an aspartate residue for
asparagine.
Additionally, a series of isolates was obtained
from a hypogammaglobulinaemic vaccinee (222f) who excreted a
vaccine derived type 3 poliovirus for a prolonged period
after administration of a monovalent type 3 Sabin vaccine
(MacCallum 1971)~ Six of these strains proved to be
different from each other on the basis of Tl oliogonucleotide
maps of their R~A, but all failed to react with antibody 138
(P Minor unpublished). Sequencing studies revealed that the
~2~i79~ :~7
amino acid at codon 288 from the N-terminus of VPl was
aspartate for all six excreted strains and asparagine for the
parental Sabin vaccine virus.
The findings with recombinant virus, mutant virus
and excreted strains strongly im~ly that antibody 138
recognises a strain specific antigenic site encompassing
codons 286-288 from the N-terminus of VPl.
2. Identification of the antigenic site coded for by VPl
codons 286-290 and of the subsidiary antigenic site
Next, monoclonal antibodies were prepared by the
fusion of splenocytes from immunised Balb/C mice with myeloma
cells as described (Ferguson et al 1984). The mice were
immunised with antigenically abnormal virus, either an
antigenically drifted strain (P3/23127/Finland/84) implicated
in an outbreak of poliomyelitis in Finland ~Lenikki et al
1985) or poliovirus type 3 Sabin strain which had been
treated with trypsin as described (Fricks et al 1985).
Hybridoma supernatants were screened using a modified single
radical diffusion assay (antigen blocking test) and
monoclonal antibodies were generally used as ascites prepared
in syngeneic mice (Ferguson et al 1984). Immunisation
schedules and other protocols were similar in all ~usions~
Monoclonal antibodies ~enerated from animals
immunised with both types of virus were able to neutralise
both untreated virus and all mutants having substitution
within the principal antigenic site, the region in the VPl
:~2~ 7
-27-
capsid protein from amino acids 89 to 100. Mutants were
selected with four of these antibodies as follows. Antigenic
variants were selected by plaque formation on He~2c cells by
virus treated with antibody under an agar overlay containing
the antibody as described (Minor et al 1983). Putative
mutants were subject to two cycles of selection, and small
working pools grown up from the secondary plaque plugs.
The antigenic patterns of reaction of the mutants
picked are shown in Table 3, together with two mutants which
have been previously described. Mutant 1 had an amino acid
substitution in VPl at position 98, where a glycine residue
was found instead of an arginine. Mutant 2 had a mutation in
VPl at position 287, where an aspartate residue was found in
place of an asparagine. The thirteen mutants ell into three
distinct non overlapping groups, implying the existence of
three independent antigenic sites.
The genomic RNA of mutants 3 to 13 was sequenced by
primer extension, through five regions corresponding to areas
containing antigenically significant mutations in type 1 or
type 3 poliovirus. These included regions coding for
residues 89 to 100, 220 to 222 and 286 to 290 of VPl,
residues 50 to 80 of VP3 and residues 160 to 180 of VP2. The
eesul~s are presented in Table 4. Mutants 2 to 8 had single
base s~bstitutions resulting in predicted amino acid changes
in VPl at residLes 287 and 290, and in VP3 at residues ~8,
59, 77 and 79. It was notable that the reactions of
~2~7~
-28-
antibodies 1023, 840, 251, 557, 1084 and 1007 were affected
both by mutations within VP3 at residues 58 and 59 and by
mutations within VPl at residues 287 and 290. It is believed
that the second antigenic site comprising residues 286 to 290
of VPl and the subsidiary antigenic site comprising residues
58,59,77 and 79 form a single operationally distinct
antigenic site.
