Title: Vaccines for blocking
transmission of plasmodium vivax
United States Patent: 7,407,658
Issued: August 5, 2008
Inventors: Kaslow; David C.
(Wayne, PA), Tsuboi; Takafumi (Ehime, JP), Torii; Motomi (Ehime, JP)
Assignee: The United States
of America as represented by the Secretary of the Department of Health and
Human Services (Washington, DC)
Appl. No.: 11/611,779
Filed: December 15, 2006
Web Seminars -- Pharm/Biotech/etc.
The present invention relates novel
methods and compositions for blocking transmission of Plasmodium vivax
which cause malaria. In particular, Pvs25 and Pvs28 polypeptides,
variants, including deglycosylated forms, and fusion proteins thereof, are
disclosed which, when administered to a susceptible organism, induce an
immune response against a 25 kD and 28 kD protein, respectively, on the
surface of Plasmodium vivax zygotes and ookinetes. This immune response in
the susceptible organism can block transmission of malaria.
Description of the
BRIEF SUMMARY OF THE INVENTION
The present invention relates to methods for preventing transmission of
malaria, particularly Plasmodium vivax. The invention relates to methods for
eliciting an immune response against parasites responsible for malaria.
These methods comprise administering to a susceptible organism a
pharmaceutical composition comprising Pvs28 polypeptides (such as SEQ ID
NO:2), including partially or completely deglycosylated Pvs28 polypeptides,
Pvs25 polypeptides(such as SEQ ID NO:4), variants thereof, or Pvs25/Pvs28
fusion proteins(such as SEQ ID NO:5), in an amount sufficient induce an
immune response against a 25 kD and 28 kD protein, respectively, on the
surface of Plasmodium vivax zygotes and ookinetes. The immune response in
the susceptible organism can block transmission of malaria.
The invention also relates to methods of preventing transmission of malaria
comprising administering to a susceptible organism a pharmaceutical
composition comprising a recombinant virus or expression cassette encoding a
Plasmodium vivax polypeptide, including Pvs28 polypeptides (including
partially or completely deglycosylated Pvs28 polypeptides), Pvs25
polypeptides, or Pvs25/Pvs28 fusion proteins, in an amount sufficient to
block transmission of the disease.
The invention further relates to pharmaceutical compositions comprising a
pharmaceutically acceptable carrier and a Pvs28 polypeptide(including
partially or completely deglycosylated Pvs28 polypeptides), a Pvs25
polypeptide, or a Pvs25/Pvs28 fusion protein, as described herein.
The invention also relates to isolated nucleic acids comprising nucleotide
sequences encoding Pvs28 polypeptides (including partially or completely
deglycosylated Pvs28 polypeptides), Pvs25 polypeptides, or Pvs25/Pvs28
fusion proteins. These nucleic acids may be isolated from, for instance, P.
vivax. The sequences are typically contained in an expression vector for
recombinant expression of the proteins. The sequences can also be
incorporated into recombinant viruses, vectors or expression cassettes for
use as nucleic acid vaccines, including "naked DNA" vaccines, for
recombinant expression of the proteins in vivo. In another embodiment, the
nucleic acids of the invention comprise a pharmaceutical excipient and are
injected into a host, e.g., as "naked" DNA vectors or "expression cassettes"
injected into muscle, to express recombinant protein in vivo, to induce
transmission blocking antibodies against encoded polypeptides.
The invention also provides a composition comprising an isolated nucleic
acid molecule encoding a Plasmodium vivax Pvs28 polypeptide lacking at least
one N-linked glycosylation site. In alternative embodiments, the nucleic
acid encodes a polypeptide comprising a sequence as set forth in SEQ ID
NO:2, excepting that the amino acid residue corresponding to residue 130 of
SEQ ID NO:2 is not an asparagine residue; and the amino acid residue
corresponding to residue 130 of SEQ ID NO:2 is glutamine.
The invention also provides a composition comprising an isolated Plasmodium
vivax Pvs28 polypeptide lacking at least one N-linked glycosylation site. In
alternative embodiments, the polypeptide comprises a sequence as set forth
in SEQ ID NO:2, excepting that the amino acid residue corresponding to
residue 130 of SEQ ID NO:2 is not an asparagine residue; and, the amino acid
residue corresponding to residue 130 of SEQ ID NO:2 is glutamine.
The invention further provides a method of inducing a transmission blocking
immune response in a mammal, comprising administering a partially or
completely deglycosylated Pvs28 polypeptide, or a nucleic acid encoding such
a polypeptide, to a mammal.
Pvs28 (including partially or completely deglycosylated Pvs28) as an
immunogenic carrier is provided for by the invention. Pvs28, administered
with a second composition, provides a superior antigenic response to the
second composition. Thus, the invention relates to an immunogenic
composition capable of eliciting an immunogenic response directed to an
epitope comprising an isolated Pvs28 and an isolated molecule comprising the
epitope. The invention is also directed to methods of eliciting an
immunogenic response directed to an epitope comprising administering an
isolated Pvs28 and an isolated molecule comprising the epitope. The Pvs28
and the second molecule can be chemically linked or joined together as
recombinant fusion proteins.
In one embodiment, the Pvs28-containing fusion protein is a Pvs25-Pvs28
fusion protein. The Pvs28 polypeptide can be designed to be partially or
completely deglycosylated, as described herein. The sexual stage malarial
proteins Pvs25 and Pvs28, in the form of a Pvs25-Pvs28 fusion protein, will
generate transmission-blocking antibodies against both Pvs25 and Pvs28.
These fusion proteins have enhanced antigenic properties, as compared to use
of either alone as an immunogen.
The invention also provides for Pvs25/Pvs28, Pvs25, partially or completely
deglycosylated Pvs28, and Pvs28 fusion proteins, and the nucleic acids
encoding such polypeptides, further comprising non-malarial sequences. For
example, a Pvs25, Pvs28, or Pvs25/Pvs28 polypeptide of the invention can
further comprise epitope tags, enzyme cleavage sequences, leader sequences,
sequences which cause the polypeptides to be transported to a particular
intracellular organelle, and the like. For example, as discussed below,
inclusion of yeast alpha mating pheromone signal sequence in a fusion
protein of the invention allows for secretion of the expressed Pvs25 or
Pvs28. These fusion proteins can provide for simplified manufacturing of
In one class of embodiments, the Pvs25-Pvs28 fusion protein includes an N
terminal Pvs25 domain and a C terminal Pvs28 domain. This arrangement of
Pvs25 and Pvs28 in a fusion protein provides superior antigenic and
transmission blocking properties for the fusion protein. In one preferred
embodiment, the C terminal Pvs28 domain includes the carboxyl terminal
region of Pvs28. Exemplar fusion proteins are provided in the examples set
forth herein, and conservative modifications thereof.
Typically, the Pvs25-Pvs28 fusion proteins of the invention include a
flexible linker separating the Pvs25 and Pvs28 domains. An exemplar flexible
linker is the amino acid sequence GGGPGGG (SEQ ID NO:15).
In one embodiment, the fusion proteins (as Pvs25 and Pvs28) are produced
recombinantly. The recombinant proteins of the invention can be expressed,
e.g., in vitro, in prokaryotic or in eukaryotic systems. In alternative
embodiments, bacterial, yeast, insect, plant, mammalian, or other expression
systems can be used.
In another embodiment, a nucleic acid encoding a fusion protein of the
invention is optimized for expression in a particular expression system,
such as preferred codon usage in bacteria or partial or complete
deglycosylation by mutation for yeast expression, thereby facilitating
recombinant expression and manufacturing of the polypeptide of the
invention. For example, Pvs25 and Pvs28 consist of four epidermal growth
factor-like (EGF) domains (similar domains are found in the related Pfs25
and Pfs28 Plasmodium polypeptides). These EGF domains comprise structural
domains in the molecules. In alternative embodiments, the immunogen includes
one or more domains in a variety of permutations and orientations. As
domains may require disulfide bonds to create and maintain structural
integrity, alternative embodiments encompass various expression systems that
faithfully recreate these disulfide linkages.
In another embodiment, the Pvs25, Pvs28 or Pvs25-Pvs28 fusion protein
sequences can be mutated or altered, e.g., using site-specific mutational
methodologies. For example, in one embodiment, the Pvs25 and Pvs28 sequences
are mutated to eliminate one, several or all potential glycosylation sites.
Such mutations can facilitate recombinant expression and manufacturing of
the polypeptides of the invention, as in yeast expression systems. The
partially or completely deglycosylated Pvs polypeptides of the invention
are, in some circumstances, better immunogens, i.e., administration of these
forms enhance the antigenicity of the polypeptide. For example, in one
embodiment, an amino acid residue at position 130 of Pvs28 is altered to
remove a potential glycosylation site.
