Title: Malaria immunogen and vaccine
United States Patent: 6,942,866
Issued: September 13, 2005
Inventors: Birkett; Ashley J. (Escondido, CA)
Assignee: Apovia, Inc. (San Diego, CA)
Appl. No.: 931325
Filed: August 15, 2001
A chimeric, carboxy-terminal truncated hepatitis B virus nucleocapsid
protein (HBc) is disclosed that contains an immunogen for inducing the
production of antibodies to malarial proteins. An immunogenic malarial
epitope is expressed between residues 78 and 79 of the HBc immunogenic loop
sequence. The chimer preferably contains a malaria-specific T cell epitope
and is preferably engineered for both enhanced stability of self-assembled
particles and enhanced yield of those chimeric particles. Methods of making
and using the chimers are also disclosed.
BRIEF SUMMARY OF THE INVENTION
The present invention contemplates an immunogen for inducing antibodies
to the malaria-causing parasite, Plasmodium, and particularly the
species P. falciparum and P. vivax, and a vaccine comprising
that immunogen dispersed in a physiologically tolerable diluent. A
contemplated immunogen is a recombinant hepatitis B virus core (HBc) protein
chimer molecule with a length of about 140 to about 310 amino acid residues
that contains four peptide-linked amino acid residue sequence domains from
the N-terminus that are denominated Domains I, II, III and IV.
The first domain, Domain I, comprises about 71 to about 85 amino acid
residues whose sequence includes at least the sequence of the residues of
position 5 through position 75 of HBc.
The second domain, Domain II, comprises about 18 to about 58 amino acid
residues peptide-bonded to residue 75 of which (i) a sequence of HBc is
present from HBc positions 76 through 85 and (ii) a sequence of 8 to about
48 residues that constitute a B cell epitope of the CS protein of a species
of the parasite Plasmodium that is peptide-bonded between the HBc
residues of positions 78 and 79.
The third domain, Domain III, is an HBc sequence from position 86 through
position 135 peptide-bonded to residue 85.
The fourth domain, Domain IV, comprises (i) zero to fourteen residues of a
HBc amino acid residue sequence from position 136 through 149 peptide-bonded
to the residue of position 135 of Domain III, (ii) zero to three cysteine
residues, (iii) fewer than three arginine or lysine residues, or mixtures
thereof adjacent to each other, and (iv) up to about 100 amino acid residues
in a sequence heterologous to HBc from position 150 to the C-terminus, with
the proviso that at least five amino acid residues are present of the amino
acid residue sequence from position 136 through 149; i.e., residues of
positions 136-140, when (a) zero cysteine residues are present and (b) fewer
than about five heterologous amino acid residues are present, thus, Domain
IV contains at least 5 residues.
In preferred embodiments, the immunogen is in the form of self-assembled
particles and the Plasmodium B cell epitope is that of P.
falciparum or P. vivax. It is also preferred that the HBc
sequence of Domain I includes the residues of position 1 through position 75
with no additional residues at the N-terminus. It is further preferred that
a contemplated immunogen contain one cysteine residue within Domain IV in an
amino acid residue sequence heterologous to that of HBc from position 150 to
the C-terminus. It is particularly preferred that that heterologous sequence
comprise a T cell epitope from the same species of Plasmodium as the B cell
Another embodiment comprises an inoculum or vaccine that comprises an above
HBc chimer particle that is dissolved or dispersed in a pharmaceutically
acceptable diluent composition that typically also contains water. When
administered in an immunogenic effective amount to an animal such as a
mammal or bird, an inoculum induces antibodies that immunoreact specifically
with the chimer particle or the conjugated (pendently-linked) hapten. The
antibodies so induced also preferably immunoreact specifically with (bind
to) an antigen containing the hapten, such as a protein where the hapten is
a peptide or a saccharide where the hapten is an oligosaccharide.
The present invention has several benefits and advantages.
A particular benefit of the invention is that its use as a vaccine provides
extraordinary antibody titers against the Plasmodium species of the B
An advantage of the invention is that those very high antibody titers have
been produced with the aid of an adjuvant approved for use in humans.
Another benefit of the invention is that the recombinant immunogen is
prepared easily and using well known cell culture techniques.
Another advantage of the invention is that the immunogen is easily prepared
using well known recombinant techniques.
Yet another benefit of the invention is that a preferred immunogen exhibits
greater stability at elevated temperatures than to other HBc chimers.
Yet another advantage of the invention is that a contemplated immunogen is
substantially free of nucleic acids.
DETAILED DESCRIPTION OF THE INVENTION
The present invention contemplates an immunogen and a vaccine comprising
that immunogen against the malaria parasite, particularly those that infect
humans; i.e., P. falciparum and P. vivax. Historically, one of
the main shortfalls of peptide-based vaccines has been the lack of
persistence of antibody following immunization. As discussed hereinafter,
using the HBc chimer immunogen applied to P. falciparum vaccine
development, high titers of neutralizing antibody are maintained for more
than 6 months in mice following a 2-dose immunization regimen. This is
consistent with and superior to the protection studies in the P. yoelii
model using a similar but differently constructed immunogen, in which
immunity obtained from challenge infection was evident 3 months after
immunization using an immunogen different from that used here. [Schodel et
al., Behring Inst. Mitt., 1997(98): p. 114-119.]
A contemplated immunogen is a recombinant hepatitis B virus core (HBc)
protein chimer molecule with a length of about 140 to about 310 and
preferably about 155 to 235 amino acid residues that contains four
peptide-linked amino acid residue sequence domains from the N-terminus that
are denominated Domains I, II, III and IV.
(a) Domain I comprises about 71 to about 85 amino acid residues whose
sequence includes at least the sequence of the residues of position 5
through position 75 of HBc.
(b) Domain II comprises about 11 to about 58 amino acid residues
peptide-bonded to residue 75. This sequence includes (i) a sequence of HBc
from HBc positions 76 through 85 and (ii) a sequence of 8 to about 48
residues that constitute a B cell epitope of the circumsporozoite protein of
a species of the parasite Plasmodium that is peptide-bonded between
the HBc residues of positions 78 and 79.
(c) Domain III is an HBc sequence from position 86 through position 135 that
is peptide-bonded to residue 85.
d) Domain IV comprises (i) zero to fourteen residues of a HBc amino acid
residue sequence from position 136 through 149 peptide-bonded to the residue
of position 135 of Domain III, (ii) zero to three cysteine residues, (iii)
fewer than three arginine or lysine residues, or mixtures thereof adjacent
to each other, and (iv) up to 100, and preferably up to 25, amino acid
residues in a sequence heterologous to HBc from position 150 to the
C-terminus, with the proviso that at least five amino acid residues of the
amino acid residue sequence from position 136 through 149; i.e., residues of
positions 136-140, are present when (a) zero cysteine residues are present
and (b) fewer than about five heterologous amino acid residues are present.
In examining the length of a contemplated HBc chimer, such a recombinant
protein can have a length of about 140 to about 310 amino acid residues.
Preferably, that length is about 155 to about 235 residues. More preferably,
the length is about 165 to about 210 residues. Most preferably, the length
is about 190 to about 200 residues. These differences in length arise from
changes in the length of Domains I, II and IV.
HBc chimers having a Domain I that contains more than a deletion of the
first three amino-terminal (N-terminal) residues have been reported to
result in the complete disappearance of HBc chimer protein in E. coli
cells. Pumpens et al., (1995) Intervirology, 38:63-74. On the other
hand, a recent study in which an immunogenic 23-mer polypeptide from the
influenza M2 protein was fused to the HBc N-terminal sequence reported that
the resultant fusion protein formed particles when residues 1-4 of the
native HBc sequence were replaced. Neirynck et al. (October 1999) Nature
Med., 5(10):1157-1163. Thus, the art teaches that particles can form
when an added amino acid sequence is present peptide-bonded the one of
residues 1-5 of HBc, whereas particles do not form if no additional sequence
is present and more than residues 1-3 are deleted from the N-terminus of HBc.
An N-terminal sequence peptide-bonded to one of the first five N-terminal
residues of HBc can contain a sequence of up to about 25 residues that are
heterologous to HBc. Exemplary sequences include a B cell or T cell epitope
such as those discussed hereinafter, a sequence of another (heterologous)
protein such as β-galactosidase as can occur in fusion proteins as a result
of the expression system used, or another hepatitis B-related sequence such
as that from the Pre-S1 or Pre-S2 regions or the major HbsAg immunogenic
Domain I preferably has the sequence of residues of positions 1 through 75
of HBc, and is free of added residues at the amino-terminus (N-terminus).
Domain I is also therefore preferably free of deletions of residues of
Domain II, which is peptide-bonded to residue 75, contains the sequence of
HBc residues of positions 76 through 85, and has a malarial B cell epitope
whose length is 8 through about 28 residues peptide-bonded between residues
78 and 79. Preferred malarial B cell epitopes are discussed hereinafter.
Preferred malarial B cell epitopes for insertion between residues 78 and 79
of a recombinant HBc chimer are enumerated in Table A, below.
|Malarial B Cell Epitopes
||SEQ ID NO: 1
||SEQ ID NO: 2
||SEQ ID NO: 3
||SEQ ID NO: 4
||SEQ ID NO: 5
||SEQ ID NO: 6
||SEQ ID NO: 7
||SEQ ID NO: 8
||SEQ ID NO: 9
||SEQ ID NO: 10
||SEQ ID NO: 11
||SEQ ID NO: 12
||SEQ ID NO: 13
||SEQ ID NO: 14
||SEQ ID NO: 15
||SEQ ID NO: 16
||SEQ ID NO: 17
||SEQ ID NO: 18
||SEQ ID NO: 19
||SEQ ID NO: 20
||SEQ ID NO: 21
Domain III contains the sequence of HBc position 86 through position 135
peptide-bonded at its N-terminus to residue 85.