Example 2: Synthesis of Cys-Glu-Val-Asp-Asn-Glu-
Gln-Pro-Thr-Thr-Arg-Ala-Gln-Lys-Leu-Phe-Ala-Met-Gly-Val-Asp-
Tyr-Arg-Asn-Asn-~eu-Asp-Pro-Leu-Cys (Peptide 1) and
Cys-Gly-Val-Asp-Tyr-Arg-Asn-Asn-Leu-Asp-Pro-Leu-Cys tPeptide
2)
The required peptides were synthesised by the
Fmoc-polyamide mode of solid phase peptide synthesis (Brown
et al 1983 and references cited therein). The general
protocol was as follows:
Polydimethylacrylamide gel resin (a copolymer of
dimethylacrylamide-ethylenebisacrylamide-acryloylsarcosine
methyl ester) containing 0.3 milliquivalents of sarcosine per
gram resin, was treated with ethylenediamine overnight.
After thorough washing, the acid labile linkage agent,
4-hydroxymethylphenoxyacetic acid, was added as its
symmetrical anhydride. After thorough washing this afforded
the low loading acid labile resin that was used to prepare
the peptides under discussion.
Fmoc-amino acids were coupled tin a twelve fold
i2~4~7
-29-
excess) as their preformed symmetrical anhydrides: the
Fmoc-amino acid (2 equiv) was dissolved in dichloromethane
with a few drops of N,N-dimethylformamide (DMF) if required
to aid dissolution. N,N-Dichlorohexylcarbodiimide tDCC) (1
equiv) was added and the mixture stirred at room temperature
for 10 minutes. The precipitated ~,N-dicyclohexylurea (DCU)
was filtered off, the filtrate evaporated to dryness and the
residue dissolved in DMF. This solution was added to the
deprotected and washed resin and the coupling reaction
allowed to proceed.
Asparagine and glutamine residues were added as
follows: l-hydroxybenzotriazole (1 equiv) and DCC (1 e~uiv)
were dissolved in DMF at 0C. After stirring for ten minutes
at 0C a solution of Fmoc-asparagine (or glutamine) (1 equiv)
lS in DMF was added. This mixture was stirred for a further ten
minutes at 0C and then the entire mixture added to the resin
and coupling allowed to proceed.
A typical synthetic cycle was as follows:
Reagent Duration Operation
20 DMF 5 x 1 min Wash
20% Piperidine/DMF 1 x 3 + 1 x 7 min Deprotection
DM~ 10 x 1 min Wash
Preformed symmetrical
anhydride or active
ester 60 - 120 min Coupling
DMF 5 x 1 min Wash
8~7~ ~
-30-
The completeness of coupling at each stage was
monitored using ninhydrin and trimethylbenzenesulphonic
acid test reagents.
The coupling of the first residue to the
S derivatised resin was carried out in the presence of
N,N-dimethylaminopyridine (DMAP) (0.1 equiv).
As the C-terminal dodecapeptide sequence ~as
common to both peptides, the synthesis was carried out on
twice the scale, half being used to continue the first
peptide and cysteine being added to the other half to give
the second peptide.
The quantities used were as follows:
Acid Labile Resin (0.1 g; 0.3 mequiv g 1); for
each cycle, Fmoc-amino acid ~3.6 mmol) and DCC (0.38 g; 1.8
lS mmol); for the first cycle, DMAP ~0.022 g; 0.18mmol); and,
for asparagine and glutamine residues HOBT (0~24 g; 1.8
mmol). The cycles were carried out on the following basis:
Fmoc-amino acid-OHQuantities Coupling Time
Fmoc-Cys(Trt)-OH2.10 g; 3.6 mmol1 hour
20 Fmoc-Leu-OH 1.28 g; 3.6 mmol 1 hour
Fmoc-Pro-OH 1.22 g 3.6 mmol 1 ho~r
Fmoc-Asp(0But)-OH1.48 g; 3.6 mmol1 hour
Fmoc-~eu-OH 1.28 g; 3.6 mmol 1 hour
Fmoc-Asn-OH 0.64 g; 1.8 mmol 1 hour
25 Fmoc-Asn-OH 0.64 g; 1.8 mmol 3 hour
Fmoc-Arg(Mtr)-OH2.20 g; 3.6 mmol1 hour
:~2~37'1~'7
-31-
Fmoc-Tyr(Bu )-OH 1.64 g; 3.6 mmol 1 hour
Fmoc-Asp(OBu )-OH 1.48 g; 3.6 mmol 1 hour
Fmoc-Val-OH 1.22 g; 3.6 mmol 1 hour
Fmoc-Gly-OH 1.08 g; 3.6 mmol 1 hour
Resin split in half after deprotection, to half
was added:
Boc-Cys(Trt)-OH 0.83 g; 1.8 mmol 1 hour
giving the second peptide.
