EP1019435A1

EP1019435A1 - Amplification of response from expressed recombinant protein

Info

Publication number: EP1019435A1
Application number: EP97906880A
Authority: EP
Inventors: Eugene A. Davidson; Shutong Yang
Original assignee: Georgetown University
Current assignee: Georgetown University
Priority date: 1996-01-29
Filing date: 1997-01-29
Publication date: 2000-07-19
Also published as: JP2000504330A; AU2248997A; KR19990082085A; EP1019435A4; AU724621B2; WO1997027216A1

Abstract

The present invention relates to a method for amplifying the response from a protein expressed from a recombinant vaccinia vector. The present invention may be used to amplify the intended effect of expressed proteins in any method where such expression is desired, for example, in vaccine production, vaccination and gene therapy. The present invention also relates to synthetic peptides, nucleic acid sequences, preferably DNA sequences and constructs comprising a vector linked to a DNA segment which encodes a protein containing a signal peptide at its N-terminus and an anchor sequence at its C-terminus. These expression vectors are generally useful for expressing polypeptides in a variety of animal cells.

Description

Amplification of Response from Expressed Recombinant Protein

Field of the Invention

The present invention relates to a method for amplify¬ ing the response from a protein expressed from an expression vector, preferably, a recombinant vaccinia vector. The pres¬ ent invention may be used to amplify the intended effect of expressed proteins in any method where such expression is desired, for example, in vaccine production, vaccination and gene therapy. The present invention also relates to synthetic peptides, nucleic acid sequences, preferably DNA sequences and constructs comprising a vector linked to a DNA segment which encodes a protein containing a signal peptide at its N- terminus and an anchor sequence at its C-terminus. These expression vectors are generally useful for expressing polypeptides in a variety of animal cells.

By encoding the protein in an expression vector such that the N-terminus contains a signal peptide and the C- terminus contains an anchor sequence, the protein which is expressed will elicit a response or intended effect which is significantly amplified in comparison to proteins which do not contain a signal sequence or anchor sequence. The present method may be used to amplify the effects of an expressed protein for a variety of purposes including vaccine produc¬ tion, vaccination and treatment of disease such as in gene therapy, among others.

More particularly, the present invention relates to a method for amplifying the therapeutic or immunogenic effect of proteins which are expressed pursuant to gene therapy techni¬ ques or in vaccines which rely upon a protein or peptide as an antigenic determinant.

In particular embodiments associated with vaccine therapy or disease prevention, the present invention relates to certain vaccines which are useful for the prevention and treatment of a number of diseases including those caused by various microbes including bacteria, viruses, fungi and protozoa, among others. In particular, the present method may be used to amplify antigenic proteins expressed in vaccines to Herpes simplex, cytomegalovirus, hepatitis and malaria caused by Plasmodiu falciparum in humans, among numerous others.

This work was supported by DARPA grant number N-00014- 90. The government retains certain rights in the invention.

Background of the Invention

Preventing or treating a variety of disease states, including microbial infections, has long been a challenging health problem. Many disease states and infections, espe¬ cially those of a viral variety, are not readily treatable with antimicrobial agents. Those diseases which are treatable with antimicrobials, often become resistant to the therapy after time. Although there has been steady progress in the last few years to treat these infections, several problems still must be overcome, including the selection of an appropriate delivery system vehicle and the expression of the protein.

In most instances, the development of vaccines for use against a number of diseases has involved the inclusion of an adjuvant to boost the immunogenic effect of the antigenic proteins presented in the vaccine.

Herpes simplex is the causative viral agent of genital and other infections (generally of the mouth, commonly called cold sores) . The incidence of Herpes simplex has increased several fold in the last two decades. The need for an effec¬ tive vaccine is substantial. It has been established that neutralizing antibodies directed against the virus recognize surface glycoproteins of the organism with two such glycoproteins (Glycoproteins B and D) functioning as the predominant antigens. Recombinant glycoproteins have been produced in Chinese Hamster Ovary (CHO) cells and have been shown to provide a measure of protection for guinea pigs against a direct challenge. The extent of protection varies and is dependent on adjuvant given in coad inistration with the glycoproteins, however. A number of herpes glycoproteins have also been expressed in baculovirus (an insect-based cell system) and several of these have also shown protective immunizing potential when administered with Freund's adjuvant. The inflammatory properties of this adjuvant preclude its use in humans and mitigate strongly against its use in laboratory test animals. The capsid proteins from the virus may be readily expressed by recombinant vaccinia virus.

Human cytomegalovirus is a pathogen which can cause morbidity as well as mortality, particularly in imrriunocompromised individuals. In pregnant women, infection may result in improper development of the motor system in the fetus. The virus is widespread in the population with an incidence as high as 1% and a downstream incidence of several thousand birth defects yearly. The acquisition of immunity in women prior to conception should sharply reduce the incidence of adverse effects. It has been established that infected invidiuals are able to mount an immune response that includes both humoral and cellular components. Trials have been con¬ ducted with an attenuated strain of the virus as an immunogen. Attempts have been made to confer protection by ad nistration of a protein antigen combined with an adjuvant, but the results to date have been less than acceptable.

Hepatitis is a widespread, debilitating and sometimes fatal disease. There are a number of forms of viral hepatitis which infect humans- typically hepatitis A, B, and C, etc. The first recombinant vaccine product approached for human use was derived from the B strain of hepatitis and is an adjuvant based formulation. There still exist difficulties in maximiz¬ ing the immune response of this material and continued attempts at improving the adjuvant mediated response are presently being investigated. One commercial hepatitis vac¬ cine is made from material produced in yeast and admixed with alum. Such products may lack the information imparted to the viral glycoprotein by expression in a mammalian host cell (e.g. , complex glycosylation which may confer specific con¬ formational properties, modulate processing and increase stability) . The overriding problem in the development of infec¬ tious disease vaccines is to accurately identify protective antigens and present them effectively to the host. Formula¬ tions which depend on the use of adjuvant to promote the immunogenic effect risk adverse reactions which may preclude their use. It is important to emphasize that a vaccine preparation that is effective only when used with an adjuvant is one in which presentation to immune cells is intrinsically inefficient. This overwhelming shortcoming is perhaps the major reason why many prior art vaccines have failed to become commercializable.

In one aspect of the present invention, a general pro¬ cedure is offered for overcoming the shortcomings of the prior art attempts to produce vaccines against a host of diseases caused by microbes, including viruses, bacteria, protozoa and fungi, among others. Vaccines according to the present inven¬ tion present antigenic proteins in a fashion such that they are more likely to create an immunogenic effect.

Many genetic diseases are manifest in the patient's inability to produce adequate amounts of one or more critical protein or polypeptide. Many neurological, muscular or hematopoeitic disorders and related diseases often trace their causes to a deficiency in the ability of the individual to produce a critical protein or polypeptide in sufficient amounts. A viable treatment option lies in the reinstatement of the biosynthesis or production of the deficient protein. Alternatively, gene therapy techniques involving the adminis¬ tration of a protein expression vector to the patient suffer¬ ing from genetic disease represents a viable approach to treatment.