In other embodiments, the different domains of the immunogenic composition
are joined, or linked, together by chemical means. In further embodiments,
the domains of the immunogenic compositions are derived from natural
The Pvs25-Pvs28 fusion protein, when administered to a mammal, elicits the
production of at least two classes of antibodies: antibodies which
specifically bind to Pvs25, and antibodies which specifically bind to Pvs28.
In preferred embodiments, the administration of the fusion proteins of the
invention elicit a transmission blocking immune response. Immunological
enhancers and pharmaceutically acceptable carriers are optionally added to
the fusion protein to enhance the immunogenicity of the fusion protein and
to facilitate delivery of the fusion protein to a mammal. For example, in
alternative embodiments, adjuvants such as alum are added.
Immunogenic compositions comprising the fusion proteins of the invention
elicit transmission blocking antibodies in a variety of mammals, including
humans and other primates, and mice and other rodents.
Cells expressing the nucleic acids and polypeptide of the invention are a
feature of the invention. For example, recombinant cells such as yeast cells
can be used to express the Pvs25-Pvs28 fusion protein of the invention. Cell
lines containing a nucleic acid encoding the immunogenic polypeptides and
fusion proteins in an expression vector are also disclosed.
The invention provides methods of inducing a transmission blocking antibody
in a mammal. In the methods, the Pvs25-Pvs28 fusion protein, or a nucleic
acid encoding the fusion protein is administered to a mammal, which produces
transmission blocking antigens. Administration is typically performed
intramuscularly, intradermally, or subcutaneously. An adjuvant such as alum
is optionally administered with the fusion protein or nucleic acid.
The present invention relates to novel compositions and methods for blocking
transmission of malaria, particularly Plasmodium vivax. The invention
provides agents which are capable eliciting antibodies and antiserum
(generated by administration of the compositions of the invention) which,
when ingested by the mosquito, are capable of inhibiting the life cycle of
the disease-causing parasite in the mosquito midgut. The agents include
Plasmodium vivax Pvs 25 and Pvs28 polypeptides (including partially and
completely deglycosylated forms), nucleic acids encoding these polypeptides,
and fusion proteins thereof, that are useful for inducing antibodies that
block transmission of the parasite. The invention also provides the isolated
antibodies generated by these polypeptides. These nucleic acid and
polypeptide compositions are useful as vaccines against malaria.
This invention further relates to an immunogenic composition capable of
eliciting an immunogenic response directed to an epitope comprising an
isolated Pvs25 or Pvs28 and an isolated molecule comprising the epitope. The
invention is also directed to methods of eliciting an immunogenic response
directed to an epitope comprising administering an isolated Pvs28 and an
isolated molecule comprising the epitope. In one embodiment, the Pvs28 is
acting as an immunogenic carrier to a hapten epitope to elicit, stimulate or
augment a humoral immune response to the epitope.
The fusion proteins of the invention (optionally used with an adjuvant such
as alum) can be used to block transmission of a number of parasites
associated with malaria. Examples of parasites whose transmission is blocked
by the materials and compositions of the invention include the causative
parasites for malaria. Four species of the genus Plasmodium infect humans,
P. vivax, P. ovale, P. malariae, and P. falciparum. P. falciparum is the
most prevalent cause of malaria in humans: Other Plasmodium species infect
other animals. For instance, P. gallinaceum is responsible for avian
The present invention relates to recombinant viruses and vaccines comprising
nucleic acid sequences which encode malaria parasite (Plasmodium vivax)
Pvs25 and Pvs28 polypeptides, including fusion proteins and deglycosylated
forms (SEQ ID NOs: 1 to 5). These polypeptides are naturally expressed by
Plasmodium during its mosquito-infective, sexual stage. Because naturally
expressed Pvs polypeptides are expressed in malaria parasite oocytes and
zygotes, recombinant forms can be used to induce an immune response to the
sexual stage of the parasite.
These Pvs25- and Pvs28-expressing malaria parasite sexual stages occur only
in the mosquito host and not in the human. This invention includes
compositions and methods for eliciting human antibodies which, when ingested
by the mosquito during its feeding process, block the development of malaria
in the mosquito. Blocking the sexual development of the malaria parasite in
the mosquito reduces the vector's ability to further transmit the disease to
a second human host.
The human antiserum generated by the compositions and methods of the
invention, when ingested by the mosquito, significantly reduces the numbers
of malaria parasite oocysts within the insect. Significant public health
benefits are attained by the vaccines' ability to elicit antibodies which,
upon mosquito ingestion, significantly decrease the number of oocysts
capable of maturing into infectious sporozoites. A vaccine is still very
useful when it generates an antiserum that decreases the numbers of oocysts
in the mosquito, thus reducing the numbers of parasites transmitted by the
mosquito. To be useful, it is not necessary that the ingested antisera
render the mosquito completely incapable of transmitting the malaria
parasite to a second person (i.e., completely inhibit sexual development of
The use of sexual stage polypeptides as a transmission blocking antigens are
described, e.g., in U.S. Pat. No. 5,217,898 to Kaslow and Barr directed to
Pfs25 as a transmission blocking antigen, and U.S. Pat. No. 5,527,700 to
Kaslow and Duffy, directed to Pfs28 as a transmission blocking antigen.
The Pvs25-Pvs28 fusion proteins of the invention have several surprising
properties. The fusion protein is more efficient in producing transmission
blocking antibodies, e.g., in mice, than Pvs25 or Pvs28 alone. This is true
despite the fact a mixed dose of Pvs25 and Pvs28 will not induce a higher
level of transmission blocking antibody activity than either Pvs25 or Pvs28
alone. Second, less fusion protein is required as an immunogen than either
Pvs25 or Pvs28 alone. Third, titers of transmission blocking antibodies will
remain high for a longer period of time when the antigen is a Pvs25-Pvs28
fusion protein than either Pvs25 or Pvs28 alone. In a preferred aspect, the
invention provides a nucleic acid with yeast preferred codons for encoding
and expressing the fusion protein in yeast.
Pvs28 and Pvs25 Polypeptides
The present invention includes immunogenic Pvs 25 and Pvs28 polypeptides and
fragments derived from these proteins, and partially or completely
deglycosylated forms of these polypeptides, that are useful for inducing an
immune response when the proteins are injected into a human or other host
animal. An exemplary polynucleotide sequence for a Pvs25 of the invention is
shown in SEQ ID NO:3, FIG. 3 (see Original Patent). An exemplary amino acid
sequence for a Pvs25 polypeptide of the invention is shown in SEQ ID NO:4,
FIG. 4 (see Original Patent). An exemplary polynucleotide sequence for a
Pvs28 of the invention is shown in SEQ ID NO:1, FIG. 1 (see Original Patent).
An exemplary amino acid sequence for a Pvs28 polypeptide of the invention is
shown in SEQ ID NO:2, FIG. 2 (see Original Patent).
In another embodiment, the immunogenic composition, comprising an isolated
Pvs28 and an isolated molecule comprising the epitope, is capable of
eliciting or augmenting an immunogenic response directed to the epitope. The
Pvs28 can act as a immunological "carrier" to boost, augment or increase the
cellular or humoral response to the epitope. The antibodies that arise from
the immune response block transmission of the parasite by interfering with
the portion of the parasite's life cycle that occurs in the mosquito. For
example, purified polypeptides having an amino acid sequence substantially
identical to a subsequence of Pvs28 may be used; including partially or
completely deglycosylated forms of Pvs28.
The antibodies or T cells that arise from administration of Pvs28, Pvs25 or
Pvs28-Pvs25 fusion proteins (e.g., as in a polypeptide vaccine, or a vaccine
comprising nucleic acid encoding these polypeptides, such as a virus or
vector) generate an immune response by blocking transmission of the parasite
malaria by interfering with the portion of the parasite's life cycle that
occurs in the mosquito. Pvs 25 and Pvs28 are similar in structure to other
known ookinete antigens such as Pfs25 and Pfs28, respectively. All four
proteins comprise a putative secretory signal sequence, followed by four EGF-like
domains and a terminal hydrophobic transmembrane region without a
cytoplasmic tail. Although the four proteins share the six-cysteine motif of
the EGF-like domains, the functions of these proteins may be very different.
EGF-like domains have been recognized in a range of proteins that have
diverse functions (Davis (1990) New Biol. 2:410-419).