To the other half of the resin was added:
10 Fmoc-Met-OH 0.67 g; 1.8 mmol 1 hour
Fmoc-Ala-OH 0.56 g; 1.8 mmol 1 hour
Fmoc-Phe-OH 0.74 g; 1.8 mmol 1 hour
Fmoc-Leu-OH 0.64 g; 1.8 mmol 1 hour
Fmoc-Lys(Boc)-OH 0.84 g; 1.8 mmol 1 hour
Fmoc-Gln-OH 0.33 g; 0.9 mmol 1 hour ~ 2
hour~
Fmoc-Ala-OH 0.56 g; 1.8 mmol 1 hour
Fmoc-Arg(Mtr)-OH 1.10 g; 1.8 mmol 1 hour
Fmoc-Thr(Bu )-OH 0.72 g: 1.8 mmol 1 hour
20 Fmoc-Thr(Bu )-OH 0.72 g; 1.8 mmol 1 hour
Fmoc-Pro-OH 0.61 g; 1.8 mmol 1 hour
Fmoc-Gln-OH 0.33 g; 0.9 mmol 5 hoùrs
Fmoc-Glu(OBut)-OH 0.77 g; 1.8 mmol 1 hour
Fmoc-Asn-OH 0.32 g; 0.9 mmol 1 1/2
hours
Fmoc-Asp(OBut)_OH 0.74 g; 1.8 mmol 1 hour
~287~ ~7
Fmoc-Val-OH 0.61 g, 1.8 mmol l hour
Fmoc-Glu(OBu )-OH 0.77 g; 1.8 mmol l hour
Boc-Cys(Trt)-OH 0.83 9; 1.8 mmol l hour
After washing both peptide resins were shrunk by washing
with dichloromethane and diethyl ether.
Peptide l
The peptide was cleaved from the resin and the
side chain protecting groups were removed by treating the
peptide resin with 95~ trifluoroacetic acid (TFA)/5% ethane
dithion (EDT) (3 x 1 hour). After filtration, evaporation
of each fragment afforded a residue which on trituration
with diethyl ether afforded three white solids (104 m~, 80
mg and 41 mg). Hplc (uBondpak C18; linear gradient 5 - 95%
0.1% TFA/CH3 CN - 0.1~ TFA/H20 over 20 minutes) showed the
product to consist of four major compounds, i.e. Mtr
protecting groups still present. The Mtr
(~-methoxy-2,3,6-trimethylbenzenesulphonyl) group used for
the peotection of the arginine side chain is cleaved
considerably more slowly than the t-butyl based side chain
protecting groups used for other functional residues and,
therefore, extended treatment with trifluoroacetic acid was
necessary to facilitate its removal. The three Eractions
were therefore combined and re-treated with TFA for a
further five hours. ~Iplc showed one major peak and several
smaller ones. The combined materials were dissolved in 10%
acetic acid and then subjected to exclusion chromatography
on Sephadex G-25 Superfine eluted with 10~ acetic acid.
The eluate was monitored at 254 nm and fractions
~2t37~'~'7
-33-
corresponding to the required compound were combined and
lyophilised af~ording the product as a white fluffy solid
(130 mg). This compound did not give a molecular ion when
subjected to fast atom bombardment mass spectrometry.