In the past, live vaccinia virus was used as a vaccine to eradicate smallpox successfully, and a recombinant vaccinia virus expressing viral antigens has been shown to induce a strong antibody response in immunized animals, conferring pro¬ tection against disease (Arita, I., Nature, 1979, 279, 293- 298) . Furthermore, it has been shown in animal models that co-presentation of potential immunogens with highly immunogenic vaccinia virus proteins can elicit a strong immune response against that specific immunogen (Moss and Flexner, Annals of the New York Academy of Sciences. 86-103; Mackett and Smith, J. Gen. Virol. , 1986, 67, 2067-2082; Houard, et al., J. Gen. Virol.. 1995, 76, 421-423; Fujii, et al., J. Gen. Virol. , 1994, 75, 1339-1344; Rodrigues, et al. J. Immunol.. 1994, 153, 4636-4648) . Therefore, the utilization of live recombinant vaccinia virus as a vaccine might overcome many problems of antigen expression and delivery presently encountered in the preparation of recombinant proteins in E. coli, yeast or insect expression systems. A panel of transfer vectors have been constructed that allow insertion of foreign genes into several sites within the 180kb vaccinia virus genome (Earl and Moss, Current Protocols in Molecular Biology, 1993, 16.17.1-16.17.16) . Also, it has been reported that >25kb of foreign DNA can be inserted into the vaccinia virus genome (Smith and Moss, Gene. 1983, 25, 21-28) . The correct processing (Chakrabarta, et al.. Nature, 1986, 320, 535-537) and the appropriate post-translational modification (Hu, et al.. Nature. 1986, 320, 537-540; Ball, et al., Proc. Natl. Acad. Sci. USA. 1986, 83, 246-250; de la Salle, et al., Nature, 1985, 316, 268-270), transport and secretion (Ball, et al., Proc. Natl. Acad. Scienc. USA_f 1986, 83, 246-250 and Langford, et al., Mol. Cell. Biol. 1986, 6, 3191-3199) are dictated by the primary structure of the expressed protein. In addition, a recombinant vaccinia virus vaccine has the advantage of being relatively inexpensive and easily stored, transported and delivered, features which are particularly important in developing countries, e.g. where malaria and other diseases are most prevalent.

Summary of the Invention

The present invention relates to a method for enhanc¬ ing the biological or pharmacological effect of an expressed protein or peptide in an animal patient. In the present invention, an expression vector containing a gene or other nucleic acid sequence which encodes for a protein or peptide sequence which exhibits an intended pharmacological effect is introduced into the patient. The expressed protein or peptide sequence also contains an anchor peptide sequence at its C- ter inus and optionally, a signal sequence at its N-terminus. Preferably, in a particularly preferred embodiment, the expressed protein or peptide sequence is immunogenic and con¬ tains both a signal peptide at its N-terminus and an anchor peptide sequence at its C-terminus.

After the expression vector is introduced into an animal patient, preferably a mammal, such as a human, the vec¬ tor will express the desired protein containing the signal peptide and anchor peptide in the patient and produce an intended pharmacological effect. It is unexpectedly found that the inclusion of a signal peptide sequence at the N- terminus of an expressed peptide sequence and an anchor sequence at the C-terminus of the expressed peptide sequence produces a significantly enhanced improvement in the resulting pharmacological effect obtained from the expressed protein. When an immunogenic protein or peptide is expressed along with a signal and anchor protein, the immunogenic effect is often substantially greater than when the protein or peptide is expressed without a signal or anchor peptide.

A method for significantly enhancing the pharmacologi¬ cal effect of an expressed protein in a patient comprises the steps of incorporating into an expression vector a DNA (nucleic acid) sequence which encodes for a peptide sequence which comprises a pharmacologically active protein or peptide to be expressed which normally is not expressed with a signal peptide and/or anchor peptide, in combination with an anchor peptide at the C-terminus of the expressed peptide, optionally including a signal peptide at the N-terminus of said expressed peptide and administering the expression vector to the patient. The method may be very broadly applied. The present invention is applicable for enhancing the biological or pharmacological effect of virtually any protein which can be incorporated into an expression vector and exhibits its biological or pharmacological effect outside of the cell in which the protein is expressed. The method is applicable essentially to any protein or peptide for which cell surface expression is an important functional event including immogenic proteins and peptides, receptors, including the insulin receptor in the case of insulin-resistant diabetes, hormone receptors and growth factor receptors, among numerous others. The present invention is particularly applicable to the production of vaccines which elicit enhanced immunogenic activity, without the need to coadminister an adjuvant. The present invention also finds utility in gene therapy techni¬ ques, among numerous other biological and therapeutica appli¬ cations.

By following the method steps of the present inven¬ tion, the pharmacological results obtained from the expressed protein or peptide are significantly enhanced in comparison to the results obtained from the expression of the same protein without the signal peptide and anchor peptide. In general, the incorporation of a signal and anchor peptide will produce an effect which is at least two-fold greater than the response which is produced by the peptide which does not contain a sig¬ nal and anchor peptide. In preferred cases the effect will be 5-fold greater, more preferably at least about 10-fold greater and, in certain cases, the effect may be even 100-fold or more greater. For antigens, this enhancement is measured in a man¬ ner based on direct comparison of elicited antibodies in the recipient host. These antibodies are directed against the protein/peptide core structure and quantitated by ELISA. In the case of the therapeutic aspect of this invention, expres¬ sion of the protein of interest may be quantitated by use of a specific antibody in a standard capture ELISA or by measure¬ ment for example, of a specific biological function (such as, for example, enzyme activity or other activity) .

In the expression vector according to the present invention, the nucleic acid coding for the desired protein domain is expressed in the patient by the expression vector after administration. The peptide as a complete protein or sub-fragment thereof which is then expressed in the patient provides a desired therapeutic effect in the patient or, in the case of a vaccine approach, raises a humoral and/or cell- mediated response to the antigenic protein or peptide, which response provides the effect of protecting the vaccinated patient from a subsequent infection by a microbe containing the expressed protein or peptide. In preferred embodiments according to the present invention, the expression vector con¬ tinues to express the protein or peptide in the patient for a period of days, months or even years, thus reinforcing the therapeutic effect or immunogenic response of the patient to the expressed peptide.

The peptide which is expressed by the expression vec¬ tor contains an anchor peptide. Preferred embodiments also advantageously comprise a signal peptide and an anchor peptide sequence. It has been found that the addition of a signal and anchor peptide to the expressed peptide according to the pres¬ ent invention unexpectedly enhances the therapeutic or immunogenic effect in the patient of the expressed protein. It is an unexpected result that the inclusion of a signal and anchor protein with the expressed peptide according to the present invention will produce a significantly greater therapeutic or immunogenic response than the peptide will pro¬ duce alone or in the case of vaccines, alone or in combination with an adjuvant.

The vaccine aspect of the present invention is particularly advantageous over the prior art in that it avoids the need for the co-administration of an adjuvant in order to enhance the immunogenic effect of the antigenic protein or peptide. In the case of the immunogenic aspect of the present invention, it is an unexpected result that the inclusion of a signal and anchor sequence in the antigenic peptide sequence expressed by the expression vector will produce an immunogenic response which is at least two-fold and may be as much as 100 fold or more greater than the immnogenic response which is produced by the antigenic peptide which does not contain a signal or anchor peptide sequence. The overriding problem in the development of infec¬ tious disease vaccines is to accurately identify protective antigens and present them effectively to the host. Formula¬ tions that are dependent on the use of adjuvant risk adverse reactions that may preclude their use. A vaccine preparation which is effective only when used with an adjuvant is one in which presentation to immune cells is intrinsically ineffi¬ cient. In the present invention, the general procedure offered overcomes this shortcoming. In the case of the pro¬ duction of vaccines, the inclusion of the genetic information for correct pathway traverse in an infected host cell with a means to immobilize the produced protein in the plasma mem¬ brane of that same cell insures the availability to the host immune cells even if the cell dies.