Included among the polypeptides of the present invention are proteins that
are variants of the native proteins constructed by in vitro or in vivo
techniques, including recombinant or synthetic techniques. One skilled in
the art will appreciate, for instance, that for certain uses it would be
advantageous to produce a Pvs25 or a Pvs28 polypeptide that is lacking one
of its structural characteristics. For example, one may remove the
transmembrane domain to obtain a polypeptide that is more soluble in aqueous
Alternatively, the invention provides partially and completely
deglycosylated variants, such as the genetically engineered Pvs28 of the
invention in which the amino acid at position 130 does not encode an
asparagine, and thus cannot be a putative site for N-linked glycosylation.
In an exemplary sequence, the nucleic acid of the invention was modified to
encode glutamine, and the Pvs28 variant polypeptide of the invention was
modified to be glutamine at residue 130. However, any putative amino acid
site of N-- or O-linked glycosylation (and the nucleic acid which encodes
such a site, or motif) can be modified to alternatively be (or encode, in
the case of the nucleic acid) any amino acid residue incapable of acting as
a glycosylation signal.
The Pvs28 and Pvs25 proteins of the invention may be purified from parasites
isolated from infected host organisms. For a review of standard techniques
see, e.g., Methods in Enzymology, "Guide to Protein Purification", M.
Deutscher, ed. Vol. 182 (1990); Scopes, R. K., Protein Purification:
Principles and Practice, 2nd ed., Springer Verlag, (1987). For instance,
Pvs25 and Pvs28 polypeptides can be purified using affinity chromatography,
SDS-PAGE, and the like. Illustrative examples of methods for purifying
Pvs25, Pvs28 and fusion proteins thereof of the invention are described
below. Methods for purifying desired proteins are well known in the art and
are not presented in detail here.
If the protein mixture is complex, an initial salt fractionation can
separate many of the unwanted host cell proteins (or proteins derived from
the cell culture media) from the recombinant protein of interest. The
preferred salt is ammonium sulfate. Ammonium sulfate precipitates proteins
by effectively reducing the amount of water in the protein mixture. Proteins
then precipitate on the basis of their solubility. The more hydrophobic a
protein is, the more likely it is to precipitate at lower ammonium sulfate
concentrations. A typical protocol is to add saturated ammonium sulfate to a
protein solution so that the resultant ammonium sulfate concentration is
between 20-30%. This will precipitate the most hydrophobic of proteins. The
precipitate is discarded (unless the protein of interest is hydrophobic) and
ammonium sulfate is added to the supernatant to a concentration known to
precipitate the protein of interest. The precipitate is then solubilized in
buffer and the excess salt removed if necessary, either through dialysis or
diafiltration. Other methods that rely on solubility of proteins, such as
cold ethanol precipitation, are well known to those of skill in the art and
can be used to fractionate complex protein mixtures.
Size Differential Filtration
If the size of the protein of interest is known or can be estimated from the
cDNA sequence, proteins of greater and lesser size can be removed by
ultrafiltration through membranes of different pore size (for example,
Amicon or Millipore membranes). As a first step, the protein mixture is
ultrafiltered through a membrane with a pore size that has a lower molecular
weight cut-off than the molecular weight of the protein of interest. The
retentate of the ultrafiltration is then ultrafiltered against a membrane
with a molecular cut off greater than the molecular weight of the protein of
interest. The recombinant protein will pass through the membrane into the
filtrate. The filtrate can then be chromatographed.
Proteins can be separated on the basis of their size, net surface charge,
hydrophobicity and affinity for ligands. In addition, antibodies raised
against proteins can be conjugated to column matrices and the proteins
immunopurified. All of these general methods are well known in the art. See
Scopes (1987) supra. Chromatographic techniques can be performed at any
scale and using equipment from many different manufacturers (e.g., Pharmacia
Biotech). Protein concentrations can be determined using any technique,
e.g., as in Bradford (1976) Anal. Biochem. 72:248-257.
Amino Acid Sequence Determination
Illustrative amino acid sequences of the Pvs28 and Pvs25 and fusion proteins
of this invention can be determined by, for example, Edman degradation, a
technique which is well known in the art. In addition to the internal
sequencing (see also Hwang (1996) J. Chromatogr. B. Biomed. Appl.
686:165-175), N-terminal sequencing can be performed by techniques known in
the art. For C-terminal sequence determination, a chemical procedure for the
degradation of peptides and analysis by matrix-assisted-laser-desorption
ionization mass spectrometry (MALDI-MS) can be used Thiede (1997) Eur. J.
Molecular Weight/Isoelectric Point Determination
The molecular weight of a protein can be determined by many different
methods, all known to one of skill in the art. Some methods of determination
include: SDS gel electrophoresis, native gel electrophoresis, molecular
exclusion chromatography, zonal centrifugation, mass spectroscopy, and
calculation from sequencing. Disparity between results of different
techniques can be due to factors inherent in the technique. For example,
native gel electrophoresis, molecular exclusion chromatography and zonal
centrifugation depend on the size of the protein. The proteins that are
cysteine rich can form many disulfide bonds, both intra- and intermolecular.
SDS gel electrophoresis depends on the binding of SDS to amino acids present
in the protein. Some amino acids bind SDS more tightly than others,
therefore, proteins will migrate differently depending on their amino acid
composition. Mass spectroscopy and calculated molecular weight from the
sequence in part depend upon the frequency that particular amino acids are
present in the protein and the molecular weight of the particular amino
acid. If a protein is glycosylated, mass spectroscopy results will reflect
the glycosylation but a calculated molecular weight may not.
The isoelectric point of a protein can be determined by native gel (or disc)
electrophoresis, isoelectric focussing or in a preferred method, by
calculation given the amino acid content of the protein (see, for example,
Wehr (1996) Methods Enzymol. 270:358-374; Moorhouse (1995) J. Chromatogr. A.
717:61-69, describing capillary isoelectric focusing).
Pvs25-Pvs28 Fusion Proteins
The present invention includes immunogenic polypeptides which comprise
polypeptide subsequences derived from both Pvs28 and Pvs25, including the
exemplary fusion protein of the invention SEQ ID NO:5 (see FIG. 5 (see Original Patent))
and deglycosylated forms. These polypeptides are useful for inducing an
immune response when the fusion protein is injected into a human, mouse or
other host animal. The antibodies that arise from the immune response block
transmission of the malarial parasite by interfering with the portion of the
parasite's life cycle that occurs in the mosquito.
The fusion proteins typically include an immunogenic domain, or epitope,
from a Pvs25 and an immunogenic domain, or epitope from a Pvs28 (including
deglycosylated forms). The immunogenic domains, or epitopes, are peptide and
polypeptide subsequences of the corresponding polypeptides which are
sufficient to elicit an immunogenic response (antibody or T cell response)
against the domain when administered to a mammal (e.g., a mouse or a human).
In one embodiment, the immunogenic domain can elicit the production of an
antibody which recognizes the corresponding full length protein. For
example, if the immunogenic domain is a Pvs25 subsequence, the domain (epitope)
elicits the production of an antibody which specifically binds to Pvs25.
Similarly, if the immunogenic domain is a Pvs28 subsequence, the domain
preferably elicits the production of an antibody which specifically binds to
To elicit the production of an antibody, the immunogenic domain is typically
at least about 3-10 amino acids in length, because the protein recognition
site on an antibody typically recognizes an amino acid of about 3-10 amino
acids in length. More often, the immunogenic domain is longer than 10 amino
acids, and the domain optionally includes the full length sequence of the
corresponding protein (i.e., in one embodiment, the Pvs25-Pvs28 fusion
protein comprises the complete sequence of both Pvs25 and Pvs28).
Ordinarily, only a fraction of the full length protein is included. In one
embodiment, about 10% of the full length Pvs25 is included in the fusion
protein. In another embodiment, about 20% of the full length Pvs25 is
included in the fusion protein. In yet another embodiment, about 30% of the
full length protein is included. In still another embodiment, about 40% of
the full length Pvs25 is included in the fusion protein. Optionally, as much
as about 50% of the full length Pvs25 is included in the fusion protein.
Occasionally, as much as about 60% of the full length Pvs25 is included in
the fusion protein. In some embodiments, as much as about 70% of the full
length Pvs25 is included in the fusion protein. In one class of embodiments,
as much as about 80% of the full length Pvs25 is included in the fusion
protein. As much as about 90% of the full length Pvs25 is optionally
included in the fusion protein. As already mentioned, the entire full length
Pvs25 protein is optionally incorporated into the fusion protein.