Peptide 2
The peptide was cleaved from the resin by
treating with 95~ TFA/5% EDT (3 x 1 hour). ThiS afforded
three white solids (70 mg, 61 mg and 42 mg). Hplc (same
conditions as above) showed two major peaks again
indicating Mtr groups present. The three fractions were
combined and re-treated with TFA to give a white solid (149
mg). Hplc showed the product to be essentially
homogeneous. FAB-mass spectrometry gave a sharp molecular
ion at 1481, this being consistent with a molecular weight
15 of 1480.
Example 3: Preparation o~ Cys-Ile-Pro-Phe-Asp-Leu-Ser-Ala-
Thr-Lys-L~s-Asn-Thr-Met-Glu-Met-Tyr-Cys (Peptide 3) and
Cys-Ile-Pro-Leu-Asn-Leu-Glu-Ser-Thr-Lys-Arg-Asn-Thr-Met-Asp
-Met-Tyr-Cys (Peptide 4)
These two peptides comprise amino acid residues
58 to 61 of the VP3 capsid protein of type 1 Sabin
poliovirus (Peptide 3) and type 3 Sabin poliovirus (Peptide
4). Both peptides are composed of residues S3 to 68 with
Cys residues at each terminus. The peptides were
synthesised according to the procedure of Example 2. Hplc
showed each product to be essentially homogeneous.
FAB-mass spectrometry gave a sharp molecular ion at 2097
for Peptide 3, this being consisten~ with a molecular
121~7'~
-34-
weight of 2096, and at 2134 for Peptide 4, this being
consistent with a molecular weight of 2133.
Example 4: Measu,rement of Specific Antibody Responses
The specific antibody responses of laboratory
rabbits to peptides 1 and 2 were measured in respect to:
1. antibody to uncoupled peptide (1 or 2) detected by
enzyme-linked immunoabsorbent assay (E~ISA), and
2. antibody to polioviruses of types 1, 2 and 3 as
detected by antigen blocking assays against poliovirus
C antigen measured in single-radial-diffusion (SRD)
tests in gels.
Coupling of peptide of bovine thyroglobulin
1 ml of O.lM sodium phosphate buffer pH 7.5 was
added to a glass vial containing 30 mg of bovine
thyroglobulin (BTG, Sigma) or 30 mg of keyhole limpet
haemocyanin. The dissolved material was transferred with 1
ml sodium phosphate buffer washing to a second vial
containing 10 mg of peptide (1 or 2) to give a final volume
of 2 ml peptide-BTG solution. The vial was wrapped in
aluminium foil to exclude light. A solution of 2~
glutaraldehyde was made in 0.1 M sodium phosphate buffer pH
7.5 and 200 ul, added to the peptide-BTG solution in four
lots of 50 ul, shaking between additions, and then left for
2S 1 hour at room temperature with intermittent shaking. The
solution was then dialysed against one litre of phosphate
lZ8~7~47
-35-
buffered saline (PsS) at 4 overnight, and then against 1
litre of fresh PBS for a further eight hours. Coupled
peptide was stored at -70C until required.
Coupled oligopeptides used for immunization of experimental
animals
Synthetic oligopeptides 1 and 2 were conjugated
separately to bovine thyrogobulin (BTG) as described above.
The preparations used for immunization contained 500 ug/ml
of peptide 1 or 2 and 1500 ug/ml of BTG suspended in
phosphate buffered saline (pH 7.2).
Immunization schedule for experimental animals
Young (5-6 months of age) healthy rabbits were
injected intramuscularly with an initial dose of 0.5 ml
(500 ug) coupled peptide mixed with an equal volume of
Freunds complete adjuvant (FCA, Bacto) and subsequently
injected with booster doses (0.5 ml) 500 ug coupled peptide
according to the following schedule. Serum samples for
analysis were collected at intervals up to 76 days after
the first injection.