Methods of treating diseases, inducing an immunogenic response in a patient, or vaccinating a patient against dis¬ ease are also contemplated by the present invention. In this method, a patient is administered an amount of an expression vector, preferably a vaccinia virus vector, effective to express the protein or peptide of interest such that the patient begins producing the desired protein or peptide in amounts which treat the indicated ailment, or alternatively, produces an immunogenic response to the expressed protein or peptide. In the case of a vaccine, the immunogenic response generated preferably will be "substantially protective", i.e., the vaccine will protect the patient from some of the more severe symptoms and physiological states of the disease, including the death of the patient from the disease.

The present invention also relates to dosage forms of the expression vector as a treatment modality such as a pharmaceutical agent or as a vaccine. Dosage forms according to the present invention may be used for example, for treating a disease such as a genetic disease in which a protein or pep¬ tide to be expressed is normally not produced or for inducing an immunogenic response to a protein or peptide in order to produce a protective effect against a disease. Methods of vaccinating a patient against numerous microbial infections, especially including bacterial and viral infections such as Ly e disease (Borrelia burgdorferi as the causative spirochete) , Herpes simplex, cytomegalovirus and hepatis (A,B,C, etc.), among numerous others, are also con¬ templated by the present invention. In this method, a patient is vaccinated against a microbial infection by administering an immunogenic response producing effective amount of an expression vector, preferably a vaccinia virus vector capable of expressing an antigenic protein or peptide of a disease- causing microbe.

The present invention also relates to chimeric peptide sequences which incorporate both the expressed protein or pep¬ tide and the anchor peptide. Optionally, the chimeric peptide sequence comprises expressed protein or peptide, anchor pep¬ tide and signal peptide. These chimeric peptides may be util¬ ized as therapeutic or immunogenic agents either alone, or after expression from an appropriate expression vector.

The present invention may be used to treat numerous diseases including autoimmune diseases, genetic diseases in which proteins are produced in diminished concentrations or not at all, cancer, viral infections, protozoal infections, other microbial infections

Detailed Description of the Invention

The following terms shall be used throughout the instant specification in an effort to describe the present invention.

The term "patient" is used to describe an animal, including a mammal and especially including a human patient which is administered a dosage form of an expression vector according to the present invention. In the present invention, the expression vector encodes for a desired protein or peptide and expresses the encoded protein or peptide in the patient. The expressed protein or peptide may provide a therapeutic or immunogenic effect in the treated patient.

The term "chimeric peptide" or "chimeric peptide sequence" is used to describe the non-natural peptide sequences according to the present invention which comprise the expressed protein or peptide, an anchor peptide and/or a signal peptide. As noted by the use of this term, chimeric peptides are synthetic peptides produced by an expression vec¬ tor which contain a desired target protein or peptide or a portion of a target protein or peptide (which, in nature, is not normally found in combination with a signal and/or anchor sequence) in combination with such a signal and/or anchor pep¬ tide sequence.

The term "expression vector" is used to describe the means by which nucleic acid, including DNA, cDNA, RNA or variants thereof, more preferably DNA fragments, encoding for a specific peptide or protein, may be introduced into the patient or the patient's tissue in order to express or produce the desired protein. Such vectors include any vectors into which a nucleic acid sequence encoding for the desired protein or peptide, anchor peptide sequence and optionally, signal protein sequence may be inserted (along with any required or optional operational elements) into a host organism and repli¬ cated. Preferred vectors are those which are capable of expressing peptide or protein sequences in eukaryotic cells and whose restriction sites are well known and which contain the required operational elements for expression of the desired protein or peptide sequence. In the present inven¬ tion, the vector is preferably a vaccinia virus vector, adenovirus vector or herpes virus vector which has the capa¬ city to infect a mammalian cell and express or synthesize proteins utilizing the host's biosynthetic mechanism. In such cases, the viral vector used for delivery should optimally be one which infects cells but which does not cause lysis due to replication (i.e., an attenuated or partially disabled virus selected from among adenovirus, vaccinia virus and herpes viruses, among similar types) . According to the vector approach in the present inven¬ tion, the vector will infect the host cells and, using the host cells' biosynthetic pathways, synthesize the protein or peptide encoded by the vector. Any immunizing vehicle which has a detailed genetic and human use history may be used as the expression vector in the present invention. The preferred expression vector is a viral vector, more preferably, a vac¬ cinia virus vector, for example, as described by Earl and Moss, Current Protocols in Molecular Biology. 1993, 16.17.1- 16.17.16) and Smith and Moss, Gene. 1983, 25, 21-28. However, any vaccinia or other viral vector which may be used in the above-described manner may be appropriate for use in the pres¬ ent invention. In the case of orally administered therapeutic products, where expression of the protein will occur in the gastrointestinal tract, the use of a bacterial expression vec¬ tor, for example, a Salmonella expression vector, may be preferred.

In order to express the desired protein or peptide sequence, the expression vector should contain at least one promoter, at least one operator, at least one terminator codon, and any other sequences which are necessary for the efficient transcription and subsequent translation of the nucleic acid from the vector. These operational elements are well known to those of ordinary skill in the art. In preferred embodiments according to the present invention, the expression vectors will advantageously comprise at least one origin of replication which is recognized by the host organism along with at least one selectable marker and at least one promoter sequence capable of initiating transcription of the nucleic acid (preferably, DNA) sequence.

The terms "expressed peptide", "expressed polypeptide" and "expressed protein" are used to describe target peptides and proteins which are expressed by the expression vector according to the present invention in order to treat a disease or condition or generate an immunogenic response or a pro¬ tective effect against a disease. Preferred expressed proteins and peptides include any protein or peptide for which cell surface expression is an important functional event. All of these proteins, peptides or polypeptides normally (i.e., in nature) are not found combined with signal and/or anchor sequences. The target peptides and proteins may be found in nature, but not in combination with a signal and/or anchor sequence.

Preferred proteins and polypeptides according to the present invention include, for example, hormones, growth fac¬ tors, clotting factors, enzymes, neuroproteins, apolipoproteins, tumor suppressors, antigens, including endogenous or exogenous tumor antigenic proteins and peptides, bacterial surface proteins and peptides, viral surface proteins and peptides, parasitic including protozoal cell sur¬ face proteins and peptides, fungal surface proteins and pep¬ tides and viral reverse transcriptase and related viral specific enzymes, among numerous others. Other preferred proteins include receptors such as the insulin receptor in the case of insulin-resistant diabetes, hormone receptors, growth factor receptors.

The term "vaccine" is used throughout the specifica¬ tion to describe a preparation intended for active immunologi¬ cal prophylaxis. In the present invention, vaccines comprise an expression vector, preferably a vaccinia virus expression vector which expresses an antigenic protein or peptide from a disease-causing microbe after administration of the expression vector to an animal, such as a mammal. The method of administering the vaccine according to the present invention may vary and include intravenous, buccal, oral, transdermal and nasal, among others, but intramuscular or subcutaneous administration is the most common method of administration. In this procedure for making a vaccine for humans and other animals, the DNA encoding for the desired protein or peptide to be expressed is incorporated into the expression vector by generally available methods known in the art or otherwise as described in greater detail hereinbelow. The vector is then formulated as an immunogenic dosage form as a vaccine and administered to the patient by one or more of the above- described methods to protect the patient against disease.

The terms "amino-terminus" or "N-terminus" and "amino- terminal" are used to describe that portion of the expressed protein or peptide (such term including the amino-terminus amino acid) according to the present invention which is at or near the amino terminal end of the protein or peptide. It is at or about the 5' end of the DNA sequence encoding the amino- terminus of the expressed protein or peptide (generally directly upstream or in some cases slightly upstream from the 5' end of the sequence encoding the protein or peptide) and at the N-terminus of the expressed protein or peptide where the sequence encoding for the signal protein or peptide is found.