Similarly, in one embodiment, about 10% of the full length Pvs28is included
in the fusion protein. In another embodiment, about 20% of the full length
Pvs28 is included in the fusion protein. In yet another embodiment, about
30% of the full length protein is included. In still another embodiment,
about 40% of the full length Pvs28 is included in the fusion protein.
Optionally, as much as about 50% of the full length Pvs28 is included in the
fusion protein. Occasionally, as much as about 60% of the full length Pvs28
is included in the fusion protein. In some embodiments, as much as about 70%
of the full length Pvs28 is included in the fusion protein. In one class of
embodiments, as much as about 80% of the full length Pvs28 is included in
the fusion protein. As much as about 90% of the full length Pvs28 is
optionally included in the fusion protein. As already mentioned, the entire
full length Pvs28 protein is optionally incorporated into the fusion
The portion of the Pvs25 or Pvs28 protein from which the immunogenic domain,
or epitope, is selected is optionally optimized for maximum immunogenicity
for the induction of transmission blocking vaccines. Any combination of
Pvs25 and Pvs28 subsequences (epitopes) can be combined. Any combination of
complete or partially deglycosylated subsequences can be combined. In
alternative embodiments, the Pvs25 and Pvs28 epitopes can be in alternating
or sequential patterns. For example, in one embodiment, the carboxyl
terminal portion of Pvs28 is included. Embodiments also include those
derived from fusion proteins in which about 10-20 amino acids are deleted or
added to the particular Pvs25 or Pvs28 subsequences described. The added or
deleted amino acids are added or deleted by reference to the corresponding
full length sequence, e.g., where the subsequence is derived from Pvs25, a
10-20 amino acid sequence derived from Pvs25 is optionally added to either
end of the subsequence.
The fusion proteins optionally includes additional features such as a
flexible linker between Pvs25 and Pvs28 domains. The linkers can facilitate
the independent folding of the Pvs25 and Pvs28 proteins. Preferred flexible
linkers are amino acid subsequences which are synthesized as part of a
recombinant fusion protein. In one embodiment, the flexible linker is an
amino acid subsequence comprising a proline such as Gly.sub.3-Pro-Gly.sub.3
(SEQ ID NO:15). In other embodiments, a chemical linker is used to connect
synthetically or recombinantly produced Pvs25 and Pvs28 subsequences. Such
flexible linkers are known to persons of skill in the art. For example,
poly(ethylene glycol) linkers are available from Shearwater Polymers, Inc.
Huntsville, Ala. These linkers optionally have amide linkages, sulfhydryl
linkages, or heterofunctional linkages.
In addition to flexible linkers, the fusion proteins optionally include
polypeptide subsequences from proteins which are unrelated to Pvs25 or
Pvs28, e.g., a sequence with affinity to a known antibody to facilitate
affinity purification, detection, or the like. Such detection- and
purification-facilitating domains include, but are not limited to, metal
chelating peptides such as polyhistidine tracts and histidine-tryptophan
modules that allow purification on immobilized metals, protein A domains
that allow purification on immobilized immunoglobulin, and the domain
utilized in the FLAGS extension/affinity purification system (Immunex Corp,
Seattle Wash.). The inclusion of a cleavable linker sequences such as Factor
Xa or enterokinase (Invitrogen, San Diego Calif.) between the purification
domain and Pvs25 or Pvs28 protein(s) may be useful to facilitate
purification. One such expression vector provides for expression of a fusion
protein comprising the sequence encoding a Pvs25 or Pvs28 of the invention,
or a fusion protein thereof, and nucleic acid sequence encoding six
histidine residues followed by thioredoxin and an enterokinase cleavage site
(for example, see Williams (1995) Biochemistry 34:1787-1797). The histidine
residues facilitate detection and purification while the enterokinase
cleavage site provides a means for purifying the desired protein(s) from the
remainder of the fusion protein. Technology pertaining to vectors encoding
fusion proteins and application of fusion proteins are well described in the
patent and scientific literature, see e.g., Kroll (1993) DNA Cell. Biol.,
An exemplary fusion of Pvs25 to Pvs28 by a flexible linker is represented by
the polypeptide of FIG. 5, SEQ ID NO:5, whose individual domains are:
a Pvs25 sequence (with or without a signal sequence or anchor)
-- see Original Patent.
with a flexible linker, e.g.:
GGGPGGG (SEQ ID NO:15); and
a Pvs28 sequence (with or without signal sequence or anchor)
-- see Original Patent.
The fusion protein (and a Pvs25 or Pvs28 polypeptide) can also include a
secretory signal sequence, e.g., in mammalian cell expression: Ig secretion
signal or tPA signal sequence; or a pre-pro secretion signal, e.g., in
Included among the polypeptides of the present invention are fusion proteins
that have subsequences which are homologues or allelic variants of Pvs28 or
Pvs25. Such homologues, also referred to as Pvs28 or Pvs25 polypeptides,
respectively, include variants of the native proteins constructed by in
vitro techniques, and proteins from parasites related to P. vivax and P.
falciparum. For example, one skilled in the art will appreciate that for
certain uses it is advantageous to produce a Pvs28 or Pvs25 polypeptide
subsequence that is lacking a structural characteristic; e.g., one may
remove a transmembrane domain (to obtain a polypeptide that is more soluble
in aqueous solution) or a glycosylation site (to obtain a polpeptide that is
more antigenic under certain conditions).
One of skill will appreciate that many conservative variations of the fusion
proteins and nucleic acid which encode the fusion proteins yield essentially
identical products. For example, due to the degeneracy of the genetic code,
"silent substitutions" (i.e., substitutions of a nucleic acid sequence which
do not result in an alteration in an encoded polypeptide) are an implied
feature of every nucleic acid sequence which encodes an amino acid. As
described herein, sequences are preferably optimized for expression in a
particular host cell used to produce the fusion protein (e.g., yeast).
Similarly, "conservative amino acid substitutions," in one or a few amino
acids in an amino acid sequence are substituted with different amino acids
with highly similar properties (see, the definitions section, supra), are
also readily identified as being highly similar to a particular amino acid
sequence, or to a particular nucleic acid sequence which encodes an amino
acid. Such conservatively substituted variations of any particular sequence
are a feature of the present invention.
One of skill will recognize many ways of generating alterations in a given
nucleic acid sequence, which optionally provides alterations to an encoded
protein. Such well-known methods include site-directed mutagenesis, PCR
amplification using degenerate oligonucleotides, exposure of cells
containing the nucleic acid to mutagenic agents or radiation, chemical
synthesis of a desired oligonucleotide (e.g., in conjunction with ligation
and/or cloning to generate large nucleic acids) and other well-known
techniques. See, Giliman and Smith (1979) Gene 8:81-97; Roberts et al.
(1987) Nature 328:731-734 and Sambrook, Innis, Ausbel, Berger, Needham
VanDevanter and Mullis (below).
Most commonly, amino acid sequences are altered by altering the
corresponding nucleic acid sequence and expressing the polypeptide. However,
polypeptide sequences are also optionally generated synthetically on
commercially available peptide synthesizers to produce any desired
polypeptide (see, Merrifield, and Stewart and Young, supra).
One can select a desired nucleic acid or polypeptide of the invention based
upon the sequences and constructs provided and upon knowledge in the art
regarding malaria generally. The life-cycle, genomic organization,
developmental regulation and associated molecular biology of malaria strains
have been the focus of research since the advent of molecular biology.
Moreover, general knowledge regarding the nature of proteins and nucleic
acids allows one of skill to select appropriate sequences with activity
similar or equivalent to the nucleic acids, vectors and polypeptides
disclosed herein. The definitions section herein describes exemplar
conservative amino acid substitutions.
Finally, most modifications to nucleic acids and polypeptides are evaluated
by routine screening techniques in suitable assays for the desired
characteristic. For instance, changes in the immunological character of a
polypeptide can be detected by an appropriate immunological assay.
Modifications of other properties such as nucleic acid hybridization to a
target nucleic acid, redox or thermal stability of a protein, hydrophobicity,
susceptibility to proteolysis, or the tendency to aggregate are all assayed
according to standard techniques.
Pvs25 Pvs28 and Pvs25-Pvs28 Nucleic Acids
Another aspect of the present invention relates to the cloning and
recombinant expression (using expression cassettes, plasmids, vectors,
recombinant viruses, and the like) of Pvs 25 and Pv28 proteins, variants
(i.e. deglycosylated forms) construction of Pvs25-Pvs28 fusion proteins, as
described above. The recombinantly expressed proteins can be used in a
number of ways. For instance, they can be used as transmission-blocking
vaccines or as immunogens to raise antibodies, as described below. In
addition, oligonucleotides from the cloned genes can be used as probes to
identify homologous, allelic and variant species of Pvs polypeptides in
Plasmodium vivax, Plasmodium sp., and in other species.