Domain IV comprises (i) zero to fourteen residues of a HBc amino acid
residue sequence from position 136 through 149 peptide-bonded to the residue
of position 135 of Domain III, (ii) zero to three cysteine residues, (iii)
fewer than three arginine or lysine residues, or mixtures thereof adjacent
to each other, and (iv) up to about 100 amino acid residues in a sequence
heterologous to HBc from position 150 to the C-terminus (typically as one or
more T cell epitopes), with certain provisos. Although Domain IV can contain
up to about 100 residues that are heterologous to HBc from position 150
through the C-terminus, this domain needs no residues in addition to those
recited before to provide an effective immunogen.
However, when the chimeric protein ends at HBc residue 135, desired,
particularly immunogenic particles do not form even when a C-terminal
cysteine is present. On the other hand, desired particles do form when
residues of positions 136-140 are present with or without an added
C-terminal cysteine or when (a) one cysteine residue is present and (b)
about five heterologous amino acid residues are also present peptide-bonded
to HBc residue 135. Put differently, Domain IV can end at HBc residue 135 so
long as at least five heterologous residues are present and a cysteine
residue is also present. Otherwise, Domain IV ends at least at HBc residue
140. Thus, Domain IV contains at least 5 amino acid residues.
It is preferred that Domain IV contain up to fourteen residues of an HBc
sequence from position 136 through position 149 peptide-bonded to residue
135; i.e., an HBc sequence that begins with the residue of position 136 that
can continue through position 149. Thus, if the residue of position 148 is
present, so is the sequence of residues of positions 136 through 147, or if
residue 141 is present, so is the sequence of residues of positions 136
In one embodiment, Domain IV comprises a sequence of HBc from residue 136
through 140 peptide-bonded to the residue of position 135 of Domain III. The
remainder of Domain IV contains (i) zero to nine residues of a HBc amino
acid residue sequence from position 141 through 149 peptide-bonded to the
position 136-140 sequence, (ii) zero to three cysteine residues, (iii) fewer
than three arginine or lysine residues, or mixtures thereof adjacent to each
other, and (iv) up to 50 amino acid residues, and more preferably up to
about 25 residues, in a sequence that constitutes a T cell epitope of the
same species of Plasmodium as the B cell epitope peptide-bonded to
the final HBc amino acid residue present in the chimer or a cysteine
residue. Thus, for example, the T cell epitope can be bonded to the carboxy-terminal-most
HBc residue such as residue 149, or to a cysteine residue that is bonded to
that final HBc residue.
Domain IV can also contain zero to three cysteine residues and those Cys
residues are present within about 30 residues of the carboxy-terminus
(C-terminus) of the chimer molecule. Preferably, one cysteine (Cys) residue
is present, and that Cys is preferably present as the carboxy-terminal
(C-terminal) residue, unless a malarial T cell epitope is present as part of
Domain IV. When such a T cell epitope is present, the preferred Cys is
preferably within the C-terminal last five residues of the HBc chimer.
Preferred malarial T cell epitopes are discussed hereinafter.
The presence of the above-discussed cysteine residue(s) provides an
unexpected enhancement of the ability of the chimer molecules to form
immunogenic particles, as well as unexpected thermal stability to an
immunogen particle (discussed hereinafter). Thus, a preferred HBc chimer
immunogen tends to be stable to decomposition at 37° C. to a greater extent
than does a similar chimer lacking that cysteine residue.
Domain IV contains fewer than three arginine or lysine residues, or mixtures
thereof adjacent to each other. Arginine and lysines are present in the
C-terminal region of HBc that extends from position 150 through the
C-terminus of the native molecule. That region is sometimes referred to in
the art as the "protamine" or "arginine-rich" region of the molecule and is
thought to bind to nucleic acids. A contemplated HBc chimer molecule and
particle are substantially free of bound nucleic acids.
The substantial freedom of nucleic acid binding can be readily determined by
a comparison of the absorbance of the particles in aqueous solution measured
at both 280 and 260 nm; i.e., a 280/260 absorbance ratio. The contemplated
particles do not bind substantially to nucleic acids that are oligomeric
and/or polymeric DNA and RNA species originally present in the cells of the
organism used to express the protein. Such nucleic acids exhibit an
absorbance at 260 nm and relatively less absorbance at 280 nm, whereas a
protein such as a contemplated chimer absorbs relatively less at 260 nm and
has a greater absorbance at 280 nm.
Thus, recombinantly expressed HBc particles or chimeric HBc particles that
contain the arginine-rich sequence at residue positions 150-183 (or 150-185)
exhibit a ratio of absorbance at 280 nm to absorbance at 260 nm (280:260
absorbance ratio) of about 0.8, whereas particles free of the arginine-rich
nucleic acid binding region of naturally occurring HBc such as those that
contain fewer than three arginine or lysine residues or mixtures thereof
adjacent to each other, or those having a native or chimeric sequence that
ends at about HBc residue position 140 to position 149, exhibit a 280:260
absorbance ratio of about 1.2 to about 1.6.
Chimeric HBc particles of the present invention are substantially free of
nucleic acid binding and exhibit a 280:260 absorbance ratio of about 1.2 to
about 1.6, and more typically, about 1.4 to about 1.6. This range is due in
large part to the number of aromatic amino acid residues present in Domains
II and IV of a given chimeric HBc particle. That range is also in part due
to the presence of the Cys in Domain IV of a contemplated chimer, whose
presence can diminish the observed ratio by about 0.1 for a reason that is
The contemplated chimer HBc particles are more stable in aqueous buffer at
37° C. over a time period of about two weeks to about one month than are
particles formed from a HBc chimer containing the same peptide-linked Domain
I, II and III sequences and an otherwise same Domain IV sequence in which
the one to three cysteine residues [C-terminal cysteine residue(s)] are
absent or a single C-terminal residue present is replaced by another residue
such as an alanine residue. Stability of various chimer particles is
determined as discussed hereinafter.
Thus, for example, particles containing a heterologous malarial epitope in
Domain II [e.g. (NANP)4] and a single cysteine residue C-terminal
to residue valine 149 is more stable than otherwise identical particles
assembled from chimer molecules whose C-terminal residue is valine 149.
Similarly, particles containing the above malarial B cell epitope in Domain
II and the universal malarial T cell epitope that contains a single cysteine
near the C-terminus are more stable than are otherwise identical particles
in which that cysteine is replaced by an alanine residue.
A contemplated particle containing a C-terminal cysteine residue is also
typically prepared in greater yield than is a particle assembled from a
chimer molecule lacking a C-terminal cysteine. This increase in yield can be
seen from the mass of particles obtained or from integration of traces from
analytical gel filtration analysis using Superose® 6 HR as discussed
hereinafter and shown in Tables 9A and 9B.
Although the T cell help afforded by HBc is highly effective in enhancing
antibody responses (i.e. B cell-mediated) to 'carried' epitopes following
vaccination, HBc does not activate malaria-specific T cells, except in
restricted individuals for whom the B cell epitope is also a T cell epitope.
To help ensure universal priming of malaria-specific T helper cells, in
addition to B cells, one or more malaria-specific T helper epitopes is
preferably incorporated into a contemplated immunogen and is located in
Domain IV of the immunogen.
A particularly preferred recombinant HBc chimer includes a T cell epitope of
the same Plasmodium species as the B cell epitope. Thus, where the B
cell epitope of Domain II is that of P. falciparum, the T cell
epitope is also that of P. falciparum, and the like. Using this
matching strategy, T cells are primed to the same species as that to which
antibodies are initially induced by the B cell epitope. Particularly
preferred T cell epitopes present as a part of Domain IV are enumerated in
Table B, below.
|Malarial Universal T Cell Epitope
||SEQ ID NO:24
||SEQ ID NO:25
||SEQ ID NO:26
A plurality of the above or another T cell epitopes can be present in
Domain IV or another B cell epitope can be present. In preferred practice,
Domain IV has up to about 50 residues in a sequence heterologous to HBc.
Most preferably, that sequence is up to about 25 residues and includes one
of the universal T cell epitopes shown in Table B, above.
A contemplated recombinant HBc chimer molecule is typically present and is
used in an immunogen or vaccine as a self-assembled particle. These
particles are comprised of 180 to 240 chimer molecules that separate into
protein molecules in the presence of disulfide reducing agents such as
2-mercaptoethanol, and the individual molecules are therefore thought to be
bound together into the particle primarily by disulfide bonds. These
particles are similar to the particles observed in patients infected with
HBV, but these particles are non-infectious. Upon expression in various
prokaryotic and eukaryotic hosts, the individual recombinant HBc chimer
molecules assemble in the host into particles that can be readily harvested
from the host cells.
The amino acid sequence of HBc from residue position 1 through at least
position 140 is preferably present in a contemplated chimer molecule and
particle. The sequence from position 1 through position 149 is more
preferably present. A malarial B cell epitope is present between residues 78
and 79 and a single cysteine residue or a malarial T cell epitope containing
a cysteine residue is preferably present as a C-terminal addition to the HBc
sequence as part of Domain IV. A contemplated recombinant HBc chimer is
substantially free of bound nucleic acid. A preferred chimer particle that
contains an added Cys residue at or near the C-terminus of the molecule is
also more stable at 37° C. than is a similar particle that does not contain
that added Cys.