The terms "carboxy-terminus" and "carboxy-terminal" are used to describe that portion of the expressed protein or peptide (such term including the carboxy-terminus amino acid) according to the present invention which is at or near the carboxyl terminus of the protein or peptide. The DNA sequence encoding the carboxy-terminus is at or near the 3' terminus of the sequence encoding the expressed protein or peptide. It is in the proximity of the 3' end of the DNA sequence encoding the carboxy-terminal region of the expressed protein or pep¬ tide (generally directly downstream or in some cases slightly downstream from the 3' end of the sequence encoding the protein or peptide) and at the Carboxy terminus of the expressed protein or peptide where the anchor protein or pep¬ tide is generally found.

In certain vaccines, the carboxy-terminus or the region in proximity to the carboxy-terminus of the expressed protein or peptide may represent a preferred target for the development of humoral and/or cell mediated response because of the degree of specificity of the immune response which can be elicited against such a protein segment. Thus certain preferred immunogenic proteins or peptides for use in the inventive vaccines may reside at the carboxy-terminus or the region in proximity to the carboxy-terminus of the complete immunogenic protein.

The term "signal peptide" "signal sequence" or "signal protein" is used to describe a 7-30 unit amino acid peptide sequence, preferably about a 15-26 unit amino acid peptide sequence, which is generally found at or near the N-terminus of the expressed protein or peptide which is used in the pres¬ ent invention in order to substantially enhance the biological activity of the protein or peptide expressed in the patient according to the present invention. Signal sequences generally contain hydrophobic peptide sequences of between about 7 and 30 amino acid units, more preferably, about 15 to 26 amino acid units, even more preferably about 16 to 24 amino acid units and most preferably about 18 to 20 amino acids units appear to be essential for the targeting of protein chains (generally, secretory proteins) to membranes within the cell. These hydrophobic sequences are of sufficient length to cross the lipid bilayer of the cell membranes. Signal sequences serve as organizers for the cellular traffic of mac¬ romolecules. These proteins are believed to play a central role in the translocation of polypeptide chains across mem¬ branes. In the present invention, the incorporation of a sig¬ nal protein sequence at the amino terminus of the protein or peptide sequence expressed by the vaccinated patient is asso¬ ciated with the substantial enhancement in the biological activity (including the therapeutic effect of immunogenicity) associated with the expressed protein or peptide. In the present invention, signal sequences which are known in the art may be used in the present invention. For example, although it may be possible to utilize yeast or lower trophic order signal sequences, clearly mammalian signal sequences are preferred for use in mammals and the specific species signal sequences are most preferred for use in the desired mammalian species to be treated. Thus, in providing for an expressed protein or polypeptide in humans, a human signal sequence is most preferred.

Signal sequences for use in the present invention generally contain three regions, a first or c region at the carboxy end of the peptide (which serves as the cleavage site for a signal peptidase enzyyme) , comprising about 5 to 7 amino acid residues which tend to be highly polar but uncharged; a second or h region which is N-terminal to the c region, generally about 7 to 13 amino acid residues in length and highly hydrophobic (comprised primarily of Leu, Ala, Met, Val, lie, Phe, and Trp amino acids, but may contain an occasional Pro, Gly, Ser or Thr amino acid residue) ; and a third region or n-region of highly variable length and composition, but generally carrying a net positive charge contributed by the N- terminus (negative charges contributed from acidic residues are also known) and any charged residues. Between the c region and the h region are between 1 and 3 amino acid residues which tend to be small and uncharged (Ala, Gly, Ser, others) . Synthetic homopolymeric h regions comprised of amino acids selected from the group consisting of leucine, isoleucine, phenylalanine, valine, alanine and tryptophan, preferably leucine, isoleucine and phenylalanine may be used in the signal proteins according to the present invention. See generally, von Heijne, European Journal of Biochemistry. (1983) , 133, pp. 17-21.

The signal sequences which are used in the present invention preferably encompass eukaryotic signal sequences, preferably between 7 and 30 amino acid units in length, preferably between 15 and 26 units, more preferably between about 16 and 26 amino acids, even more preferably between 18 and 20 amino acid units. In the present invention, the c region of the signal peptide should be more polar and the boundary between the h and c regions between residues -5 and - 6, or -7 or -8 (counting from the position of cleavage of the signal sequence- i.e., the first amino acid of the mature or expressed protein or peptide is +1) is between 1 and 3 amino acid residues which tend to be small and uncharged (Ala, Gly, Ser, others) . Position preferences in the h/c for amino acids are as follows:

-10 most preferably leucine or alternatively, isoleucine, valine, alanine, or phenylalanine; -9 most preferably leucine, alternatively, isoleucine, alanine, valine, phenylalanine;

-8 most preferably leucine, alternatively isoleucine, alanine, valine, glycine, phenylalanine;

-7 most preferably alanine, alternatively, leucine, isoleucine, valine, phenylalanine;

-6 most preferably valine, alternatively leucine, valine, isoleucine, phenylalanine, alanine;

-5 most preferably proline, alternatively glycine, alanine, leucine, valine;

-4 most preferably glycine, alternatively proline, leucine, alanine, valine;

-3 most preferably alanine, alternatively valine;

-2 most preferably leucine, alternatively phenylalanine;

-1 most preferably alanine, alternatively glycine.

In the signal sequences used in the present invention, the h region may vary in length as well. The n region is polar, contains positively charged amino acids (predominantly lysine and arginine) and varies with the overall length of the signal peptide as described above. The c region extends from residues -1 to -5 of the signal peptide/expressed or mature protein. In terms of location of the c, h and n regions, the c region is N-terminus to the expressed or mature protein, the h region is N-terminus to c region (with a 1-3 amino acid boundary between the c and h region) and the n region is a positively charged N-terminus to the h region. In sum, the n region is variable in length and generally positively charged (with a preferred charge of +2) , the h region is hydrophobic and variable in length and the c region preferably contains about five (5-7) generally polar amino acids.

The end of the hydrophobic domain (i.e., the boundary between the hydrophobic residues enumerated above) should preferably be at positions -6/-5. Overall, the signal sequence should comprise a 5 to 10 unit residue initial sequence (beginning with methionine) followed by at least a seven residue sequence (as described above) and an additional amino acids from 1 to 10 residues in length. A typical sequence for the region noted about is:

ILLLLAV.

The signal sequence used should be characteristic of the cell type used for expression of the protein. Thus, in veterinary applications, the signal sequence used should be mammalian in character. Most mammalian signal sequences will have significant efficacy in expressing proteins or peptides in other mammalian cells. Human signal sequences are preferably used for human applications.

The term "anchor protein" or "anchor peptide sequence" is used to describe proteins or peptides which are anchored to the external surface of the plasma membrane generally by covalent bonding to glycans containing phosphatidyl inositol. These structures to which the anchor protein or peptide is bonded are often referred to as glycosyl phosphatidylmositols or GPIs. In all cells, anchor proteins covalently bonded to GPIs are found on the external face of the plasma membrane of cells or on the lumenal surface of secretory vesicles.

In the present invention an "anchor protein" or "anchor peptide" comprises a peptide sequence preferably of about 15-35 residues in length which is generally expressed at the carboxy-terminus of the protein or peptide expressed by the expression vector according to the present invention (3' end of the DNA sequence expressing the desired protein or pep¬ tide and carboxyl terminus of the expressed protein or pep- tide) .

In the present invention, many of the proteins or pep¬ tides which are expressed in the patient and in particular, the immunogenic proteins or peptides of vaccines according to the present invention which are expressed in the patients pro¬ duce a biological or immunogenic response in the patient which is substantially enhanced when an anchor peptide is incor¬ porated at the carboxy terminus of that protein or peptide. The inclusion of a signal protein at or in the proximity of the N-terminus, in addition to the anchor peptide at the carboxy-terminus of the expressed protein, is associated with an unexpected enhancement in the biological effects of the expressed protein. This is especially true where the expressed protein is antigenic or immunogenic in nature.