Thus, the invention relies on routine techniques in the field of recombinant
genetics, well known to those of ordinary skill in the art and well
described in the scientific and patent literature, e.g., basic texts
disclosing the general methods of use in this invention include Berger and
Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume
152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al.,
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Publish., Cold
Spring Harbor, NY 2nd ed. (1989) (Sambrook); and Current Protocols in
Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, Greene
Publishing Associates, Inc. and John Wiley & Sons, Inc. (1995 Supplement).
Product information from manufacturers of biological reagents and
experimental equipment also provide information useful in known biological
methods. Such manufacturers include, e.g., the SIGMA chemical company (Saint
Louis, MO), R&D systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology
(Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem
Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), Glen Research,
Inc., GIBCO BRL Life Technologies, Inc. (Gaithersberg, Md.), Fluka
Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),
Invitrogen, San Diego, Calif., and Applied Biosystems (Foster City, Calif.),
as well as many other commercial sources known to one of skill.
In summary, the manipulations necessary to prepare nucleic acid segments
encoding the polypeptides and introduce them into appropriate host cells
involve 1) purifying the polypeptide from the appropriate sources, 2)
preparing degenerate oligonucleotide probes corresponding to a portion of
the amino acid sequence of the purified proteins, 3) screening a cDNA or
genomic library for the sequences which hybridize to the probes, 4)
constructing vectors comprising the sequences linked to a promoter and other
sequences necessary for expression and 5) inserting the vectors into
suitable host cells or viruses.
After isolation of the desired protein as described above, the amino acid
sequence of the N-terminus is determined and degenerate oligonucleotide
probes, designed to hybridize to the desired gene, are synthesized. Amino
acid sequencing is performed and oligonucleotide probes are synthesized
according to standard techniques as described, e.g., in Sambrook or Ausubel.
Genomic or cDNA libraries are prepared according to standard techniques as
described, e.g., in Sambrook or Ausubel. To construct genomic libraries,
large segments of genomic DNA are generated by random fragmentation and are
ligated with vector DNA to form concatemers that can be packaged into the
appropriate vector. Two kinds of vectors are commonly used for this purpose,
bacteriophage lambda vectors and plasmids.
To prepare cDNA, mRNA from the parasite of interest is first isolated.
Eukaryotic mRNA has at its 3' end a string of adenine nucleotide residues
known as the poly-A tail. Short chains of oligo d-T nucleotides are then
hybridized with the poly-A tails and serve as a primer for the enzyme,
reverse transcriptase. This enzyme uses RNA as a template to synthesize a
complementary DNA (cDNA) strand. A second DNA strand is then synthesized
using the first cDNA strand as a template. Linkers are added to the
double-stranded cDNA for insertion into a plasmid or phage vector for
propagation in E. coli.
cDNA can also be prepared using PCR (see below for further discussion PCR).
PCR is used to produce high-quality cDNA from nanograms of total or poly A+
RNA. For example, the CapFinder.TM. PCR cDNA Synthesis Kit (Clonetech, Palo
Alto, Calif.) was used to identify and isolate cDNA from Plasmodium. This
technique utilizes long-distance PCR (Barnes (1994) Proc. Natl. Acad. Sci.
USA 91:2216-2220, Cheng (1994) Proc. Natl. Acad. Sci. USA 91:5695-5699) to
generate high yields of representative, double-stranded cDNA. See also,
e.g., Zhu (July 1996) CLONTECHniques XI(3):12-13; CLONTECHniques (October
1995) X(4):2-5; and CLONTECHniques (January 1996) XI(1):2-4.
Identification of clones in either genomic or cDNA libraries harboring the
desired nucleic acid segments is performed by either nucleic acid
hybridization or immunological detection of the encoded protein, if an
expression vector is used. The bacterial colonies are then replica plated on
solid support, such as nitrocellulose filters. The cells are lysed and
probed with either oligonucleotide probes described above or with antibodies
to the desired protein.
Other methods well known to those skilled in the art are used to identify
desired genes, i.e., various species of Pvs25 and Pvs28 of the invention.
For example, the presence of restriction fragment length polymorphisms (RFLP)
between wild type and mutant strains lacking a Pvs25 or Pvs28 polypeptide
can be used.
Oligonucleotides can be used to identify and detect Pvs25 and Pvs28 using a
variety of hybridization techniques and conditions. For example,
amplification techniques, such as the polymerase chain reaction (PCR) can be
used to amplify the desired nucleotide sequence. One of skill in the art
will appreciate that, whatever amplification method is used, if a
quantitative result is desired, care must be taken to use a method that
maintains or controls for the relative frequencies of the amplified nucleic
acids. Suitable amplification methods include, but are not limited to:
polymerase chain reaction, PCR (PCR PROTOCOLS, A GUIDE TO METHODS AND
APPLICATIONS, ed. Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES
(1995), ed. Innis, Academic Press, Inc., NY ("Innis "); and, U.S. Pat. Nos.
4,683,195 and 4,683,202 describe this method), ligase chain reaction (LCR)
(Wu (1989) Genomics 4:560; Landegren (1988) Science 241:1077; Barringer
(1990) Gene 89:117); transcription amplification (Kwoh Proc. Natl. Acad. Sci.
USA, 86:1173 (1989)); and, self-sustained sequence replication (Guatelli
(1990) Proc. Natl. Acad. Sci. USA, 87:1874); Q Beta replicase amplification
(Smith (1997) J. Clin. Microbiol. 35:1477-1491, automated Q-beta replicase
amplification assay; Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA
polymerase mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario);
see also Berger (1987) Methods Enzymol. 152:307-316, Sambrook, and Ausubel,
as well as Mullis (1987) U.S. Pat. Nos. 4,683,195 and 4,683,202; Arnheim
(1990) C&EN 36-47; Lomell J. Clin. Chem., 35:1826 (1989); Van Brunt,
Biotechnology, 8:291-294 (1990); Wu (1989) Gene 4:560; Sooknanan (1995)
Biotechnology 13:563-564. Methods for cloning in vitro amplified nucleic
acids are described in Wallace, U.S. Pat. No. 5,426,039.
The invention provides for amplification and manipulation or detection of
the products from each of the above methods to prepare DNA encoding a Pvs25
or Pvs28 protein specie. In PCR techniques, oligonucleotide primers
complementary to the two borders of the DNA region to be amplified are
synthesized an used (see, e.g., Innis). PCR can be used in a variety of
protocols to amplify, identify, isolate and manipulate nucleic acids
encoding Pvs25 or Pvs 28. In these protocols, appropriate primers and probes
for identifying and amplifying DNA encoding Pvs25 or Pvs 28 polypeptides and
fragments thereof are generated that comprise all or a portion of any of the
DNA sequences listed herein. PCR-amplified sequences can also be labeled and
used as detectable oligonucleotide probes, but such nucleic acid probes can
be generated using any synthetic or other technique well known in the art,
as described above. The labeled amplified DNA or other oligonucleotide or
nucleic acid of the invention can be used as probes to further identify and
isolate Pvs25 or Pvs 28 protein isoforms or alleles or Pvs25 or Pvs 28 from
various cDNA or genomic libraries.
The present invention also provides RACE-based methods for isolating Pvs25
or Pvs 28 nucleic acids from any organism (RACE is another PCR-based
approach for DNA amplification). Briefly, this technique involves using PCR
to amplify a DNA sequence using a random 5' primer and a defined 3' primer
(5' RACE) or a random 3' primer and a defined 5' primer (3' RACE). The
amplified sequence is then subcloned into a vector where can be sequenced
and manipulated using standard techniques. The RACE method is well known to
those of skill in the art and kits to perform RACE are commercially
available, e.g. Gibco BRL, Gaithersburg, Md., #18374-058 (5' RACE) or
#18373-019 (3' RACE), see also Lankiewicz (1997) Nucleic Acids Res
25:2037-2038; Frohman (1988) Proc. Natl. Acad. Sci. USA 85:8998; Doenecke
(1997) Leukemia 11:1787-1792.
For 5' RACE, a primer, the gene-specific primer, is selected near the 5' end
of the known sequence oriented to extend towards the 5' end. The primer is
used in a primer extension reaction using a reverse transcriptase and mRNA.