20 Day 0 0.5 ml coupled peptide + FCA serum sample
Day 20 serum sample
Day 24 0.5 ml coupled peptide + FCA
Day 28 serum sample
Day 41 serum sample
25 Day 44 0.5 ml coupled peptide
~2874~7
-36-
Day 55 serum sample
Day 76 serum sample
Enzyme immunoassays (ELISA) for antibody to oligopeptide
Enzyme immunoassays were carried out to
investigate the immune response of the rabbit to the
peptide. Rabbit sera were examined for antibody which
bound to oligopeptide linked to polyvinyl plates by
glutaraldehyde. The bound antibody was detected b~ the
addition of anti rabbit antibodies which were coupled to
biotin followed by streptavidin biotinylated horse radish
peroxidase complexes. On addition of substrate for the
horseradish peroxidase (5-aminosalicylic acid) a
colorimetric change takes place, the intensity of which is
proportion to the amount of antibody bound to peptide.
Ninety-six well Microelisa plates (Dynatech)
were coated with oligopeptide (10 ug/ml). After incubation
overnight at 4 centigrade plates were washed x5 with PBS
containing 0.5~ Tween 20 (Koch-Light Laboratories,
Colnbrook, Berks). Dilutions of rabbit sera in PBS were
addded to wells and incubated for 2 hours at 37C. Plates
were washed x5 with phosphate buffered saline containing
0.5~ Tween 20 and donkey anti rabbit Ig linked to Biotin
(Amersham International) diluted in PBS added. After 1
hour at 37 degrees centigrade the biotinylated antibody was
removed, the plates washed x 5 with phosphate buffered
saline containing Tween 20. Streptavidin biotinylated
....
-37-
horseradish peroxidase complexes were added and the plates
incubated at 37C for 30 minutes. The plates were washed
x3 in Pss containing 0.5~ Tween 20 and x2 with Pss and the
substrate, 5-aminosalicylic acid (80 ug in 100 ml PBS was
added to each well. The plates were incubated at 37
degrees centigrade until colour developed. Optical density
was read on a Titertek multiscan set at 492 nm. The
machine was 'blanked' using substrate and serum dilutions
considered positive if the optical density was greater than
that of 1:100 dilution of normal rabbit serum collected
from the animal prior to immunization of the peptide.
Antigen blocking assays for antibody to poliovirus anti~en
employing single radial diffusion (SRD) in gel
The rabbit sera were tested in SRD
antigen-blocking tests to determine their reactivities with
C antigen of poliovirus type 1 or 3. The method used was a
modification of the autoradiographic SRD method of Schild
et al (1980) as described elsewhere (Ferguson et al 1982).
Briefly, [35S]-methionine labelled 80S 'C' peaks of
poliovirus antigen from sucrose gradients were mixed with
the test monoclonal antibody before adding to wells in
agarose gels containing low concentrations of hyperimmune
anti-poliovirus type 3 serum. Diffusion or radiolabelled
antigen in the gel after 24-48h was detected by
autoradiography. Test antibody which reacts with 'C'
antigen inhibits its diffusion into the gel compared with
~Z~37447
-38-
control antigen treated with phosphate buffered saline
alone. The antigen blocking titres are assessed as the
dilution of serum which significantly reduces the zone size
in comparison with zones produced with control antigen
mixed with phosphate buffered saline.
RESULTS
Induction of antibody to homologous peptide
The titres of antibody to peptide 2 determined by
ELISA assay are shown in Table 5 for representative
animals. Twenty one days following the initial
immunization with peptide all animals had readily
demonstrable antibody to homologous peptide. The titres
had increased by day 41 and in later serum samples
following booster doses of the oligopeptides. No
anti-peptide antibody was detected in prebleeds from any
animal. Table 6 shows ELISA titres to peptide 1. This
peptide is peptide SlOa of GB-A-2 128 621 plus peptide 2
linked together and the titres of antibody reached with
each peptide are also given.