The carboxy-terminus of the expressed protein or pep¬ tide residue is modified by attachment of a glycolipid anchor, which serves to anchor the modified protein or peptide to the cell surface. The peptide residue to which the GPI anchor is added is always one of small amino acids, such as glycine, aspartic acid, asparagine, alanine, serine and cysteine. These occur at the carboxyl terminus of the protein/peptide of interest and thus can be specified by inclusion of the appropriate codons in the DNA fragment to be added to the cDNA sequence specifying the protein/peptide of interest. In addi¬ tion, the two residues downstream of the anchor addition site are usually small.

The cleavage/anchor addition site resides in a domain of three small amino acid residues, although the central of the three residues has less stringent steric requirements. In order to be certain that functionally or immunologically important amino acids at or near the carboxyl terminus of the protein/peptide target are not compromised, several additional amino acids (preferably, polar ones such as lysine or arginine as well as threonine, alanine and proline) to make up a total of up to 10 residues are inserted in such an orientation so that the small, polar segment is at the carboxyl terminus. The remainder of the addition signal sequence will contain from 15 to 35 amino acids with a hydrophobic domain at the extreme carboxyl terminus. This domain should extend for 15- 25 amino acids and will include amino acids such as valine, leucine, isoleucine, alanine, pphenylalanine, but may also contain proline and glycine as well as tryptophan. A typical such sequence is as follows:

TACDLAPPAGTTD AAHPGRSWPALLPLLAGTLLLLETATAP

The small sequence is in bold face with the left por¬ tion represeting the terminus of the protein and the D residue the site of GPI addition. The right hand portion is that cleaved during GPI addition with the underlined sequence indicating the hydrophobic terminus.

In the present invention, the anchor peptide may have a cleavable N-terminal sequence, which directs the peptide to the endoplasmic reticulum and the cellular trafficking pathway where the GPI anchor is added. As described above, the anchor peptide also has a predominantly hydrophobic sequence at the extreme carboxy terminus which generally ranges in size from about 15 to about 35, more preferably about 15 to 30, and even more preferably about 15 to 25 amino acid residues, signals the addition of the GPI anchor and is cleaved off concurrent with GPI addition. It is the hydrophobicity rather than the sequence itself which is important for anchor addition. Essentially any hydrophobic amino acid sequence of at least about 15 to about 35, more preferably about 15 to 30 amino acid residues would be capable of directing the addition of a GPI anchor. Anchor addition is generally a transamidation reaction in which the free ethanolamine amino group of the GPI precursor attacks (by way of nucleophilic addition) a peptide bond at the target amino acid, which becomes the C-terminal amino acid.

Generally, in the expressed anchor peptide sequence, just upstream of the hydrophobic sequence to which the GPI anchor is added is a hydrophilic spacer (usually about 5-10 residues) which contains hydrophilic amino acids. The residue to which the GPI anchor is added (the "anchor addition site") is an amino acid residue within this hydrophilic spacer selected from the group consisting of glycine, aspartic acid, arginine, asparagine, alanine, serine and cysteine. In addi¬ tion, the two residues downstream from the anchor addition site are also usually small amino acid residues apparently to minimize steric hindrance at the anchor addition site.

Preferably, the GPI portion is preassembled and added as a single unit to a specific amino acid residue near the carboyxl terminus of the expressed protein or peptide. Thus, the carboxyl terminal region may be characterized by the presence of a C-terminal signal peptide whichis preferably ten to thirty amino acids in length and provides the information needed to add the GPI anchor. The actual amino acid residue to which the GPI structure is attached is called the omega site and this residue should be glycine, alanine, cysteine, serine, asparagine or aspartic acid. The omega +1 site (towards the carboxyl terminus of the expressed, unprocessed protein) preferably is selected from glycine, alanine, cysteine, serine, asparagine, aspartic acid, glutamate and threonine. The omega +2 site is alanine or glycine. The omega +2 site is followed by a hinge or spacer of ideally 5 to 7 amino acids that preferably contains charged amino acids and proline; this is followed in turn by a preferably hydrophobic sequence of amino acids which terminate the carboxyl signal peptide.

The overall structure of the anchor peptide may be summarized as a 15-35 amino acid peptide at the carboxyl terminus of the expressed protein or peptide. This anchor peptide sequence (reading from the terminus towards the amino end) begins with a hydrophobic stretch of amino acids of vari¬ able length, followed by a sequence of preferably 5-7 amino acids which contains charged residues, followed by three amino acids (either glycine or alanine at the omega +2 site) ; any of glycine, alanine, cysteine, serine, asparagine, aspartic acid, glutamate and threonine at the +1 omega site; and any of glycine, alanine, serine, cysteine, aspartic acid or asparagine at the omega site.

It is noted that in the present invention, while the signal peptide sequence is generally found at the N-terminus (directly at the N-terminus or removed as much as 1,000 or more amino acids from the N-terminus) and the anchor peptide sequence is generally found at the carboxy-terminus of the expressed protein or peptide, the signal peptide may be found at or near the carboxy terminus of the expressed target protein or peptide.

In the present invention, anchor sequences which are known in the art may be used in the present invention. For example, although it may be possible to utilize yeast or lower trophic order anchor sequences, clearly mammalian anchor sequences are preferred for use in mammals and the specific species signal sequences are most preferred for use in the desired mammalian species to be treated. Thus, in providing for an expressed protein or polypeptide in humans, a human anchor sequence is most preferred.

The term "effective amount" refers to an amount or concentration of recombinant expression vector effective to express or produce quantities of a protein or peptide which is therapeutically effective to treat a disease or immunogeni- cally effective to produce a protective immunogenic response with respect to a disease, i.e. , vaccinate a patient against disease. In general, an effective amount of the expression vector which is administered to a patient will vary depending upon a number of factors associated with that patient, includ¬ ing whether the patient produces any of the peptide or protein to be expressed, whether the patient has previously has been exposed to the peptide or protein to which an immunogenic reaction is desired. An effective amount of vaccinia virus can be determined by varying the dosage of the product and measuring the resulting concentrations of protein or peptide produced. In the case of vaccines, the cellular and humoral immune and/or therapeutic responses, may be measured, prior to administration. In general terms, in humans, an effective amount of a recombinant expression vector may vary over a wide range and generally, as in the case of viral vaccinia vectors, an effective amount represents approximately IO³ to about IO⁸ plaque-forming units, more preferably about IO⁴ to about IO⁷ plaque forming units. In the case of vaccines, a preferred effective amount ranges from about IO⁴ to about IO⁷ plaque forming units, more preferably about 1 X IO⁶ to about 5 X IO⁶ plaque-forming units (determined by assay, as described herein) . It is noted that the above described range of administered vaccinia virus is chosen to enhance the likelihood of producing sufficient quanitities of protein or peptide without producing a toxic response. In the case of vaccines, an effective concentration is that which is capable of eliciting an immunogenic response without causing the very infection in the patient for which he or she is being vac¬ cinated.

It is noted that the chimeric peptide according to the present invention may be administered directly rather than in an expression vector in amounts which are therapeutically effective to treat a disease or immunogenically effective to produce a protective immunogenic response with respect to a disease, i.e., vaccinate a patient against disease.