After the RNA is optionally removed, the specifically-primed cDNA is either:
1) "tailed" with deoxynucleotide triphosphates (dNTP) and dideoxyterminal
transferase, then a primer that is complimentary to the tail with a 5' end
that provides a unique PCR site and the first gene-specific primer is used
to PCR amplify the cDNA. Subsequent amplifications are usually performed
with a gene-specific primer nested with respect to the first primer, or 2)
an oligonucleotide that provides a unique PCR site is ligated to an end of
the cDNA using RNA ligase; then a primer complimentary to the added site and
the first gene-specific primer is used to PCR amplify the cDNA, with
subsequent amplifications usually performed with a gene-specific primer
nested with respect to the first primer. Amplified products are then
purified, usually by gel electrophoresis then sequenced and examined to see
contain the additional cDNA sequences desired.
For 3' RACE, an oligo dT-primer is annealed to the poly-A tails of an mRNA
and then extended by a reverse transcriptase. Usually the oligo dT primer
has a 5' end that provides a unique PCR site. The RNA is then removed,
optionally, or dissociated, and the cDNA is amplified with a primer to the
oligo dT tail and a gene-specific primer near the 3' end of the known
sequence (oriented towards the 3' end). Subsequent amplifications are
usually performed with a gene-specific primer nested with respect to the
first primer. Amplified products are then purified, usually by gel
electrophoresis then sequenced and examined to see contain the additional
cDNA sequences desired.
Sequences amplified by PCR can be purified from agarose gels and cloned into
an appropriate vector according to standard techniques.
Standard transfection methods are used to produce prokaryotic, mammalian,
yeast or insect cell lines which express large quantities of the Pvs25 or
Pvs 28 polypeptide, which is then purified using standard techniques, as
described above. See, e.g., Colley (1989) J. Biol. Chem. 264:17619-17622;
and Scopes, supra.
The polypeptides of the present invention can be readily designed and
manufactured utilizing various recombinant DNA or synthetic techniques well
known to those skilled in the art. For example, the polypeptides can vary
from the naturally-occurring sequence at the primary structure level by
amino acid, insertions, substitutions, deletions, and the like. These
modifications can be used in a number of combinations to produce the final
modified protein chain.
The amino acid sequence variants can be prepared with various objectives in
mind, including immunogenicity, facilitating purification, and preparation
of the recombinant polypeptide. Design of completely or partially
deglycosylated polypeptides improve the antigenicity of the immunogenic
composition, as discussed above. Modified polypeptides can also be useful
for modifying plasma half life, improving therapeutic efficacy, and
lessening the severity or occurrence of side effects during therapeutic use.
The amino acid sequence variants are usually predetermined variants not
found in nature but exhibit the same, or improved (in the case of
deglycosylation variants) immunogenic activity as naturally occurring, Pvs25
and Pvs28 polypeptides. For instance, polypeptide fragments comprising only
a portion (usually at least about 60-80%, typically 90-95%) of the primary
structure may be produced. For use as vaccines, polypeptide fragments are
typically preferred so long as at least one epitope capable of eliciting
transmission blocking antibodies remains. In the construction of
deglycosylation variants, amino acid motifs which act as N-linked or
O-linked glycosylation signals (which are well known in the art, see, e.g.,
Kakinuma (1997) J Biol Chem 272:28296-28300) are modified to forms (motif
variants) that are not recognized as glycosylation sites in the expression
systems in which the recombinant form is produced.
The nucleotide sequences used to express the polypeptides of the invention
and to transfect the host cells can be modified according to standard
techniques to yield Pvs25-Pvs28, Pvs25 or Pvs28 polypeptides, fusion
proteins, variants or fragments thereof, with a variety of desired
properties. For example, the invention also provides for Pvs25 and Pvs28
which have been modified in a site-specific manner to modify or delete any
or all functions or epitopes. Site-specific mutations can be introduced into
Pvs25 and Pvs28-encoding nucleic acid by a variety of conventional
techniques, well described in the scientific and patent literature. For
example, one rapid method to perform site-directed mutagenesis efficiently
is the overlap extension polymerase chain reaction (OE-PCR) (Urban (1997)
Nucleic Acids Res. 25 :2227-2228). Other illustrative examples include:
site-directed mutagenesis by overlap extension polymerase chain reaction (OE-PCR),
as in Urban (1997) Nucleic Acids Res. 25:2227-2228; Ke (1997) Nucleic Acids
Res 25:3371-3372, and Chattopadhyay (1997) Biotechniques 22:1054-1056,
describing PCR-based site-directed mutagenesis "megaprimer" method; Bohnsack
(1997) Mol. Biotechnol. 7:181-188; Ailenberg (1997) Biotechniques
22:624-626, describing site-directed mutagenesis using a PCR-based staggered
re-annealing method without restriction enzymes; Nicolas (1997)
Biotechniques 22:430-434, site-directed mutagenesis using long primer-unique
site elimination and exonuclease III. See Gillman (1979) Gene 8:81-97;
Roberts (1987) Nature 328:731-734.
In general, modifications of the sequences encoding the homologous
polypeptides may be readily accomplished by a variety of well-known
techniques, such as site-directed mutagenesis, described above. One of
ordinary skill will appreciate that the effect of many mutations is
difficult to predict. Thus, most modifications are evaluated by routine
screening in a suitable assay for the desired characteristic. For instance,
the effect of various modifications on the ability of the polypeptide to
elicit transmission blocking can be easily determined using the mosquito
feeding assays, described below. In addition, changes in the immunological
character of the polypeptide can be detected by an appropriate competitive
binding assay. Modifications of other properties such as redox or thermal
stability, hydrophobicity, susceptibility to proteolysis, or the tendency to
aggregate are all assayed according to standard techniques.
The particular procedure used to introduce the genetic material into the
host cell for expression of the Pvs 25 and Pvs28 polypeptide is not
particularly critical. Any of the well known procedures for introducing
foreign nucleotide sequences into host cells may be used. These include the
use of calcium phosphate transfection, spheroplasts, electroporation,
liposomes, microinjection, plasma vectors, viral vectors and any of the
other well known methods for introducing cloned genomic DNA, cDNA, synthetic
DNA or other foreign genetic material into a host cell (see, e.g., Sambrook,
Ausubel, supra). It is only necessary that the particular procedure utilized
be capable of successfully introducing at least one gene into the host cell
which is capable of expressing the gene.
The particular vector used to transport the genetic information into the
cell is also not particularly critical. Any of the conventional vectors used
for expression of recombinant proteins in prokaryotic and eukaryotic cells
may be used.
Expression vectors for mammalian cells typically contain regulatory elements
from eukaryotic viruses. SV40 vectors include pSVT7 and pMT2. Vectors
derived from bovine papilloma virus include pBV-1MTHA, and vectors derived
from Epstein Bar virus include pHEBO, and p2O5.
Other exemplary vectors include pMSG, pAV009/A.sup.+, pMTO10/A.sup.+,
pMAMneo-5, bacculovirus pDSVE, and any other vector allowing expression of
proteins under the direction of the SV-40 early promoter, SV-40 later
promoter, metallothionein promoter, murine mammary tumor virus promoter,
Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown
effective for expression in eukaryotic cells.
The expression vector typically contains a transcription unit or expression
cassette that contains all the elements required for the expression of the
Pvs28 or Pvs25 polypeptide DNA in the host cells. A typical expression
cassette contains a promoter operably linked to the DNA sequence encoding a
Pvs28 or Pvs25 polypeptide and signals required for efficient
polyadenylation of the transcript. The term "operably linked" as used herein
refers to linkage of a promoter upstream from a DNA sequence such that the
promoter mediates transcription of the DNA sequence. The promoter is
preferably positioned about the same distance from the heterologous
transcription start site as it is from the transcription start site in its
natural setting. As is known in the art, however, some variation in this
distance can be accommodated without loss of promoter function.
The DNA sequence encoding the Pvs28 or Pvs25 polypeptide will typically be
linked to a cleavable signal peptide sequence to promote secretion of the
encoded protein by the transformed cell. Additional elements of the cassette
may include selectable markers, enhancers and, if genomic DNA is used as the
structural gene, introns with functional splice donor and acceptor sites.
Enhancer elements can stimulate transcription up to 1,000 fold from linked
homologous or heterologous promoters. Enhancers are active when placed
downstream from the transcription initiation site. Many enhancer elements
derived from viruses have a broad host range and are active in a variety of
tissues. For example, the SV40 early gene enhancer is suitable for many cell
types. Other enhancer/promoter combinations that are suitable for the
present invention include those derived from polyoma virus, human or murine
cytomegalovirus, the long term repeat from various retroviruses such as
murine leukemia virus, murine or Rous sarcoma virus and HIV. See, Enhancers
and Eukaryotic Expression, Cold Spring Harbor Pres, Cold Spring Harbor, N.Y.