In addition to the before-discussed N- and C-truncations and insertion of
malarial epitopes, a contemplated chimer molecule can also contain
conservative substitutions in the amino acid residues that constitute HBc
Domains I, II, III and IV. Conservative substitutions are as defined before.
More rarely, a "nonconservative" change, e.g., replacement of a glycine with
a tryptophan is contemplated. Analogous minor variations can also include
amino acid deletions or insertions, or both. Guidance in determining which
amino acid residues can be substituted, inserted, or deleted without
abolishing biological activity can be found using computer programs well
known in the art, for example LASERGENE software (DNASTAR Inc., Madison,
The HBc portion of a chimer molecule of the present invention [the portion
having the HBc sequence that has other than a sequence of an added epitope,
or heterologous residue(s) that are a restriction enzyme artifact] most
preferably has the amino acid residue sequence at positions 1 through 149 of
subtype ayw that is shown in FIG. 6 (SEQ ID NO:170) (see Original Patent), when present. Somewhat
less preferred are the corresponding amino acid residue sequences of
subtypes adw, adw2 and adyw that are also shown in FIG. 6 (SEQ ID NOs:171,
172 and 173) (see Original Patent). Less preferred still are the sequences of woodchuck and ground
squirrel at aligned positions 1 through 149 that are the last two sequences
of FIG. 6 (SEQ ID NOs:174 and 168) (see Original Patent). As noted elsewhere, portions of
different sequences from different mammalian HBc proteins can be used
together in a single chimer.
When the HBc portion of a chimer molecule of the present invention has other
than a sequence of a mammalian HBc molecule at positions 1 through 149, when
present, because one or more conservative substitutions has been made, it is
preferred that no more than 10 percent, and more preferably no more than 5
percent, and most preferably no more than 3 percent of the amino acid
residues are substituted as compared to SEQ ID NO:170 from position 1
through 149. A contemplated chimer of 149 HBc residues can therefore contain
up to about 15 residues that are different from those of SEQ ID NO:170 at
positions 1 through 149, and preferably about 7 or 8 residues. More
preferably, up to about 5 residues are different from the ayw sequence (SEQ
ID NO:170) at residue positions 1-149. Where a HBc sequence is truncated
further at one or both termini, the number of substituted residues is
proportionally different. Deletions elsewhere in the molecule are considered
conservative substitutions for purposes of calculation.
A contemplated chimeric immunogen is prepared using the well known
techniques of recombinant DNA technology. Thus, sequences of nucleic acid
that encode particular polypeptide sequences are added and deleted from the
precursor sequence that encodes HBV.
As was noted previously, the HBc immunodominant loop is usually recited as
being located at about positions 75 through 85 from the amino-terminus
(N-terminus) of the intact protein. The malarial B cell epitope-containing
sequence is placed into that immunodominant loop sequence of Domain II. That
placement substantially eliminates the HBc immunogenicity and antigenicity
of the HBc loop sequence, while presenting the malarial B cell epitope in an
extremely immunogenic position in the assembled chimer particles.
One of two well-known strategies is particularly useful for placing the
malarial B cell sequence into the loop sequence at the desired location
between residues 78 and 79. A first, less successful strategy is referred to
as replacement in which DNA that codes for a portion of the loop is excised
and replaced with DNA that encodes a malarial B cell sequence. The second
strategy is referred to as insertion in which a malarial B cell sequence is
inserted between adjacent residues in the loop.
Site-directed mutagenesis using the polymerase chain reaction (PCR) is used
in one exemplary replacement approach to provide a chimeric HBc DNA sequence
that encodes a pair of different restriction sites, e.g. EcoRI and SacI, one
near each end of the immunodominant loop-encoding DNA. Exemplary residues
replaced are 76 through 81. The loop-encoding section is excised, a desired
malarial B cell epitope-encoding sequence flanked on each side by
appropriate HBc sequence residues is ligated into the restriction sites and
the resulting DNA is used to express the HBc chimer. See, for example, Table
2 of Pumpens et al., (1995) Intervirology, 38:63-74 for exemplary
uses of a similar technique.
Alternatively, a single restriction site or two sites can be encoded into
the region, the DNA cut with a restriction enzyme(s) to provide "sticky" or
ends, and an appropriate sticky- or blunt-ended heterologous DNA segment
ligated into the cut region. Examples of this type of sequence replacement
into HBc can be found in the work reported in Schodel et al., (1991) F.
Brown et al. eds., Vaccines 91, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y., pp. 319-325, Schodel et al., Behring Inst. Mitt.,
1997(98): p. 114-119 and Schodel et al., J. Exp. Med., (1994) 180(3):
p. 1037-4, the latter two papers discussing the preparation of vaccines
against P. yoelii and P. berghei, respectively.
It has surprisingly been found that the insertion position within the HBc
immunogenic loop and the presence of loop residues are of import to the
activity of the immunogen. Thus, as is illustrated hereinafter, placement of
a malarial B cell epitope between HBc residue positions 78 and 79 provides a
particulate immunogen that is ten to one thousand times more immunogenic
than placement of the same immunogen in an excised and replaced region
between residues 76 and 81. In addition, placement of the same malarial
immunogen between residues 78 and 79 as compared to between residues 77 and
78 provided an unexpected enhancement of about 15-fold. Thus, a replacement
strategy that results in a net removal of residues from the immunodominant
loop is not used herein.
Insertion is therefore preferred. In an illustrative example of the
insertion strategy, site-directed mutagenesis is used to create two
restriction sites adjacent to each other and between codons encoding
adjacent amino acid residues, such as those at residue positions 78 and 79.
This technique adds twelve base pairs that encode four amino acid residues
(two for each restriction site) between formerly adjacent residues in the
Upon cleavage with the restriction enzymes, ligation of the DNA coding for
the malarial sequence and expression of the DNA to form HBc chimers, the HBc
loop amino acid sequence is seen to be interrupted on its N-terminal side by
the two residues encoded by the 5′ restriction site, followed toward the
C-terminus by the malarial B-cell epitope sequence, followed by two more
heterologous, non-loop residues encoded by the 3′ restriction site and then
the rest of the loop sequence. This same strategy is also preferably used
for insertion into Domain IV of a T cell epitope or one or more cysteine
residues that are not a part of a T cell epitope. A similar strategy using
an insertion between residues 82 and 83 is reported in Schoedel et al.,
(1990) F. Brown et al. eds., Vaccines 90, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. pp. 193-198.
More specifically, this cloning strategy is illustrated schematically in
FIGS. 2A, 2B and 2C (see Original Patent). In FIG. 2A, a DNA sequence that encodes a C-terminal
truncated HBc sequence (HBc149) is engineered to contain adjacent EcoRI and
SacI sites between residues 78 and 79. Cleavage of that DNA with both
enzymes provides one fragment that encodes HBc positions 1-78 3′-terminated
with an EcoRI sticky end, whereas the other fragment has a 5′-terminal SacI
sticky end and encodes residues of positions 79-149. Ligation of a synthetic
nucleic acid having a 5′ AATT overhang followed by a sequence that encodes a
desired malarial B cell epitope and a AGCT 3′overhang provides a HBc chimer
sequence that encodes that B cell epitope flanked on each side by two
heterologous residues (GI and EL, respectively) between residues 78 and 79,
while destroying the EcoRI site and preserving the SacI site.
A similar strategy is shown in FIG. 2B for insertion of a cysteine-containing
sequence, such as a particularly preferred T cell epitope such as that
referred to as PF/CS326-345 (Pf-UTC). Here, EcoRI and HindIII restriction
sites were engineered in to the HBc DNA sequence after amino acid residue
position 149. After digestion with EcoRI and HindIII, a synthetic DNA having
the above AATT 5′overhang followed by a T cell epitope-encoding sequence, a
stop codon and a 3′ AGCT overhang were ligated into the digested sequence to
form a sequence that encoded HBc residues 1-149 followed by two heterologous
residues (GI), the stop codon and the HindIII site.
PCR amplification using a forward primer having a SacI restriction site
followed by a sequence encoding HBc beginning at residue position 79,
followed by digestion with SacI and HindIII provided a sequence encoding HBc
positions 79-149 plus the two added residues and the T cell epitope at the
C-terminus. Digestion of the construct of FIG. 2B with SacI and ligation
provided the complete gene encoding a desired recombinant HBc chimer
immunogen having the sequence, from the N-terminus, of HBc positions 1-78,
two added residues, the malarial B cell epitope, two added residues, HBc
positions 79-149, two added residues, and the T cell epitope that is shown
in FIG. 2C.
It is noted that the preferred use of two heterologous residues on either
side of (flanking) a B cell or T cell epitope is a matter of convenience. As
a consequence, one can also use zero to three or more added residues that
are not part of the HBc sequence on either or both sides of an inserted
sequence. One or both ends of the insert and HBc nucleic acid can be "chewed
back" with an appropriate nuclease (e.g. S1 nuclease) to provide blunt ends
that can be ligated together. Added heterologous residues that are neither
part of the inserted B cell or T cell epitopes nor a part of the HBc
sequence are not counted in the number of residues present in a recited
It is also noted that one can also synthesize all or a part of a desired
recombinant HBc chimer nucleic acid using well-known synthetic methods as is
discussed and illustrated in U.S. Pat. No. 5,656,472 for the synthesis of
the 177 base pair DNA that encodes the 59 residue ribulose bis-phosphate
carboxylase-oxygenase signal peptide of Nicotiana tabacum. For
example, one can synthesize Domains I and II with a blunt or "sticky" end
that can be ligated to Domains III and IV to provide a construct that
expresses a contemplated HBc chimer that contains zero added residues to the
N-terminal side of the B cell epitope and zero to three added residues on
the C-terminal side or at the Domain II/III junction or at some other
A nucleic acid sequence (segment) that encodes a previously described HBc
chimer molecule or a complement of that coding sequence is also contemplated
herein. Such a nucleic acid segment is present in isolated and purified form
in some preferred embodiments.