Induction of antibody to poliovi_us
a) antigen blocking antibody
Antibody specific for empty virions (C antigen)
may be detected by antigen blocking assays employing the
single radial diffusion test (Schild et al 1980, Ferguson
et al 1982). This method was applied to sera obtained from
animals immunized with peptides 1 and 2 coupled to BTG.
lZ87~
-39-
Table 7 shows the induction of blocking
antibodies to poliovirus type 3 C antigen in rabbits
injected with peptide 2. Antibody first appeared between
day 17 and 31 in 3 out of 4 animals. Antisera from animals
immunised with peptide 2 were tested against viruses with
mutations in the VPl antigenic sites both of the present
invention and of GB-A-2 128 621. The results are shown in
Table 8.
Table 9 shows the induction of blocking
antibodies to poliovirus type 3 C antigen in rabbits
injected with peptide 1.
References
Brown et al 1983, J. Chem.Soc.Perkin Trans. I,
1161 et seq
Evans et al, 1983, Critical role of an eight
amino acid sequence of VPl in neutralization of poliovirus
type 3, Nature 304, 459-462.
Ferguson et al 1982, Monoclonal antibodies
specific for the Sabin vaccine strain of poliovirus, Lancet
II 122-124.
Ferguson et al 1984, Neutralisation epitopes on
poliovirus type 3 particles: an analysis using monoclonal
antibodies, J. Gen.Virol. 65, 197-201.
Fricks et al 198S, Trypsin sensitivity of the
Sabin strain of type 1 poliovirus: cleavage sites in
virions and related particles, J. Virol. 54, 856 et seq
Leinikki et al 1985, Paralytic poliomyelitis in
1;287~7
-40-
Finland, Lancet II 507
MacCallum, Hypogammaglobulinaemia in the United
~ Kin~dom, Medical Research Council special report series, No
310, pps 72-85.
Minor et al 1983, Location and primary structure
of a major antigenic site for poliovirus neutralization,
Nature 301 674-679.
Racaniello and Baltimore, Cloned poliovirus
complementary DNA in infections in mammalian cells, Science
214 916-919.
Schild et al, 1980, J. Gen. Virol. 51 157-170
Stanway et al, 1983, The nucleic acid sequence of
poliovirus type 3 Leon 12alb; comparison with type 1,
Nucleic Acids Research 11 5629-5643.
Stanway et al, 1984, Comparison of the complete
nucleotide sequences of the genomes of the neurovirulent
poliovirus P3/Leon/37 and its attenuated Sabin vaccine
derivative P3/Leon/12alb, Proc. Natl. Acad. Sci. USA 81
1539-1543.
37~a ~7
-41-
Table 1
poliovirus codon 286 287 288 289 290
type 3
Sabin Arg Asn Asn Leu Asp
Leon Lys Asn Asn Leu Asp
5 mutant a tExample 1) Arg Asp Asn Leu Asp
mutant b (Example 1) Arg Asn Asp Leu Asp
mutant c (Example 2) Arg Asn Asn Leu Glu
poliovirus codon 286 287 288 289 290
type 2
10 Sabin Lys Asp Gly Leu Thr
poliovirus codon 287 288 289 290 291 292
type 1
Sabin Lys Asp Gly Thr Leu Thr
Mahoney Lys Asp Gly Thr Leu Thr
lZ874~7
_ 42 -
Table 2
Reactions of representatives of 342 antigenic
mutant viruses with monoclonal antibodies
virus
~tr~in 25-1-14 25-4-12 22-4-4 199 194 134 208 175 204 197 165 198
_ _ _
P3 Leon
USA/l 9 37
1 r r r r r
2 r r r r r r r
3 r r r r r r r r r r r
4 r r r r r r r r r r
rrrr r r r r
6 rr r r r
7 rrrr r r r
B r r r r r
9r r r r r r r r r r
1~ r r rrr r r r rrr
11 rr rr rr
12 r rrrrr rr
13 r r r r r r r r
14 r r r rr
lSr r rrr r r rr r
16 r r rr r r rr
S~bin
Le~n
1 2.~ 1 b
A r r r r r rr r r r r
L r r rrr rr r r r
D r r r r r r r r r r r r
E r r r r r r r r r r
F r r rrr r r rr
r rrr r r r r
I r r
J r rrr r
K r r r,r r r r
L r r r r r rr
~ r r rr
N r r r r
O r r r r r r
P r r r r r r r r r
O r r
~utant viruses were said to be sensitive to antibody
if a 1:10 dilution of antibody as ascitic fluid was
able to preserve a cell sheet from challenge with 104
TCIDso of virus during incubation at 35 for 2 days.