In addition to a promoter sequence, the expression cassette should also
contain a transcription termination region downstream of the structural gene
to provide for efficient termination. The termination region may be obtained
from the same gene as the promoter sequence or may be obtained from
If the mRNA encoded by the structural gene is to be efficiently translated,
polyadenylation sequences are also commonly added to the vector construct.
Two distinct sequence elements are required for accurate and efficient
polyadenylation: GU or U rich sequences located downstream from the
polyadenylation site and a highly conserved sequence of six nucleotides,
AAUAAA, located 11-30 nucleotides upstream. Termination and polyadenylation
signals that are suitable for the present invention include those derived
from SV40, or a partial genomic copy of a gene already resident on the
Pvs25 or Pvs28 coding sequences can be inserted into a host cell genome
becoming an integral part of the host chromosomal DNA, using for example,
retroviral vectors such as SIV or HIV, see for example, Naldini (1996)
Science 272:263-267; Vanin (1997) J. Virol. 71:7820-7826; Zufferey (1997)
Nat. Biotechnol. 15:871-875, describing attenuated lentiviral vector gene
delivery in vivo; Feng (1997) Nat. Biotechnol. 15:866-870, describing stable
in vivo gene transduction via adenoviral/retroviral chimeric vector.
Nucleic acids of the invention can used in DNA immunization techniques.
Coding sequence is operably linked to expression cassettes or vectors and
injected directly as "naked" DNA into the host. The DNA can be injected
intramuscularly or intradermally. See. e.g., Donnelly (1995) Ann. NY Acad.
Sci. 772:40-46; Corr (1997) J. Immunol. 159:4999-5004; Manickan (1997) J.
Clin. Invest. 100:2371-2375. Variations of this technique use cationic
liposome-entrapped DNA vaccines (see Gregoriadis (1997) FEBS Lett.
402:107-110); immunization with naked plasmid DNA transfected in dendritic
cells (Manickan (1997) J. Leukoc. Biol. 61:125-132); and, cutaneous genetic
immunization with naked DNA (Condon (1996) Nat. Med. 2:1122-1128).
Yeast expression systems, being eukaryotic, provide an attractive
alternative to bacterial systems for some applications; for an overview of
yeast expression systems, see. e.g., Protein Engineering Principles and
Practice, eds. Cleland et al., Wiley-Liss, Inc. p 129 (1996), Barr (1988) J.
Biol. Chem. 263: 16471-16478, or U.S. Pat. No. 4,546,082. A variety of yeast
vectors are publicly available. For example, the expression vector pPICZ B (Invitrogen,
San Diego, Calif.) has been modified to create expression vectors of the
invention to express the Pvs25 or Pvs28 of the invention in yeast, such as
S. cerevisiase and Pichia pastoris. Yeast episomal plasmids comprising
inducible promoters can be used for the intracellular expression of the
Pvs25 or Pvs28 proteins of the invention. Vectors include the pYES2
expression vector (Invitrogen, San Diego, Calif.) and pBS24.1 (Boeke (1984)
Mol. Gen. Genet. 197:345); see also Jacobs (1988) Gene 67:259-269.
One embodiment uses the yeast expression vector comprising the Recombinant
Protein Expression Unit called YEpRPEU-1, -2 and -3; and pIXY154 (Immunex
Corp.). pIXY154 and YEpRPEU-3 have been used to express Pvs25, Pvs28 and
Pvs28-Q130, amutagenized form of Pvs28 which eliminates all, several, or,
one potential N-linked glycosylation site, as discussed herein.
Yeast promoters for yeast expression vectors suitable for the expression of
a Pvs25 or Pvs28 include the inducible promoter from the alcohol
dehydrogenase gene, ADH2, also called the yeast alcohol dehydrogenase II
gene promoter (ADH2P) (La Grange (1997) Appl. Microbiol. Biotechnol.
47:262-266). In one embodiment, the ADH2 promoter is modified to include a
tract of poly A to enhance the ADH2 promoter in the expression of the
polypeptides of the invention. Suitable promoters to use also include the
ADH2/GAPDH hybrid promoter as described, e.g., in Cousens (1987) Gene
In another embodiment, the Pvs25 or Pvs28 to be expressed can also be fused
at the amino terminal end to the secretion signal sequence of the yeast
mating pheromone alpha-factor (MF alpha 1S) and fused at the carboxy
terminal end to the alcohol dehydrogenase II gene terminator (ADH2T), see
van Rensburg (1997) J. Biotechnol. 55:43-53. The yeast alpha mating
pheromone signal sequence allows for secretion of the expressed Pvs25 or
Pvs28. In one embodiment, sequences are added after the KEX-2 cleavage site
to enhance cleavage of the alpha factor leader; preferred embodiments
include addition of the sequence EAEA (SEQ ID NO:22) and EAEAEAEAK (SEQ ID
Yeast cell lines suitable for the present invention include e.g., BJ 2168
(Berkeley Yeast Stock Center) as well as other commonly available lines. For
example, the yeast can be a Pichia sp., Hansenula sp., Torulopsis sp.,
Saccharomyces sp., or a Candida sp. The yeast can specifically be a Pichia
pastoris, Hansenula polymorpha, Torulopsis holmil, Saccharomyces fragilis,
Saccharomyces cerevisiae, Saccharomyces lactis, or a Candida
pseudotropicalis. In other embodiments, Saccharomyces cerevisiae cell lines
XV2181 from Immunex; and, 2905/6, VQ1 and VK1 which we have developed as our
own yeast expression hosts.
Any of a number of other well known cells and cell lines can be used to
express the polypeptides of the invention. For instance, prokaryotic cells
such as E. coli can be used. Eukaryotic cells include, Chinese hamster ovary
(CHO) cells, COS cells, mouse L cells, mouse A9 cells, baby hamster kidney
cells, C127 cells, PC8 cells, and insect cells.
Following the growth of the recombinant cells and expression of the Pvs25 or
Pvs28 polypeptide, the culture medium is harvested for purification of the
secreted protein. The media are typically clarified by centrifugation or
filtration to remove cells and cell debris and the proteins are concentrated
by adsorption to any suitable resin such as, for example, CDP-Sepharose,
Asialoprothrombin-Sepharose 4B, or Q Sepharose, or by use of ammonium
sulfate fractionation, polyethylene glycol precipitation, or by
ultrafiltration. Other routine means known in the art may be equally
suitable. Further purification of the Pvs25 or Pvs28 or fusion polypeptide
can be accomplished by standard techniques, for example, affinity
chromatography, metal affinity chromatography (IMAC) (see, e.g., Govoroun
(1997) J. Chromatogr. B. Biomed. Sci. Appl. 698:35-46; Froelich (1996)
Biochem. Biophys. Res. Commun. 229:44-49), ion exchange chromatography,
sizing chromatography or other protein purification techniques to obtain
homogeneity, as described above. The purified proteins are then used to
produce pharmaceutical compositions, as described below.
A further aspect of the invention includes antibodies against Pvs25 or Pvs28
polypeptides. The antibodies are useful for diagnostic purposes or for
blocking transmission of parasites. The antibodies of the invention may be
polyclonal or monoclonal. Typically, polyclonal sera are preferred.
Antibodies are typically tetramers of immunoglobulin polypeptides. As used
herein, the term "antibody" refers to a protein consisting of one or more
polypeptides substantially encoded by immunoglobulin genes. Immunoglobulin
genes include those coding for the light chains, which may be of the kappa
or lambda types, and those coding for the heavy chains. Heavy chain types
are alpha, gamma, delta, epsilon and mu. The carboxy terminal portions of
immunoglobulin heavy and light chains are constant regions, while the amino
terminal portions are encoded by the myriad immunoglobulin variable region
genes. The variable regions of an immunoglobulin are the portions that
provide antigen recognition specificity. The immunoglobulins may exist in a
variety of forms including, for example, Fv, Fab, and F(ab).sub.2, as well
as in single chains, e.g., Huston et al., Proc. Natl. Acad. Sci. USA,
85:5879-5883 (1988) and Bird et al., Science 242: 423-426, 1988. See,
generally, Hood et al., Immunology, Benjamin, N.Y., 2nd ed. (1984), and
Hunkapiller (1986) Nature, 323:15-16. Single-chain antibodies, in which
genes for a heavy chain and a light chain are combined into a single coding
sequence, may also be used.