In using the present method, a desired target peptide or protein is first identified and then sequenced to determine the specific amino acid sequence which is to be ultimately expressed by the administered expression vector. Appropriate amino acid sequences may be readily obtained from isolated proteins or peptides using standard sequencing techniques now commonly available in the art. Alternatively, sequences which are presently available may be obtained from a number of data bases including, for example, GCG and the Brookhaven data base, among others. Target peptide fragments which are also to be used may be easily obtained from the amino acid sequences obtained from isolated proteins.

After identifying the amino acid sequence of the target protein or peptide, the corresponding nucleic acid fragment (generally, but not exclusively, a DNA fragment) is then synthesized. The same will be true for the signal pep¬ tide sequence and anchor peptide sequence to be used. In producing the desired nucleic acid sequences which correspond to the desired protein or peptide sequences to be expressed, one of ordinary skill in the art may use well-known techniques in the art. Nucleic acid synthesizers and techniques for synthesizing DNA fragments are now readily available in the art and these will readily enable one skilled in the art to synthesize, for example, a DNA sequence corresponding to the known amino acid sequence of a protein or peptide to be expressed.

After synthesizing the appropriate DNA sequences which contain restriction digest fragments, the fragments are then cloned and then combined or alternatively, ligated together and then inserted into cloning vectors. It is preferred that the DNA fragments corresponding to the target protein or pep¬ tide to be expressed, the anchor peptide sequence and the sig¬ nal peptide sequence first be cloned and then combined after amplification. The cloning vectors which may be used to pro¬ duce adequate amounts of DNA for amplification, include for example the following cloning vectors: pBR322, pGEM3z, pSP70, pSE420, pRSET, lambdaZAP all commercially available, among others. Such restriction digest fragments may be obtained from clones isolated from eukaryotic or prokaryotic sources.

The appropriate DNA sequence (either the protein or peptide to be expressed alone or alternatively, in combination with the anchor peptide sequence and optionally, the signal sequence) is cloned in an appropriate cloning vector, iso¬ lated, for example, using gel electrophoresis or a related method and then amplified by incorporation into an amplifica¬ tion vector. The desired DNA sequence also can be amplified by standard polymerase chain reaction technique for a suffi¬ cient number of cycles to obtain a desired quantity of DNA (depending upon the amount of DNA desired, from about 5 cycles to about 40 cycles or more) . The DNA coding for the anchor peptide sequence and optionally, a signal peptide sequence may be incorporated into a vector containing the desired peptide or protein to be expressed and, after identification (selec¬ tion and screening) of the appropriate DNA fragments in posi¬ tive clones by PCR and endonuclease digestion, amplified accordingly using the same techniques.

After amplification, the nucleic acid, preferably in the form of DNA, is incorporated into a transfer vector and transfected with eukaryotic cells, for example, monkey kidney cells (BSC-1 cells) , and with wild-type vaccinia virus (WR) to produce recombinant vaccinia virus. The recombinant vaccinia virus is then purified before amplification. After amplifica¬ tion and in some cases further purification, the recombinant vaccinia virus is then administered to an animal as a therapeutic or immunogenic dosage form which expresses the desire protein or peptide, preferably, in combination with the anchor peptide and optionally, the signal peptide.

It is to be noted that it may be preferable to incor- prorate more than one copy of the DNA sequence corresponding to the desired protein or peptide in combination with the anchor peptide sequence and optionally, the signal peptide sequence and with other operational elements. In this manner multiple copies of the same target protein, in combination with anchor peptides and optionally, signal peptides, may be expressed. In one embodiment, multiple proteins may be expressed in a single polycistronic vector by placing the expression of each desired protein or peptide and anchor pep¬ tide (and optionally, signal protein) under the control of an internal ribosomal entry site or IRES. See, for example, Molla, et al.. Nature. 1992, 356:255; Jang, et al., J. of Virol.. 1989, 263:1651. Using this approach, the amount of expressed protein may be significantly increased.

Once a nucleic acid sequence encoding immunogenic chimeric protein is present in a suitable expression vector, the expression vector may then be used as an therapeutic or immunogenic dosage form or alternatively, the vector may be used for the purpose of expressing the immunogenic chimieric protein in a suitable eukaryotic cell system, for example, to promote the production of the desired peptide sequence outside of the host animal. Such eukaryotic cell systems include, for example, HeLa, L929, T2 or RMA-2, preferably T2 or RMA-S. In this method, the cells which contain the expression vector(s) are grown and then lysed in order to isolate synthetic pep¬ tides which contain the desired protein or peptide sequence in combination with the anchor peptide sequence and optionally, the signal sequence. The isolated peptide sequence may then be used directly as a therapeutic or immunogenic dosage form. Alternatively, the expression vector may be administered directly to the patient where it will express the desired protein or peptide and anchor sequence and render the intended therapeutic or immunogenic effect on the patient.

The expressed protein may be obtained from cell cul¬ ture after the cells are lysed by standard protein purifica¬ tion procedures known in the art which may include, among others, gel electrophesis, affinity and immunoaffinity chromatography, differential precipitation, molecular sieve chromatography, isoelectric focusing and ion-exchange chromatography. In the case of immunoaffinity chromatography, the protein or peptide may be purified by passage through a column containing a resin to which is bound antibodies which are specific for at least a portion of the protein or peptide.

The expressed protein or peptide containing an anchor peptide sequence and optionally, a signal peptide sequence, which is obtained from cell culture may be administered in pure or substantially pure form to a patient in need of such therapy by purifying the crude lysate from cell culture. Preferably, the expressed protein is administered in pharmaceutical dosage form as a composition or formulation comprising a therapeutically or immunogenically effective amount of the expressed protein containing anchor peptide sequence and optionally, signal peptide sequence, in combina¬ tion with a pharmaceutically acceptable additive, carrier or excipient. The formulations may be delivered in unit dosage form prepared by known methods in the art. The amount of expressed protein or peptide administered will vary depending upon the pharmokinetic parameters, severity of the disease treated or immunogenic response desired. Of course, dosages will be set by the prescribing physician considering relevant factors including the age, weight and condition of the patient including, in the case of immunogenic dosage forms, whether the patient has been previously exposed to the microorganism responsible for the disease to be vaccinated against as well as the release characteristics of the expressed protein from pharmaceutical dosage forms of the present invention.

The amount of the expressed protein which is administered according to the present invention comprises an amount effective to produce the intended effect. In the case of therapeutic dosages, the intended effect is to substan¬ tially reduce or eliminate the protein or peptide deficiency and the physiological manifestations such deficiency produces. In the case of the administration of immunogenic expressed proteins or peptides, the intended effect is to obtain an immunogenic response in the patient which provides a substan¬ tially protective effect against infection.

In certain preferred embodiments according to the present invention, the expression vector, preferably in the form of recombinant vaccinia virus, is administered directly to the patient. By administering expression vector directly to the patient, the vector will produce target protein or pep¬ tide directly in the patient, thus reducing or even eliminat¬ ing the need for constant administration of the target protein or peptide. In this embodiment, the amount of recombinant vaccinia virus which is administered to the patient is that amount effective to express sufficient protein or peptide to provide a therapeutic or immunogenic response in a patient. The expressed protein or peptide is combined with an anchor peptide sequence and optionally, a signal peptide sequence in the expression vector to substantially increase the therapeutic effect and/or immunogenicity of the expressed protein or peptide compared to protein or peptide which does not contain a signal and anchor peptide. The therapeutic aspect of the present invention provides a therapeutic effect in substantially eliminating the physiological effects associ¬ ated with a deficiency of the expressed protein or peptide, where as the immunogenic aspect of the present invention pro¬ vides a protective effect against infection.