In living organisms, the amino acid residue sequence of a protein or
polypeptide is directly related via the genetic code to the deoxyribonucleic
acid (DNA) sequence of the gene that codes for the protein. Thus, through
the well-known degeneracy of the genetic code additional DNAs and
corresponding RNA sequences (nucleic acids) can be prepared as desired that
encode the same chimer amino acid residue sequences, but are sufficiently
different from a before-discussed gene sequence that the two sequences do
not hybridize at high stringency, but do hybridize at moderate stringency.
High stringency conditions can be defined as comprising hybridization at a
temperature of about 50°-55° C. in 6×SSC and a final wash at a temperature
of 68° C. in 1-3×SSC. Moderate stringency conditions comprise hybridization
at a temperature of about 50° C. to about 65° C. in 0.2 to 0.3 M NaCl,
followed by washing at about 50° C. to about 55° C. in 0.2×SSC, 0.1% SDS
(sodium dodecyl sulfate).
A nucleic sequence (DNA sequence or an RNA sequence) that (1) itself
encodes, or its complement encodes, a chimer molecule whose HBc portion from
residue position 1 through 136, when present, is that of SEQ ID NOs: 168,
170, 171, 172, 173 or 174 and (2) hybridizes with a DNA sequence of SEQ ID
NOs: 169, 175, 176, 177, 178 or 179 at least at moderate stringency
(discussed above); and (3) whose HBc sequence shares at least 80 percent,
and more preferably at least 90 percent, and even more preferably at least
95 percent, and most preferably 100 percent identity with a DNA sequence of
SEQ ID NOs: 169, 175, 176, 177, 178 and 179, is defined as a DNA variant
sequence. As is well-known, a nucleic acid sequence such as a contemplated
nucleic acid sequence is expressed when operatively linked to an appropriate
promoter in an appropriate expression system as discussed elsewhere herein.
An analog or analogous nucleic acid (DNA or RNA) sequence that encodes a
contemplated chimer molecule is also contemplated as part of this invention.
A chimer analog nucleic acid sequence or its complementary nucleic acid
sequence encodes a HBc amino acid residue sequence that is at least 80
percent, and more preferably at least 90 percent, and most preferably is at
least 95 percent identical to the HBc sequence portion from residue position
1 through residue position 136 shown in SEQ ID NOs: 168, 170, 171, 172, 173
and 174. This DNA or RNA is referred to herein as an "analog of" or
"analogous to" a sequence of a nucleic acid of SEQ ID NOs: 169, 175, 176,
177, 178 and 179, and hybridizes with the nucleic acid sequence of SEQ ID
NOs: 169, 175, 176, 177, 178 and 179 or their complements herein under
moderate stringency hybridization conditions. A nucleic acid that encodes an
analogous sequence, upon suitable transfection and expression, also produces
a contemplated chimer.
Different hosts often have preferences for a particular codon to be used for
encoding a particular amino acid residue. Such codon preferences are well
known and a DNA sequence encoding a desired chimer sequence can be altered,
using in vitro mutagenesis for example, so that host-preferred codons are
utilized for a particular host in which the enzyme is to be expressed. In
addition, one can also use the degeneracy of the genetic code to encode the
HBc portion of a sequence of SEQ ID NOs: 168, 170, 171, 172, 173 or 174 that
avoids substantial identity with a DNA of SEQ ID Nos: 169, 175, 176, 177,
178 or 179, or their complements. Thus, a useful analogous DNA sequence need
not hybridize with the nucleotide sequences of SEQ ID NOs: 169, 175, 176,
177, 178 or 179 or a complement under conditions of moderate stringency, but
can still provide a contemplated chimer molecule.
A recombinant nucleic acid molecule such as a DNA molecule, comprising a
vector operatively linked to an exogenous nucleic acid segment (e.g., a DNA
segment or sequence) that defines a gene that encodes a contemplated chimer,
as discussed above, and a promoter suitable for driving the expression of
the gene in a compatible host organism, is also contemplated in this
invention. More particularly, also contemplated is a recombinant DNA
molecule that comprises a vector comprising a promoter for driving the
expression of the chimer in host organism cells operatively linked to a DNA
segment that defines a gene for the HBc portion of a chimer or a DNA variant
that has at least 90 percent identity to the chimer gene of SEQ ID NOs: 169,
175, 176, 177, 178 or 179 and hybridizes with that gene under moderate
Further contemplated is a recombinant DNA molecule that comprises a vector
containing a promoter for driving the expression of a chimer in host
organism cells operatively linked to a DNA segment that is an analog nucleic
acid sequence that encodes an amino acid residue sequence of a HBc chimer
portion that is at least 80 percent identical, more preferably 90 percent
identical, and most preferably 95 percent identical to the HBc portion of a
sequence of SEQ ID NOs: 168, 170, 171, 172, 173 or 174. That recombinant DNA
molecule, upon suitable transfection and expression in a host cell, provides
a contemplated chimer molecule.
It is noted that because of the 30 amino acid residue N-terminal sequence of
ground squirrel HBc does not align with any of the other HBc sequences, that
sequence and its encoding nucleic acid sequences and their complements are
not included in the above percentages of identity, nor are the portions of
nucleic acid that encode that 30-residue sequence or its complement used in
hybridization determinations. Similarly, sequences that are truncated at
either or both of the HBc N- and C-termini are not included in identity
calculations, nor are those sequences in which residues of the
immunodominant loop are removed for insertion of a heterologous epitope.
Thus, only those HBc-encoding bases or HBc sequence residues that are
present in a chimer molecule are included and compared to an aligned nucleic
acid or amino acid residue sequence in the identity percentage calculations.
Inasmuch as the coding sequences for the gene disclosed herein is
illustrated in SEQ ID NOs: 169, 175, 176, 177, 178 and 179, isolated nucleic
acid segments, preferably DNA sequences, variants and analogs thereof can be
prepared by in vitro mutagenesis, as is well known in the art and discussed
in Current Protocols In Molecular Biology, Ausabel et al. eds., John
Wiley & Sons (New York: 1987) p. 8.1.1-8.1.6, that begin at the initial ATG
codon for a gene and end at or just downstream of the stop codon for each
gene. Thus, a desired restriction site can be engineered at or upstream of
the initiation codon, and at or downstream of the stop codon so that other
genes can be prepared, excised and isolated.
As is well known in the art, so long as the required nucleic acid,
illustratively DNA sequence, is present, (including start and stop signals),
additional base pairs can usually be present at either end of the segment
and that segment can still be utilized to express the protein. This, of
course, presumes the absence in the segment of an operatively linked DNA
sequence that represses expression, expresses a further product that
consumes the enzyme desired to be expressed, expresses a product that
consumes a wanted reaction product produced by that desired enzyme, or
otherwise interferes with expression of the gene of the DNA segment.
Thus, so long as the DNA segment is free of such interfering DNA sequences,
a DNA segment of the invention can be about 500 to about 15,000 base pairs
in length. The maximum size of a recombinant DNA molecule, particularly an
expression vector, is governed mostly by convenience and the vector size
that can be accommodated by a host cell, once all of the minimal DNA
sequences required for replication and expression, when desired, are
present. Minimal vector sizes are well known. Such long DNA segments are not
preferred, but can be used.
DNA segments that encode the before-described chimer can be synthesized by
chemical techniques, for example, the phosphotriester method of Matteucci et
al. (1981) J. Am. Chem. Soc., 103:3185. Of course, by chemically
synthesizing the coding sequence, any desired modifications can be made
simply by substituting the appropriate bases for those encoding the native
amino acid residue sequence. However, DNA segments including sequences
discussed previously are preferred.
A contemplated HBc chimer can be produced (expressed) in a number of
transformed host systems, typically host cells although expression in
acellular, in vitro, systems is also contemplated. These host cellular
systems include, but are not limited to, microorganisms such as bacteria
transformed with recombinant bacteriophage, plasmid, or cosmid DNA
expression vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g. baculovirus);
plant cell systems transformed with virus expression vectors (e.g.
cauliflower mosaic virus; tobacco mosaic virus) or with bacterial expression
vectors (e.g., Ti plasmid); or appropriately transformed animal cell systems
such as CHO or COS cells. The invention is not limited by the host cell
DNA segments containing a gene encoding the HBc chimer are preferably
obtained from recombinant DNA molecules (plasmid vectors) containing that
gene. Vectors capable of directing the expression of a chimer gene into the
protein of a HBc chimer is referred to herein as an "expression vector".
An expression vector contains expression control elements including the
promoter. The chimer-coding gene is operatively linked to the expression
vector to permit the promoter sequence to direct RNA polymerase binding and
expression of the chimer-encoding gene. Useful in expressing the polypeptide
coding gene are promoters that are inducible, viral, synthetic, constitutive
as described by Poszkowski et al. (1989) EMBO J., 3:2719 and Odell et
al. (1985) Nature, 313:810, as well as temporally regulated,
spatially regulated, and spatiotemporally regulated as given in Chua et al.
(1989) Science, 244:174-181.