Results were checked by plaque assay in the presence
of dilutions of antibody.
r indicates resistance
12~7~7
Q~ ~
,v oC~ ~, ~ ~
~ 0 ~
,~ u~ r~r- C
O ~\ o ~ ~ . ~
:) ~C
o ~ ~ S
o ~ ~ ~a
E~ O
Q ~ ,~
c v u~ ~ ~ ~ E E
C . ~ ~
c c ~~ ~ ~ ~ ~ E E
O ~ o o
o E o h ~ ~r
~ ~r ~ ~ ~ co
t~ CO
a) ~
S~ ~
`~
o ~ ~ ~ C
_
O OC
Q C R
~ Q
o ~
R
o ~ ~ ~
. 43
~L2~7~
_ 4 4-
Table 4
Location of amino acid substitutions in antiqenic mutants
of type 3 poliovirus
Mutant Protein Amino acid
1 VP1Argg8 - Gly
2 VP1n287 sp
3 VP3Glu58 - Asn
4 VP3Ser59 - Asn
VP3Ser59 - Arg
6 VPlA P290
7 VP3P77 Glu
8 VP3ser79 - Leu
9 VP2hr167 Lys
VP2a 166 Ala
11 VP2172 a
12 VP2Glul72 Lys
13 VP2Asnl64 - Lys
VP2Asnl64 Thr
~Z~7447
Table 5: Antibody titres of rabbits immunised with BTG
coupled peptide 2 with homologous peptide
Rabbit Day 0Day 20 Day 41
237 <100 <100 800
238 <100 <100 1600
~3g <100 <100 200
240 <lO0 lOO 6400
Table 6: Antibody titres to peptide 1 in rabbits immunised
with BTG coupled peptide 1
Rabbit Day 0 Day 17 Day 37
256 <10~ 400 6400
257 <100 1600 12800
25~3 <100 1600 >12800
259 <100 1600 12800
able 7: Antigen blocking antibodies to Sabin type 3 C
antigen in rabbits immunised with peptide 2
Day 0 Day 17 Day 31 Day 53
237 <5 <5 5 40
238 <5 <5 20 40
239 <5 <5 10 5
240 <5 <5 <S 10
able 8: Antigen blocking titres to C antigen of various
strains of poliovirus type 3 in rabbits
immunised with peptide 2 on day 53 (after 3
doses peptide
Difference in
sequence from Rabbit
Strain SP6 237 238 239 240
SP6 - 20 40 5 10
Leon VPl 287 20 40 10 5
306 VPl 88-100 ~0 20 10 5
183 VPl 88-100 20 40 10 5
63.1.9 3 changes 10 20 10 10
VP1 88-100
able 9: Antigen blocking titres against type 3 C antigen in
animals immunised with peptide 1
Rabbit Day 0 Day 17 Day 37 Day 64
256 <5 <5 80 20
257 <5 20 >320 1280
258 <5 5 >320 ND
259 <5 <5 10 <5