The pharmaceutical compositions according to the pres¬ ent invention may comprise an effective amount of protein or peptide or alternatively, may comprise an effective amount of the expression vector, in substantially pure form or alterna¬ tively, in combination with a pharmaceutically acceptable additive, carrier or excipient. The compositions can be administered directly, or for convenience of administration, can be added to a pharmaceutically acceptable additive, excipient or carrier. Suitable pharmaceutically acceptable carriers will be apparent to those skilled in the art, and include water and other polar substances, including lower molecular weight alkanols, polyalkanols such as ethylene glycol, polyethylene glycol, and propylene glycol as well as non-polar carriers. Stabilizers and other additives, includ¬ ing anti-adsorption agents, may also advantageously be employed in the present pharmaceutical compositions.

In producing the pharmaceutical compositions according to the present invention, the active ingredient is generally brought into uniform and consistent association with the pharmaceutical additive, excipient or carrier and placed in unit dosage form, generally as a liquid, suspension or solid. Solutions or suspensions which are administered parenterally, for example, intramuscularly, intravenously, subcutaneously or intraperitoneally are generally isotonic to the blood of the patient or recipient. Such formulations may be prepared by dissolving the active component in water containing physologi- cally compatible substances such as sodium chloride, other salts, amino acids and the like and buffering the pH of the solution conpatible with physiological solutions. The dosage forms are administered in sterile form. Sterile solutions may be prepared by filtering the solution through filters designed to remove any microorganism which might contaminate a prepara¬ tion, without substantially impacting the componentry or effect of the pharmaceutical compositions.

The present invention, especially as it relates to the administration of the expressed protein directly (i.e., not in the form of an expression vector which will produce protein or peptide in the recipient) , may be administered in a manner which controls the release of the active. Controlled release preparations may be achieved through the use of a pharmaceuti¬ cally compatible polymer or mixture of polymers to complex or absorb the proteins or peptides or their derivatives or by incorporation into microcapsules, liposomes, albumin micro¬ spheres and microemulsions, among others.

Oral preparations are prepared by combining the active agent (either as expressed protein or peptide or the expres¬ sion vector) with typical oral carriers such as sucrose, lac¬ tose, magnesium stearate, starch, various carbohydrates including gums such as gum arabic, algins and carrageenan and the cellulose ethers such as methyl cellulose and hydroxypropyl cellulose, among others.

In the case of the administration of an expression vector, the route of administration may be intrasmusuclar, subcutaneous, intraperitoneal and even oral, but is preferably intravenous. Dosages of expression vector, preferably recom¬ binant vaccinia virus according to the present invention which are coadministered with carriers will often be about the same as the amount administered alone (in the absence of coad- ministration) . An expression vector may be administered sub¬ stantially in pure form or as a complex with a substance having affinity for nucleic acid and an internalizing factor vound to the substance having affinity for nucleid acid. See, for example, Wu, et al., J. Biol. Chem. , 262, 4429 (1987) and Wagner, et al., Proc. Natl.. Acad. Sci. USA, 87:3655 (1990). Polycations such as polylysine may be complexed with the expression vector for admnistration. Internalizing factors such as ligands having specificity for certain receptors on cells may be employed to direct the expression vector to desirable target cells when administered. Internalizing fac¬ tors may include, for example, transferrin and antibodies which are specific for cell surface proteins to which the expression vector is desirably targetted.

Dosages of the administered expression vectors will be set by the prescribing physician considering relevant factors including the age, weight and condition of the patient includ¬ ing whether the patient has been previously exposed to the microorganism which is the causative agent of the disease against which the patient is being vaccinated and the release characteristics of the expression vector from the pharmaceuti¬ cal dosage forms of the present invention.

In the immunization or vaccine aspect of the present invention, the dose of expression vector, preferably vaccinia virus, will depend upon the form in which it is administered. For example, the vaccine will generally contain a concentra¬ tion of virus ranging from about IO⁴ to about 10⁷ plaque form¬ ing units, preferably about 1 X IO⁶ to about 5 X IO⁶ plaque- forming units, depending upon the desired levels of expressed immunogenic protein. Thus, the concentration or amount of vaccinia virus included within the present vaccine will generally fall within this range; however, the amount of recombinant vaccinia virus used in any vaccine form will depend upon the strength of the immunogenic response elicited.

In the therapeutic aspect of the present invention, the dose of expression vector, preferably vaccinia virus, will generally range depending upon the concentration of the expressed protein desired (which usually depends upon the severity of disease and the type of protein to be expressed) and the number of copies or the concentration of protein or peptide which any given vector may produce. Generally, in the case of vaccinia virus expression vectors in this therapeutic aspect of the invention, the concentration will generally range from about IO⁴ to about 10¹⁰ plaque-forming units, more preferably within the range of about IO⁶ to about IO⁷ plaque- forming units. In this therapeutic aspect of the invention, the concentration of expression vector may be higher than in the vaccine aspect, of course depending upon the desired con¬ centration range of the expressed protein or peptide.

In determining the amount of expression vector, in particular, vaccinia virus in a given vaccine dose, the fol¬ lowing method may be used. In certain vaccine dosage forms, standard pharmaceutical carriers as described above may be included. The ratio of virus included in the vaccine will depend on the chemical nature, solubility, and stability of the virus, as well as the dosage contemplated. For parenteral administration or injection via such parenteral routes as intraperitoneal, intramuscular, subcutaneous, intrama mary or other route, sterile solutions of the vaccinia virus are prepared. Vaccines according to the present invention may also be administered intravenously. Preferably, the vaccines according to the present invention are administered via a sub¬ cutaneous route.

The dosage of the vaccine employed and the treatment schedule would follow practices normally employed for other vaccination or therapeutic regimens wherein this general method of treatment is employed. It is not anticipated that more than one therapeutic dose or vaccine initially would be required, but the possiblity of providing booster doses, in the case of vaccine is anticipated. Preferably, the dosage schedule for immunization against most microorganims involves the subcutaneous injection of at least about 1 X IO⁶ plaque- forming units of vaccinia virus.

In the therapeutic method according to the present invention, a human patient is administered with an effective amount of expression vector, preferably, recombinant vaccinia virus, such that expression of a target protein or peptide in combination with an anchor peptide sequence and optionally, a signal peptide sequence takes place. In certain instances, an additional boost of recombinant vaccinia virus may be given to promote the therapeutic or immunogenic response. Additional doses of recombinant recombinant virus may be provided to boost the initial inoculation, if needed.

The present invention contemplates, as a preferred embodiment, the incorporation of both a signal peptide sequence and anchor peptide sequence into the expressed protein or peptide. This combination is found to be particularly advantageous in producing therapeutic and immunogenic responses to the target protein or peptide. The signal peptide sequence is preferably incorporated into the target protein or peptide at or near the amino end of the target protein or peptide and the anchor peptide is generally incorporated at or near the carboxyl end of the protein or peptide. After administration of the recombinant expression vector, preferably a recombinant vaccinia virus, the protein or peptide is expressed by the vector accordingly and will contain a signal peptide sequence and an anchor peptide sequence. Thus, in the present invention, the signal and anchor peptides are preferably expressed at the amino and car¬ boxy terminus of the expressed protein or peptide, respec¬ tively. Accordingly, in preferred embodiments, the signal peptide sequence is located upstream from the expressed protein or peptide and the anchor peptide is downstream from the protein or peptide.

The following examples are provided for purposes of illustration only and are not to be viewed as a limitation of the scope of the invention.

EXAMPLES

Example 1- Herpes Simplex

It has been established that neutralizing antibodies directed against the virus recognize surface glycoproteins of the organism with two glycoproteins (Glycoproteins B and D) functioning as predominant antigens. Recombinant glycoproteins have been produced in Chinese Hamster Ovaries (CHO) cells and have been shown to provide a measure of pro- -33 -

tection for guinea pigs against a direct challenge. The extent of protection varies and generally depends upon Freund's adjuvant, which preclude its use in humans.