One preferred promoter for use in prokaryotic cells such as E. coli
is the Rec 7 promoter that is inducible by exogenously supplied nalidixic
acid. A more preferred promoter is present in plasmid vector JHEX25
(available from Promega) that is inducible by exogenously supplied
isopropyl-β-D-thiogalacto-pyranoside (IPTG). A still more preferred
promoter, the tac promoter, is present in plasmid vector pKK223-3 and is
also inducible by exogenously supplied IPTG. The pKK223-3 plasmid can be
successfully expressed in a number of E. coli strains, such as XL-1,
TB1, BL21 and BLR, using about 25 to about 100 μM IPTG for induction.
Surprisingly, concentrations of about 25 to about 50 μM IPTG have been found
to provide optimal results in 2 L shaker flasks and fermentors.
Several strains of Salmonella such as S. typhi and S. typhimurium
and S. typhimurium-E. coli hybrids have been used to
express immunogenic transgenes including prior HBc chimer particles both as
sources of the particles for use as immunogens and as live, attenuated whole
cell vaccines and inocula, and those expression and vaccination systems can
be used herein. See, U.S. Pat. Nos. 6,024,961; 5,888,799; 5,387,744;
5,297,441; Ulrich et al., (1998) Adv. Virus Res., 50:141-182; Tacket
et al., (August 1997) Infect. Immun., 65(8):3381-3385; Schodel et
al., (February 1997) Behring Inst. Mitt., 98:114-119;
Nardelli-Haefliger et al., (December 1996) Infect. Immun.,
64(12):5219-5224; Londono et al., (April 1996) Vaccine,
14(6):545-552, and the citations therein.
Expression vectors compatible with eukaryotic cells, such as those
compatible with yeast cells or those compatible with cells of higher plants
or mammals, are also contemplated herein. Such expression vectors can also
be used to form the recombinant DNA molecules of the present invention.
Vectors for use in yeasts such as S. cerivisiae or Pichia pastoris
can be episomal or integrating, as is well known. Eukaryotic cell
expression vectors are well known in the art and are available from several
commercial sources. Normally, such vectors contain one or more convenient
restriction sites for insertion of the desired DNA segment and promoter
sequences. Optionally, such vectors contain a selectable marker specific for
use in eukaryotic cells. Exemplary promoters for use in S. cerevisiae
include the S. cerevisiae phosphoglyceric acid kinase (PGK) promoter
and the divergent promoters GAL 10 and GAL 1, whereas the alcohol oxidase
gene (AOX1) is a useful promoter for Pichia pastoris.
For example, to produce chimers in the methylotrophic yeast, P. pastoris,
a gene that encodes a desired chimer is placed under the control of
regulatory sequences that direct expression of structural genes in Pichia.
The resultant expression-competent forms of those genes are introduced into
More specifically, the transformation and expression system described by
Cregg et al. (1987) Biotechnology, 5:479-485; (1987) Molecular and
Cellular Biology, 12:3376-3385 can be used. A gene for a chimer
V12.Pf3.1 is placed downstream from the alcohol oxidase gene (AOX1) promoter
and upstream from the transcription terminator sequence of the same AOX1
gene. The gene and its flanking regulatory regions are then introduced into
a plasmid that carries both the P. pastoris HIS4 gene and a P.
pastoris ARS sequence (Autonomously Replicating Sequence), which permit
plasmid replication within P. pastoris cells [Cregg et al. (1987)
Molecular and Cellular Biology, 12:3376-3385].
The vector also contains appropriate portions of a plasmid such as pBR322 to
permit growth of the plasmid in E. coli cells. The resultant plasmid
carrying a chimer gene, as well as the various additional elements described
above, is illustratively transformed into a his4 mutant of P. pastoris;
i.e. cells of a strain lacking a functional histidinol dehydrogenase gene.
After selecting transformant colonies on media lacking histidine, cells are
grown on media lacking histidine, but containing methanol as described Cregg
et al. (1987) Molecular and Cellular Biology, 12:3376-3385, to induce
the AOX1 promoters. The induced AOX1 promoters cause expression of the
chimer protein and the production of chimer particles in P. pastoris.
A contemplated chimer gene can also be introduced by integrative
transformation, which does not require the use of an ARS sequence, as
described by Cregg et al. (1987) Molecular and Cellular Biology,
Production of chimer particles by recombinant DNA expression in mammalian
cells is illustratively carried out using a recombinant DNA vector capable
of expressing the chimer gene in Chinese hamster ovary (CHO) cells. This is
accomplished using procedures that are well known in the art and are
described in more detail in Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratories
In one illustrative example, the simian virus (SV40) based expression
vector, pKSV-10 (Pharmacia Fine Chemicals, Piscataway, N.J.), is subjected
to restriction endonuclease digestion by NcoI and HindIII. A NcoI/HindIII
sequence fragment that encodes the desired HBc chimer prepared as described
in Example 1 is ligated into the expression plasmid, which results in the
formation of a circular recombinant expression plasmid denominated pSV-Pf.
The expression plasmid pSV-Pf contains an intact E. coli ampicillin
resistance gene. E. coli RR101 (Bethesda Research Laboratories,
Gaithersburg, Md.), when transformed with pSV-Pf, can thus be selected on
the basis of ampicillin resistance for those bacteria containing the
plasmid. Plasmid-containing bacteria are then cloned and the clones are
subsequently screened for the proper orientation of the inserted coding gene
into the expression vector.
The above obtained plasmid, pSV-Pf, containing the gene that encodes a
desired HBc chimer is propagated by culturing E. coli containing the
plasmid. The plasmid DNA is isolated from E. coli cultures as
described in Sambrook et al., above.
Expression of a chimer is accomplished by the introduction of pSV-Pf into
the mammalian cell line, e.g., CHO cells, using the calcium
phosphate-mediated transfection method of Graham et al. (1973) Virol.,
52:456, or a similar technique.
To help ensure maximal efficiency in the introduction of pSV-Pf into CHO
cells in culture, the transfection is carried out in the presence of a
second plasmid, pSV2NEO (ATCC #37149) and the cytotoxic drug G418 (GIBCO
Laboratories, Grand Island, N.Y.) as described by Southern et al. (1982)
J. Mol. Appl. Genet., 1:327. Those CHO cells that are resistant to G418
are cultured, have acquired both plasmids, pSV2NEO and pSV-Pf, and are
designated CHO/pSV-Pf cells. By virtue of the genetic architecture of the
pSV-Pf expression vector, a chimer is expressed in the resulting CHO/pSV-Pf
cells and can be detected in and purified from the cytoplasm of these cells.
The resulting composition containing cellular protein is separated on a
column as discussed elsewhere herein.
The choice of which expression vector and ultimately to which promoter a
chimer-encoding gene is operatively linked depends directly on the
functional properties desired, e.g. the location and timing of protein
expression, and the host cell to be transformed. These are well known
limitations inherent in the art of constructing recombinant DNA molecules.
However, a vector useful in practicing the present invention can direct the
replication, and preferably also the expression (for an expression vector)
of the chimer gene included in the DNA segment to which it is operatively
In one preferred embodiment, the host that expresses the chimer is the
prokaryote, E. coli, and a preferred vector includes a prokaryotic
replicon; i.e., a DNA sequence having the ability to direct autonomous
replication and maintenance of the recombinant DNA molecule
extrachromosomally in a prokaryotic host cell transformed therewith. Such
replicons are well known in the art.
Those vectors that include a prokaryotic replicon can also include a
prokaryotic promoter region capable of directing the expression of a
contemplated HBc chimer gene in a host cell, such as E. coli,
transformed therewith. Promoter sequences compatible with bacterial hosts
are typically provided in plasmid vectors containing one or more convenient
restriction sites for insertion of a contemplated DNA segment. Typical of
such vector plasmids are pUC8, pUC9, and pBR329 available from Biorad
Laboratories, (Richmond, Calif.) and pPL and pKK223-3 available from
Pharmacia, Piscataway, N.J.
Typical vectors useful for expression of genes in cells from higher plants
and mammals are well known in the art and include plant vectors derived from
the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens
described by Rogers et al. (1987) Meth. in Enzymol., 153:253-277 and
mammalian expression vectors pKSV-10, above, and pCI-neo (Promega Corp.,
#E1841, Madison, Wis.). However, several other expression vector systems are
known to function in plants including pCaMVCN transfer control vector
described by Fromm et al. (1985) Proc. Natl. Acad. Sci. USA,
82:58-24. Plasmid pCaMVCN (available from Pharmacia, Piscataway, N.J.)
includes the cauliflower mosaic virus CaMV 35S promoter.
The above plant expression systems typically provide systemic or
constitutive expression of an inserted transgene. Systemic expression can be
useful where most or all of a plant is used as the source to a contemplated
chimer molecule or resultant particles or where a large part of the plant is
used to provide an oral vaccine. However, it can be more efficacious to
express a chimer molecule or particles in a plant storage organ such as a
root, seed or fruit from which the particles can be more readily isolated or
One manner of achieving storage organ expression is to use a promoter that
expresses its controlled gene in one or more preselected or predetermined
non-photosynthetic plant organs. Expression in one or more preselected
storage organs with little or no expression in other organs such as roots,
seed or fruit versus leaves or stems is referred to herein as enhanced or
preferential expression. An exemplary promoter that directs expression in
one or more preselected organs as compared to another organ at a ratio of at
least 5:1 is defined herein as an organ-enhanced promoter. Expression in
substantially only one storage organ and substantially no expression in
other storage organs is referred to as organ-specific expression; i.e., a
ratio of expression products in a storage organ relative to another of about
100:1 or greater indicates organ specificity. Storage organ-specific
promoters are thus members of the class of storage organ-enhanced promoters.