Use of the Polypeptides or Nucleic Acids of the Invention to Induce Immune
The immunoglobulins, nucleic acids, and polypeptides of the present
invention are also useful as prophylactics, or vaccines, for blocking
transmission of malaria or other diseases caused by parasites. Compositions
containing the immunoglobulins, polypeptides, nucleic acids or a cocktail
thereof are administered to a subject, giving rise to an anti-Pvs25 or
anti-Pvs28 polypeptide immune response in the mammal entailing the
production of anti-Pvs25 or anti-Pvs28 polypeptide immunoglobulins. The
Pvs25 or Pvs28 polypeptide-specific immunoglobulins then block transmission
of the parasite from the subject to the arthropod vector, preventing the
parasite from completing its life cycle. An amount of prophylactic
composition sufficient to result in a titer of antiserum which, upon
ingestion by the mosquito, is capable of blocking transmission or is capable
of decreasing ability of the oocyte to mature in the mosquito (resulting in
fewer infective particles passed to the mosquitoes' next target bloodmeal),
is defined to be an "immunologically effective dose."
The isolated nucleic acid sequences coding for Pvs25 or Pvs28 polypeptides
can be used in viruses to transfect host cells in the susceptible organism,
particularly, a human. Live attenuated viruses, such as vaccinia or
adenovirus, are convenient alternatives for vaccines because they are
inexpensive to produce and are easily transported and administered. Vaccinia
vectors and methods useful in immunization protocols are well known in the
art and are described, e.g., in U.S. Pat. No. 4,722,848.
Suitable viruses for use in the present invention include, but are not
limited to, pox viruses, such as, canarypox and cowpox viruses, and vaccinia
viruses, alpha viruses, adenoviruses, and other animal viruses. The
recombinant viruses can be produced by methods well known in the art: for
example, using homologous recombination or ligating two plasmids together. A
recombinant canarypox or cowpox virus can be made, for example, by inserting
the gene encoding the Pvs25 or Pvs28, or other homologous polypeptide into a
plasmid so that it is flanked with viral sequences on both sides. The gene
is then inserted into the virus genome through homologous recombination.
A recombinant adenovirus virus can be produced, for example, by ligating two
plasmids each containing 50% of the viral sequence and the DNA sequence
encoding the Pvs25 or Pvs28 polypeptide. Recombinant RNA viruses such as the
alpha virus can be made via a cDNA intermediate using methods known in the
The recombinant virus of the present invention can be used to induce
anti-Pvs25 or anti-Pvs28 polypeptide antibodies in mammals, such as mice or
humans. In addition, the recombinant virus can be used to produce the Pvs25
or Pvs28 polypeptides by infecting host cells which in turn express the
The nucleic acids can also be used to produce other recombinant
microorganisms such as bacteria, yeast, and the like. For instance, BCG (Bacille
Calmette Guerin) vectors are described, e.g., in Stover (1991) Nature
351:456-460. A wide variety of other vectors useful for therapeutic
administration or immunization of the peptides of the invention, e.g.,
Salmonella typhi, Saccharomyces vectors and the like, will be apparent to
those skilled in the art from the description herein.
The DNA encoding the polypeptides of the invention can also be administered
to the patient. Typically, an expression cassette suitable for driving
expression in human cells is prepared. This approach is described, for
instance, in Wolff (1990) Science 247:1465-1468; U.S. Pat. Nos. 5,580,859
The present invention also relates to host cells infected with the
recombinant virus of the present invention. The host cells of the present
invention are preferably eukaryotic, such as yeast cells, or mammalian, such
as BSC-1 cells. Host cells infected with the recombinant virus express the
Pvs25 or Pvs28 polypeptides on their cell surfaces. In addition, membrane
extracts of the infected cells induce transmission blocking antibodies when
used to inoculate or boost previously inoculated mammals.
In the case of vaccinia virus (e.g., strain WR), the sequence encoding the
Pvs25 or Pvs28 polypeptides can be inserted into the viral genome by a
number of methods including homologous recombination using a transfer
vector, pTKgpt-OFIS as described in Kaslow et al., Science 252:1310-1313,
The Pvs25 or Pvs28 polypeptides or nucleic acids of the present invention
can be used in pharmaceutical and vaccine compositions that are useful for
administration to mammals, particularly humans, to block transmission of a
variety of infectious diseases. The compositions are suitable for single
administrations or a series of administrations. When given as a series,
inoculations subsequent to the initial administration are given to boost the
immune response and are typically referred to as booster inoculations.
The pharmaceutical compositions of the invention are intended for parenteral,
topical, oral or local administration. Preferably, the pharmaceutical
compositions are administered parenterally, e.g., intravenously,
subcutaneously, intradermally, or intramuscularly. Thus, the invention
provides compositions for parenteral administration that comprise a solution
of the agents described above dissolved or suspended in an acceptable
carrier, preferably an aqueous carrier. A variety of aqueous carriers may be
used, e.g., phosphate buffered saline, water, buffered water, 0.4% saline,
0.3% glycine, hyaluronic acid and the like. These compositions may be
sterilized by conventional, well known sterilization techniques, or may be
sterile filtered. The resulting aqueous solutions may be packaged for use as
is, or lyophilized, the lyophilized preparation being combined with a
sterile solution prior to administration. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to approximate
physiological conditions, such as pH adjusting and buffering agents,
tonicity adjusting agents, wetting agents and the like, for example, sodium
acetate, sodium lactate, sodium chloride, potassium chloride, calcium
chloride, sorbitan monolaurate, triethanolamine oleate, etc.
For solid compositions, conventional nontoxic solid carriers may be used
which include, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose,
sucrose, magnesium carbonate, and the like. For oral administration, a
pharmaceutically acceptable nontoxic composition is formed by incorporating
any of the normally employed excipients, such as those carriers previously
listed, and generally 10-95% of active ingredient and more preferably at a
concentration of 25%-75%.
For aerosol administration, the polypeptides or nucleic acids are preferably
supplied in finely divided form along with a surfactant and propellant. The
surfactant must, of course, be nontoxic, and preferably soluble in the
propellant. Representative of such agents are the esters or partial esters
of fatty acids containing from 6 to 22 carbon atoms, such as caproic,
octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and
oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
Mixed esters, such as mixed or natural glycerides may be employed. A carrier
can also be included, as desired, as with, e.g., lecithin for intranasal
In therapeutic applications, Pvs25 or Pvs28 polypeptides or nucleic acids of
the invention are administered to a patient in an amount sufficient to
prevent parasite development in the arthropod and thus block transmission of
the disease. An amount adequate to accomplish this is defined as a
"therapeutically effective dose." Amounts effective for this use will depend
on, e.g., the particular polypeptide or virus, the manner of administration,
the weight and general state of health of the patient, and the judgment of
the prescribing physician.
The vaccines of the invention contain as an active ingredient an
immunogenically effective amount of the Pvs25 or Pvs28 polypeptides, nucleic
acids, or recombinant virus as described herein. Useful carriers are well
known in the art, and include, e.g., thyroglobulin, albumins such as human
serum albumin, tetanus toxoid, polyamino acids such as
poly(D-lysine:D-glutamic acid), influenza, hepatitis B virus core protein,
hepatitis B virus recombinant vaccine and the like. The vaccines can also
contain a physiologically tolerable (acceptable) diluent such as water,
phosphate buffered saline, or saline, and further typically include an
adjuvant. Adjuvants such as incomplete Freund's adjuvant, aluminum
phosphate, aluminum hydroxide, or alum are materials well known in the art.
Vaccine compositions containing the polypeptides or nucleic acids of the
invention are administered to a patient to elicit a transmission-blocking
immune response against the antigen and thus prevent spread of the disease
through the arthropod vector. Such an amount is defined as an "immunogenically
effective dose." In this use, the precise amounts again depend on the
patient's state of health and weight, the mode of administration, and the
nature of the formulation.
As noted above, the Pvs25 or Pvs28 polypeptides of this invention may also
be used to make monoclonal antibodies. Such antibodies may be useful as
potential diagnostic or therapeutic agents. The polypeptides themselves may
also find use as diagnostic reagents. For example, a polypeptide of the
invention may be used to diagnose the presence of antibodies against P.
vivax in a patient. Alternatively, the polypeptides can be used to determine
the susceptibility of a particular individual to a particular treatment
regimen, and thus may be helpful in modifying an existing treatment protocol
or in determining a prognosis for an infected individual.
Claim 1 of 17 Claims
1. A composition comprising an isolated
Pvs25 polypeptide having at least 95% amino acid sequence identity to SEQ
ID NO:4, wherein the Pvs25 polypeptide induces production of antibodies in
a susceptible mammal against a 25 kD protein on the surface of Plasmodium
vivax zygotes and ookinetes, wherein the antibodies against the 25 kD
protein block the transmission of P. vivax from a mosquito.
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