Clones of the known antigenically effective herpes glycoproteins (gBl/gB2) are available. In this application, a cloned cDNA encoding the information for glycoprotein B of the herpes virus is augmented by the addition of a mammalian sig¬ nal sequence at the 5' terminus of the DNA (for effective insertion at the N-terminus of the expressed glycoprotein) and an anchor sequence at the 3' terminus of the DNA. Alterna¬ tively, a cDNA encoding for the herpes protein gK may also be used. This is accomplished by initial extension of the 5' sequence through the use of a specific oligonucleotide encompassing homology with the existing 5' sequence and a defined cleavage site for a restriction endonuclease. A similar strategy is used to append, at the 3' terminus of the cDNA, a sequence encoding for the amino acids (anchor sequence) which are recognized by the enzyme system which appends a glycosylphosphatidyl inositol chain to the carboxyl terminus of the protein. In this latter case, the restriction site is positioned at the 3' end of the sequence. The engineered cDNA construct is then ligated into a suitable transfer vector or host organism using techniques well known to those of ordinary skill in the art. A suitable transfer vector is one that can be utilized for the production of vac¬ cinia virus, for example, pSC65. A suitable host organism may be an enteric organism which can be utilized as the recipient to prepare a vehicle suitable for direct oral administration.

Recombinant vaccinia virus is then prepared by co- infection of mammalian host cells (such as BSC-1) , preferably, human host cells, with wild type vaccinia virus and the trans¬ fer vector which has, in addition to the cDNA construct of interest, sequences with homology to non-essential regions of the vaccinia genome and sequences that code for a selectable marker. Utilizing standard methods, the recombinant virus may be isolated. This will, on inoculation into a human host, infect cells and cause the production of the herpes glycoprotein in such a fashion as to maximize its interaction with cells of the human immune system. The vaccinia serves as local "recruiting" signal to the host immune cells thereby obviating the need for any adjuvant. The signal and anchor additions to the protein of interest assure proper transit of the secretory pathway of the cell and positioning of the final molecular product (glycoprotein B, for example) on the surface of the infected cell in an orientation suitable for interac¬ tion with immune cells.

Example 2- Cytomegalovirus

The acquisition of immunity prior to conception should sharply reduce the incidence of adverse effects. It has been established that infected individuals are able to mount an immune response that includes both humoral and cellular com¬ ponents; trials have been conducted with an attentuated strain of the virus as an immunogen. Attempts have been amde to con¬ fer protection by administration of a protein antigen combined with an adjuvant.

In this example, a recombinant vector is constructed which contains, in addition to the genetic information for the viral protein, a combination of signal and anchor sequences illustrated for Herpes simplex, above that will insure the correct trafficking and access to the host immune system.

The protein chose for incorporation into the expres¬ sion vector is gB. This the advantage of providing a known immune response and of being highly conserved in different strains of the virus. The cDNA for this protein is available and has previously been successfully expressed in insect cells.

The general strategy described above for Herpes simplex is followed for the gB cDNA. The region at the 5' terminus for the viral signal sequence is removed and replaced by one encoding for a mammalian signal sequence. This is accomplished by nuclease digestion, ligation with a synthetic oligonucleotide containing the sequence of interest and requisite overlap, selection of recombinant product and fur¬ ther modification at the 3' end of a sequence conferring the information needed to append a GPI anchor to the finsihed protein. This is achieved in much the same manner as for the incorporation of the signal sequence- by truncation of the DNA at the normal 3' terminus, addition of the oligonucleotide encoding the desired sequence and extension with the normal 3'untranslated sequence.

The final construct (in vaccinia or oral vector, such as a bacterial vector) would contain the coding sequence for the gB protein with the 5' and 3' additions of a signal and anchor sequence respectively. The vehicles chosen for admnistration can be viral or bacterial. The former is preferred for subcutaneous administration and the latter for oral administration.

Example 3- Hepatitis

Recombinant clones of the hepatitis B surface antigens (rHBs- in particular, HBsAg) ) cDNA are available and form the starting material for the prepartion of the re-engineered vaccine. Alternatively, another Utilizing the splicing and assembly techniques described above, a delivery organism is constructed which contains as part of its genome the DNA coding for the rHBs with the 5' and 3' additions of a signal and anchor sequence. Inoculation of host animals, (veterinary or human) with the recombinant vector results in the produc¬ tion, on the surface of infected cells, of rHBs oriented to the extracellular domain. This maximizes interaction with the host immune system and thereby confers improved protection.

This general approach can be utilized for producing recombinant expression vectors for immunogenic surface antigens (protein or peptide) from Hepatitis A and C, as well as other Hepatitis strains.

The general approach outlined above may also be used for virtually any protein or peptide sequence to be expressed, even for those proteins or peptides which have yet to be iso¬ lated.

While the invention has been described in its preferred embodiment, it is to be understood that the words which have been used are words of description rather than limitation and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.

Claims

1. A chimeric peptide sequence comprising: a) a target protein or peptide; and b) an anchor sequence peptide; and optionally, c) a signal sequence peptide.

2. The peptide sequence according to claim 1 wherein said target protein or peptide sequence is selected from the group consisting of receptors, hormones, growth factors, clot¬ ting factors, enzymes, neuroproteins, apolipoproteins, tumor suppressors, antigens, tumor antigenic proteins and peptides, bacterial surface proteins and peptides, viral surface proteins and peptides, protozoal cell surface proteins and peptides and fungal surface proteins and peptides and viral enzymes.

3. The peptide sequence according to claim 2 wherein said receptor is selected from the group consisting of an insulin receptor, hormone receptor and growth factor receptor.

4. The peptide sequence according to claim 2 wherein said enzyme is a viral enzyme.

5. The peptide sequence according to claim 4 wherein said viral enzyme is reverse transcriptase.

6. The peptide sequence according to claim 1 wherein said target protein or peptide is a viral surface protein.

7. The peptide sequence according to claim 6 wherein said viral surface protein is gB of cytomegalovirus or an immunogenic sequence thereof.

8. The peptide sequence according to claim 6 wherein said viral surface protein is rHB of Hepatitis B or an immunogenic sequence thereof.

9. The peptide sequence according to claim 6 wherein said viral surface protein is glycoprotein B of Herpes simplex virus or an immunogenic sequence thereof.

10. A pharmaceutical composition comprising the pep¬ tide sequence according to claim 1 in pure or substantially pure form.

11. The composition according to claim 9 wherein said peptide sequence is formulated in combination with a pharmaceutically acceptable additive, excipient or carrier.

12. A method of signficantly enhancing the pharmacological effect produced by a peptide expressed in a patient, comprising incorporating into an expression vector to be administered to said patient a nucleotide sequence encoding said expressed peptide, said sequence also encoding for a mam¬ malian signal peptide or a mammalian anchor peptide at the C- terminus of said expressed protein and administering said expression vector to said patient.

13. The method according to claim 12 wherein said nucleotide sequence encodes for a peptide containing both a signal peptide and an anchor peptide.

14. The method according to claim 12 wherein said expression vector is a recombinant vaccinia virus.

15. The method according to claim 12 wherein said peptide is an antigenic peptide obtained from a bacteria, virus, protozoa or fungus.

16. The method according to claim 15 wherein said peptide is a surface antigen.

17. The method according to claim 12 wherein said peptide is a surface antigen protein of a virus.

18. The method according to claim 17 wherein said virus is Herpes simplex, cytomegalovirus or a hepatitis virus.