Exemplary plant storage organs include the roots of carrots, taro or manioc,
potato tubers, and the meat of fruit such as red guava, passion fruit,
mango, papaya, tomato, avocado, cherry, tangerine, mandarin, palm, melons
such cantaloupe and watermelons and other fleshy fruits such as squash,
cucumbers, mangos, apricots, peaches, as well as the seeds of maize (corn),
soybeans, rice, oil seed rape and the like.
The CaMV 35S promoter is normally deemed to be a constitutive promoter.
However, recent research has shown that a 21-bp region of the CaMV 35S
promoter, when operatively linked into another, heterologous usual green
tissue promoter, the rbcS-3A promoter, can cause the resulting chimeric
promoter to become a root-enhanced promoter. That 21-bp sequence is
disclosed in U.S. Pat. No. 5,023,179. The chimeric rbcS-3A promoter
containing the 21-bp insert of U.S. Pat. No. 5,023,179 is a useful
root-enhanced promoter herein.
A similar root-enhanced promoter, that includes the above 21-bp segment is
the -90 to +8 region of the CAMV 35S promoter itself. U.S. Pat. No.
5,110,732 discloses that that truncated CaMV 35S promoter provides enhanced
expression in roots and the radical of seed, a tissue destined to become a
root. That promoter is also useful herein.
Another useful root-enhanced promoter is the -1616 to -1 promoter of the oil
seed rape (Brassica napus L.) gene disclosed in PCT/GB92/00416 (WO
91/13922 published Sep. 19, 1991). E. coli DH5.alpha. harboring
plasmid pRlambdaS4 and bacteriophage lambda.beta.l that contain this
promoter were deposited at the National Collection of Industrial and Marine
Bacteria, Aberdeen, GB on Mar. 8, 1990 and have accession numbers NCIMB40265
and NCIMB40266. A useful portion of this promoter can be obtained as a 1.0
kb fragment by cleavage of the plasmid with HaeIII.
A preferred root-enhanced promoter is the mannopine synthase (mas) promoter
present in plasmid pKan2 described by DiRita and Gelvin (1987) Mol. Gen.
Genet, 207:233-241. This promoter is removable from its plasmid pKan2 as
a XbaI-XbalI fragment.
The preferred mannopine synthase root-enhanced promoter is comprised of the
core mannopine synthase (mas) promoter region up to position -138 and the
mannopine synthase activator from -318 to -213, and is collectively referred
to as AmasPmas. This promoter has been found to increase production in
tobacco roots about 10- to about 100-fold compared to leaf expression
Another root specific promoter is the about 500 bp 5′ flanking sequence
accompanying the hydroxyproline-rich glycopeprotein gene, HRGPnt3, expressed
during lateral root initiation and reported by Keller et al. (1989) Genes
Dev., 3:1639-1646. Another preferred root-specific promoter is present
in the about -636 to -1 5′ flanking region of the tobacco root-specific gene
ToRBF reported by Yamamoto et al. (1991) Plant Cell, 3:371-381. The
cis-acting elements regulating expression are more specifically located by
those authors in the region from about -636 to about -299 5′ from the
transcription initiation site. Yamamoto et al. reported steady state mRNA
production from the ToRBF gene in roots, but not in leaves, shoot meristems
Still another useful storage organ-specific promoter are the 5′ and 3′
flanking regions of the fruit-ripening gene E8 of the tomato,
Lycopersicon esculentum. These regions and their cDNA sequences are
illustrated and discussed in Deikman et al. (1988) EMBO J.,
7(11):3315-3320 and (1992) Plant Physiol., 100:2013-2017.
Three regions are located in the 2181 bp of the 5′ flanking sequence of the
gene and a 522 bp sequence 3′ to the poly (A) addition site appeared to
control expression of the E8 gene. One region from -2181 to -1088 is
required for activation of E8 gene transcription in unripe fruit by ethylene
and also contributes to transcription during ripening. Two further regions,
-1088 to -863 and -409 to -263, are unable to confer ethylene responsiveness
in unripe fruit but are sufficient for E8 gene expression during ripening.
The maize sucrose synthase-1 (Sh) promoter that in corn expresses its
controlled enzyme at high levels in endosperm, at much reduced levels in
roots and not in green tissues or pollen has been reported to express a
chimeric reporter gene, β-glucuronidase (GUS), specifically in tobacco
phloem cells that are abundant in stems and roots. Yang et al. (1990)
Proc. Natl. Acad. Sci., U.S.A., 87:4144-4148. This promoter is thus
useful for plant organs such as fleshy fruits like melons, e.g. cantaloupe,
or seeds that contain endosperm and for roots that have high levels of
Another exemplary tissue-specific promoter is the lectin promoter, which is
specific for seed tissue. The lectin protein in soybean seeds is encoded by
a single gene (Le1 ) that is only expressed during seed maturation and
accounts for about 2 to about 5 percent of total seed mRNA. The lectin gene
and seed-specific promoter have been fully characterized and used to direct
seed specific expression in transgenic tobacco plants. See, e.g., Vodkin et
al. (1983) Cell, 34:1023 and Lindstrom et al. (1990) Developmental
A particularly preferred tuber-specific expression promoter is the 5′
flanking region of the potato patatin gene. Use of this promoter is
described in Twell et al. (1987) Plant Mol. Biol., 9:365-375. This
promoter is present in an about 406 bp fragment of bacteriophage LPOTI. The
LPOTI promoter has regions of over 90 percent homology with four other
patatin promoters and about 95 percent homology over all 400 bases with
patatin promoter PGT5. Each of these promoters is useful herein. See, also,
Wenzler et al. (1989) Plant Mol. Biol., 12:41-50.
Still further organ-enhanced and organ-specific promoter are disclosed in
Benfey et al. (1988) Science, 244:174-181.
Each of the promoter sequences utilized is substantially unaffected by the
amount of chimer molecule or particles in the cell. As used herein, the term
"substantially unaffected" means that the promoter is not responsive to
direct feedback control (inhibition) by the chimer molecules or particles
accumulated in transformed cells or transgenic plant.
Transfection of plant cells using Agrobacterium tumefaciens is
typically best carried out on dicotyledonous plants. Monocots are usually
most readily transformed by so-called direct gene transfer of protoplasts.
Direct gene transfer is usually carried out by electroportation, by
polyethyleneglycol-mediated transfer or bombardment of cells by
microprojectiles carrying the needed DNA. These methods of transfection are
well-known in the art and need not be further discussed herein. Methods of
regenerating whole plants from transfected cells and protoplasts are also
well-known, as are techniques for obtaining a desired protein from plant
tissues. See, also, U.S. Pat. Nos. 5,618,988 and 5,679,880 and the citations
A transgenic plant formed using Agrobacterium transformation,
electroportation or other methods typically contains a single gene on one
chromosome. Such transgenic plants can be referred to as being heterozygous
for the added gene. However, inasmuch as use of the word "heterozygous"
usually implies the presence of a complementary gene at the same locus of
the second chromosome of a pair of chromosomes, and there is no such gene in
a plant containing one added gene as here, it is believed that a more
accurate name for such a plant is an independent segregant, because the
added, exogenous chimer molecule-encoding gene segregates independently
during mitosis and meiosis. A transgenic plant containing an organ-enhanced
promoter driving a single structural gene that encodes a contemplated HBc
chimeric molecule; i.e., an independent segregant, is a preferred transgenic
More preferred is a transgenic plant that is homozygous for the added
structural gene; i.e., a transgenic plant that contains two added genes, one
gene at the same locus on each chromosome of a chromosome pair. A homozygous
transgenic plant can be obtained by sexually mating (selfing) an independent
segregant transgenic plant that contains a single added gene, germinating
some of the seed produced and analyzing the resulting plants produced for
enhanced chimer particle accumulation relative to a control (native,
non-transgenic) or an independent segregant transgenic plant. A homozygous
transgenic plant exhibits enhanced chimer particle accumulation as compared
to both a native, non-transgenic plant and an independent segregant
It is to be understood that two different transgenic plants can also be
mated to produce offspring that contain two independently segregating added,
exogenous (heterologous) genes. Selfing of appropriate progeny can produce
plants that are homozygous for both added, exogenous genes that encode a
chimeric HBc molecule. Back-crossing to a parental plant and out-crossing
with a non-transgenic plant are also contemplated.
A transgenic plant of this invention thus has a heterologous structural gene
that encodes a contemplated chimeric HBc molecule. A preferred transgenic
plant is an independent segregant for the added heterologous chimeric HBc
structural gene and can transmit that gene to its progeny. A more preferred
transgenic plant is homozygous for the heterologous gene, and transmits that
gene to all of its offspring on sexual mating.
Inasmuch as a gene that encodes a chimeric HBc molecule does not occur
naturally in plants, a contemplated transgenic plant accumulates chimeric
HBc molecule particles in a greater amount than does a non-transformed plant
of the same type or strain when both plants are grown under the same
The phrase "same type" or "same strain" is used herein to mean a plant of
the same cross as or a clone of the untransformed plant. Where alleic
variations among siblings of a cross are small, as with extensively inbred
plant, comparisons between siblings can be used or an average arrived at
using several siblings. Otherwise, clones are preferred for the comparison.
Seed from a transgenic plant is grown in the field greenhouse, window sill
or the like, and resulting sexually mature transgenic plants are
self-pollinated to generate true breeding plants. The progeny from these
plants become true breeding lines that are evaluated for chimeric HBc
molecule particle accumulation, preferably in the field, under a range of
A transgenic plant homozygous for chimeric HBc molecule particle
accumulation is crossed with a parent plant having other desired traits. The
progeny, which are heterozygous or independently segregatable for chimeric
HBc molecule particle accumulation, are backcrossed with one or the other
parent to obtain transgenic plants that exhibit chimeric HBc molecule
particle accumulation and the other desired traits. The backcrossing of
progeny with the parent may have to be repeated more than once to obtain a
transgenic plant that possesses a number of desirable traits.
An insect cell system can also be used to express a HBc chimer. For example,
in one such system Autographa californica nuclear polyhedrosis virus
(AcNPV) or baculovirus is used as a vector to express foreign genes in
Spodoptera frugiperda cells or in Trichoplusia larvae.
The sequences encoding a chimer can be cloned into a non-essential region of
the virus, such as the polyhedrin gene, and placed under control of the
polyhedrin promoter. Successful insertion of chimer sequence renders the
polyhedrin gene inactive and produces recombinant virus lacking coat
protein. The recombinant viruses can then be used to infect, for example,
S. Frugiperda cells or Trichoplusia larvae in which the HBc
chimer can be expressed. E. Engelhard et al. (1994) Proc. Natl. Acad. Sci.,
USA, 91:3224-3227; and V. Luckow, Insect Cell Expression Technology, pp.
183-218, in Protein Engineering: Principles and Practice, J. L.
Cleland et al. eds., Wiley-Liss, Inc, 1996). Heterologous genes placed under
the control of the polyhedrin promoter of the Autographa californica
nuclear polyhedrosis virus (AcNPV) are often expressed at high levels during
the late stages of infection.
Recombinant baculoviruses containing the chimeric gene are constructed using
the baculovirus shuttle vector system (Luckow et al. (1993) J. Virol.,
67:4566-4579], sold commercially as the Bac-To-Bac™ baculovirus expression
system (Life Technologies). Stocks of recombinant viruses are prepared and
expression of the recombinant protein is monitored by standard protocols
(O'Reilly et al., Baculovirus Expression Vectors: A Laboratory Manual,
W. H. Freeman and Company, New York, 1992; and King et al., The
Baculovirus Expression System: A Laboratory Guide, Chapman & Hall,
A variety of methods have been developed to operatively link DNA to vectors
via complementary cohesive termini or blunt ends. For instance,
complementary homopolymer tracts can be added to the DNA segment to be
inserted into the vector DNA. The vector and DNA segment are then joined by
hydrogen bonding between the complementary homopolymeric tails to form
recombinant DNA molecules.
Alternatively, synthetic linkers containing one or more restriction
endonuclease sites can be used to join the DNA segment to the expression
vector, as noted before. The synthetic linkers are attached to blunt-ended
DNA segments by incubating the blunt-ended DNA segments with a large excess
of synthetic linker molecules in the presence of an enzyme that is able to
catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4
Thus, the products of the reaction are DNA segments carrying synthetic
linker sequences at their ends. These DNA segments are then cleaved with the
appropriate restriction endonuclease and ligated into an expression vector
that has been cleaved with an enzyme that produces termini compatible with
those of the synthetic linker. Synthetic linkers containing a variety of
restriction endonuclease sites are commercially available from a number of
sources including New England BioLabs, Beverly, Mass. A desired DNA segment
can also be obtained using PCR technology in which the forward and reverse
primers contain desired restriction sites that can be cut after
amplification so that the gene can be inserted into the vector.
Alternatively PCR products can be directly cloned into vectors containing
T-overhangs (Promega Corp., A3600, Madison, Wis.) as is well known in the
The expressed chimeric protein self-assembles into particles within the host
cells, whether in single cells or in cells within a multicelled host. The
particle-containing cells are harvested using standard procedures, and the
cells are lysed using a French pressure cell, lysozyme, sonicator, bead
beater or a microfluidizer (Microfluidics International Corp., Newton
Mass.). After clarification of the lysate, particles are precipitated with
45% ammonium sulfate, resuspended in 20 mM sodium phosphate, pH 6.8 and
dialyzed against the same buffer. The dialyzed material is clarified by
brief centrifugation and the supernatant subjected to gel filtration
chromatography using Sepharose® CL-4B. Particle-containing fractions are
identified, subjected to hydroxyapatite chromatography, and reprecipitated
with ammonium sulfate prior to resuspension, dialysis and sterile filtration
and storage at -70° C.
Malarial Inocula and Vaccines
A before-described recombinant HBc chimer immunogen preferably in
particulate form is dissolved or dispersed in an immunogenic effective
amount in a pharmaceutically acceptable vehicle composition that is
preferably aqueous to form an inoculum or vaccine. When administered to a
host animal in need of immunization or in which antibodies are desired to be
induced such as a mammal (e.g., a mouse, dog, goat, sheep, horse, bovine,
monkey, ape, or human) or bird (e.g., a chicken, turkey, duck or goose), an
inoculum induces antibodies that immunoreact with the malarial B cell
epitope present in the immunogen. In a vaccine, those induced antibodies
also immunoreact in vivo with (bind to) the sporozoite and protect the
mammal from malarial infection by the Plasmodium species whose B cell
epitope was present in the immunogen. A composition that is an inoculum in
one animal can be a vaccine for another where the Plasmodium species
against which antibodies are raised does not infect the animal inoculated,
as where an inoculum against P. falciparum is used to raise
antibodies in mice.
The amount of recombinant HBc chimer immunogen utilized in each immunization
is referred to as an immunogenic effective amount and can vary widely,
depending inter alia, upon the recombinant HBc chimer immunogen, mammal
immunized, and the presence of an adjuvant in the vaccine, as discussed
below. Immunogenic effective amounts for a vaccine and an inoculum provide
the protection or antibody activity, respectively, discussed hereinbefore.
Vaccines or inocula typically contain a recombinant HBc chimer immunogen
concentration of about 1 microgram to about 1 milligram per inoculation
(unit dose), and preferably about 10 micrograms to about 50 micrograms per
unit dose. Immunizations in mice typically contain 10 or 20 μg of chimer
The term "unit dose" as it pertains to a vaccine or inoculum of the present
invention refers to a physically discrete unit suitable as an unitary dosage
for animals, each unit containing a predetermined quantity of active
material calculated to individually or collectively produce the desired
immunogenic effect in association with the required diluent; i.e., carrier,
or vehicle. A single unit dose or a plurality of unit doses can be used to
provide an immunogenic effective amount of recombinant HBc chimer immunogen.
Vaccines or inocula are typically prepared from a recovered recombinant HBc
chimer immunogen by dispersing the immunogen in a physiologically tolerable
(acceptable) diluent vehicle such as water, saline phosphate-buffered saline
(PBS), acetate-buffered saline (ABS), Ringer's solution or the like to form
an aqueous composition. The diluent vehicle can also include oleaginous
materials such as peanut oil, squalane or squalene as is discussed
The immunogenic active ingredient is often mixed with excipients that are
pharmaceutically acceptable and compatible with the active ingredient.
Suitable excipients are, for example, water, saline, dextrose, glycerol,
ethanol, or the like and combinations thereof. In addition, if desired, an
inoculum or vaccine can contain minor amounts of auxiliary substances such
as wetting or emulsifying agents, pH buffering agents that enhance the
immunogenic effectiveness of the composition.
A contemplated vaccine or inoculum advantageously also includes an adjuvant.
Suitable adjuvants for vaccines and inocula of the present invention
comprise those adjuvants that are capable of enhancing the antibody
responses against B cell epitopes of the chimer, as well as adjuvants
capable of enhancing cell mediated responses towards T cell epitopes
contained in the chimer. Adjuvants are well known in the art (see, for
example, Vaccine Design-The Subunit and Adjuvant Approach,
1995, Pharmaceutical Biotechnology, Volume 6, Eds. Powell, M. F., and
Newman, M. J., Plenum Press, New York and London, ISBN 0-306-44867-X).
Exemplary adjuvants include complete Freund's adjuvant (CFA) that is not
used in humans, incomplete Freund's adjuvant (IFA), squalene, squalane and
alum [e.g., Alhydrogel™ (Superfos, Denmark)], which are materials well known
in the art, and are available commercially from several sources.
Preferred adjuvants for use with immunogens of the present invention include
aluminum or calcium salts (for example hydroxide or phosphate salts). A
particularly preferred adjuvant for use herein is an aluminum hydroxide gel
such as Alhydrogel™. For aluminum hydroxide gels (alum), the chimer protein
is admixed with the adjuvant so that between 50 to 800 micrograms of
aluminum are present per dose, and preferably between 400 and 600 micrograms
Another particularly preferred adjuvant for use with an immunogen of the
present invention is an emulsion. A contemplated emulsion can be an
oil-in-water emulsion or a water-in-oil emulsions. In addition to the
immunogenic chimer protein, such emulsions comprise an oil phase of squalene,
squalane, peanut oil or the like as are well-known, and a dispersing agent.
Non-ionic dispersing agents are preferred and such materials include mono-
and di-C12-C24-fatty acid esters of sorbitan and
mannide such as sorbitan mono-stearate, sorbitan mono-oleate and mannide
mono-oleate. An immunogen-containing emulsion is administered as an
Preferably, such emulsions are water-in-oil emulsions that comprise squalene
and mannide mono-oleate (Arlacel™ A), optionally with squalane, emulsified
with the chimer protein in an aqueous phase. Well-known examples of such
emulsions include Montanide™ ISA-720, and Montanide™ ISA 703 (Seppic,
Castres, France), each of which is understood to contain both squalene and
squalane, with squalene predominating in each, but to a lesser extent in
Montanide™ ISA 703. Most preferably, Montanide™ ISA-720 is used, and a ratio
of oil-to-water of 7:3 (w/w) is used. Other preferred oil-in-water emulsion
adjuvants include those disclosed in WO 95/17210 and EP 0 399 843.
The use of small molecule adjuvants is also contemplated herein. One type of
small molecule adjuvant useful herein is a 7-substituted-8-oxo- or
8-sulfo-guanosine derivative described in U.S. Pat. Nos. 4,539,205,
4,643,992, 5,011,828 and 5,093,318, whose disclosures are incorporated by
reference. Of these materials, 7-allyl-8-oxoguanosine (loxoribine) is
particularly preferred. That molecule has been shown to be particularly
effective in inducing an antigen-(immunogen-)specific response.
Still further useful adjuvants include monophosphoryl lipid A (MPL)
available from Corixa Corp. (see, U.S. Pat. No. 4,987,237), CPG available
from Coley Pharmaceutical Group, QS21 available from Aquila
Biopharmaceuticals, Inc., SBAS2 available from SKB, the so-called muramyl
dipeptide analogues described in U.S. Pat. No. 4,767,842, and MF59 available
from Chiron Corp. (see, U.S. Pat. Nos. 5,709,879 and 6,086,901).
More particularly, immunologically active saponin fractions having adjuvant
activity derived from the bark of the South American tree Quillaja
Saponaria Molina (e.g. Quil™ A) are also useful. Derivatives of Quil™ A,
for example QS21 (an HPLC purified fraction derivative of Quil™ A), and the
method of its production is disclosed in U.S. Pat. No. 5,057,540. In
addition to QS21 (known as QA21), other fractions such as QA17 are also
3-De-O-acylated monophosphoryl lipid A is a well-known adjuvant manufactured
by Ribi Immunochem, Hamilton, Mont. The adjuvant contains three components
extracted from bacteria, monophosphoryl lipid (MPL) A, trehalose dimycolate
(TDM) and cell wall skeleton (CWS) (MPL+TDM+CWS) in a 2% squalene/Tween™ 80
emulsion. This adjuvant can be prepared by the methods taught in GB
2122204B. A preferred form of 3-de-O-acylated monophosphoryl lipid A is in
the form of an emulsion having a small particle size less than 0.2 μm in
diameter (EP 0 689 454 B1).
The muramyl dipeptide adjuvants include N-acetyl-muramyl-L-threonyl-D-
N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP),
(CGP) 1983A, referred to as MTP-PE).
Preferred adjuvant mixtures include combinations of 3D-MPL and QS21 (EP 0
671 948 B1), oil-in-water emulsions comprising 3D-MPL and QS21 (WO 95/17210,
PCT/EP98/05714), 3D-MPL formulated with other carriers (EP 0 689 454 B1),
QS21 formulated in cholesterol-containing liposomes (WO 96/33739), or
immunostimulatory oligonucleotides (WO 96/02555). Alternative adjuvants
include those described in WO 99/52549 and non-particulate suspensions of
polyoxyethylene ether (UK Patent Application No. 9807805.8).
Adjuvants are utilized in an adjuvant amount, which can vary with the
adjuvant, mammal and recombinant HBc chimer immunogen. Typical amounts can
vary from about 1 μg to about 1 mg per immunization. Those skilled in the
art know that appropriate concentrations or amounts can be readily
Inocula and vaccines are conventionally administered parenterally, by
injection, for example, either subcutaneously or intramuscularly. Additional
formulations that are suitable for other modes of administration include
suppositories and, in some cases, oral formulation or by nasal spray. For
suppositories, traditional binders and carriers can include, for example,
polyalkalene glycols or triglycerides; such suppositories may be formed from
mixtures containing the active ingredient in the range of 0.5% to 10%,
preferably 1-2%. Oral formulations include such normally employed excipients
as, for example, pharmaceutical grades of mannitol, lactose, starch,
magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and
An inoculum or vaccine composition takes the form of a solution, suspension,
tablet, pill, capsule, sustained release formulation or powder, and contains
an immunogenic effective amount of HBc chimer, preferably as particles, as
active ingredient. In a typical composition, an immunogenic effective amount
of preferred HBc chimer particles is about 1 μg to about 1 mg of active
ingredient per dose, and more preferably about 5 μg to about 50 μg per dose,
as noted before.
A vaccine or inoculum is typically formulated for parenteral administration.
Exemplary immunizations are carried out sub-cutaneously (SC)
intra-muscularly (IM), intravenusly (IV), intraperitoneally (IP) or intra-dermally
The HBc chimer particles and HBc chimer particle conjugates can be
formulated into the vaccine as neutral or salt forms. Pharmaceutically
acceptable salts, include the acid addition salts (formed with the free
amino groups of the protein or hapten) and are formed with inorganic acids
such as, for example, hydrochloric or phosphoric acids, or such organic
acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with
the free carboxyl groups can also be derived form inorganic bases such as,
for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and
such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol,
histidine, procaine, and the like.
The inocula or vaccines are administered in a manner compatible with the
dosage formulation, and in such amount as are therapeutically effective and
immunogenic. The quantity to be administered depends on the subject to be
treated, capacity of the subject's immune system to synthesize antibodies,
and degree of protection desired. Precise amounts of active ingredient
required to be administered depend on the judgment of the practitioner and
are peculiar to each individual. However, suitable dosage ranges are of the
order of several hundred micrograms active ingredient per individual.
Suitable regimes for initial administration and booster shots are also
variable, but are typified by an initial administration followed in
intervals (weeks or months) by a subsequent injection or other
Once immunized, the mammal is maintained for a period of time sufficient for
the recombinant HBc chimer immunogen to induce the production of a
sufficient titer of antibodies that bind to sporozoite. The maintenance time
for the production of anti-sporozoite antibodies typically lasts for a
period of about three to about twelve weeks, and can include a booster,
second immunizing administration of the vaccine. A third immunization is
also contemplated, if desired, at a time 24 weeks to five years after the
first immunization. It is particularly contemplated that once a protective
level titer of anti-sporozoite antibodies is attained, that the vaccinated
mammal is preferably maintained at or near that antibody titer by periodic
booster immunizations administered at intervals of about 1 to about 5 years.
The production of anti-sporozoite antibodies is readily ascertained by
obtaining a plasma or serum sample from the immunized mammal and assaying
the antibodies therein for their ability to bind to a synthetic
circumsporozoite immunodominant antigen [e.g. the P. falciparum CS
protein peptide (NANP)5 used herein] in an ELISA assay as
described hereinafter or by another immunoassay such as a Western blot as is
well known in the art. Most preferable is the use of the indirect
immunofluorescence assay (IFA), in which intact sporozoites are employed as
the capture antigen, discussed hereinafter.
It is noted that the before-described anti-CS antibodies so induced can be
isolated from the blood of the host mammal using well known techniques, and
then reconstituted into a second vaccine for passive immunization as is also
well known. Similar techniques are used for gamma-globulin immunizations of
humans. For example, antiserum from one or a number of immunized hosts can
be precipitated in aqueous ammonium sulfate (typically at 40-50 percent of
saturation), and the precipitated antibodies purified chromatographically as
by use of affinity chromatography in which (NANP)5 is utilized as
the antigen immobilized on the chromatographic column.
Inocula are preparations that are substantially identical to vaccines, but
are used in a host mammal in which antibodies to malaria are desired to be
induced, but in which protection from malaria is not desired. In one
example, a vaccine against P. falciparum of P. vivax can be
used in mice as an inoculum to induce antibody production and not be a
vaccine because neither malarial species can infect mice. Similarly, a
similar inoculum can be used in a horse or sheep to induce antibody
production against either or both malarial species for use in a passive
immunization in yet another animal such as humans.
Claim 1 of 50 Claims
1. A recombinant hepatitis B virus core (HBc) protein chimer molecule with
a length of about 140 to about 310 amino acid residues that contains four
peptide-linked amino acid residue sequence domains from the N-terminus
that are denominated Domains I, II, III and IV, wherein
(a) Domain I comprises (i) the sequence from position 1 through position
75 of HBc, or (ii) a sequence of about 85 amino acids comprising a
sequence heterologous to HBc peptide-bonded to one of the first five
N-terminal residues of HBc, and including at least the sequence of the
residues of position 5 through position 75 of HBc.
(b) Domain II comprises about 18 to about 58 amino acid residues
peptide-bonded to residue 75 of HBc, including (i) the sequence of
positions 76 through 85 of HBc, and (ii) a sequence of 8 to about 48
residues that constitute a B cell epitope of the circumsporozoite (CS)
protein of the parasite Plasmodium falciparum that is
peptide-bonded between the HBc residues of positions 78 and 79, said B
cell epitope being comprised of two to about five repeats of the amino
acid residue sequence Asn-Ala-Asn-Pro;
(c) Domain III is an HBc sequence from position 86 through position 135
peptide-bonded to residue 85; and
(d) Domain IV comprises a sequence of HBC from residue 136 through 140
peptide-bonded to the residue of position 135 of Domain III and (i) zero
to nine residues of a HBc amino acid residue sequence from position 141
through 149, (ii) zero to three cysteine residue, (iii) fewer than three
arginine or lysine residue, or mixtures thereof adjacent to each other,
and (iv) up to 100 amino acid residues in a sequence heterologous to HBc
from position 150 to the C-terminus, with the proviso that at least five
amino acid residue are present of the amino acid residue sequence from
position 136 through 149, when (a) zero cysteine residues are present and
(b) fewer than about five heterologous amino acid residues are present,
wherein no more than 10 percent of the HBc amino acid residues are
substituted as compared to SEQ ID NO:170 from position 1 through 149.
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