AU562548B2 - Creation of Inventions and Patents with Artificial Intelligence

AU562548B2 – Virus with recombinant surface proteins
– Google Patents

AU562548B2 – Virus with recombinant surface proteins
– Google Patents
Virus with recombinant surface proteins

Info

Publication number
AU562548B2

AU562548B2
AU11596/83A
AU1159683A
AU562548B2
AU 562548 B2
AU562548 B2
AU 562548B2
AU 11596/83 A
AU11596/83 A
AU 11596/83A
AU 1159683 A
AU1159683 A
AU 1159683A
AU 562548 B2
AU562548 B2
AU 562548B2
Authority
AU
Australia
Prior art keywords
virus
protein
base sequence
nucleotide base
dna
Prior art date
1982-01-11
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)

Ceased

Application number
AU11596/83A
Other versions

AU1159683A
(en

Inventor
Renato Dulbecco
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

ANIMAL VACCINE RESEARCH CORP

Original Assignee
ANIMAL VACCINE RESEARCH CORP
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
1982-01-11
Filing date
1983-01-07
Publication date
1987-06-11

1983-01-07
Application filed by ANIMAL VACCINE RESEARCH CORP
filed
Critical
ANIMAL VACCINE RESEARCH CORP

1983-07-28
Publication of AU1159683A
publication
Critical
patent/AU1159683A/en

1987-06-11
Application granted
granted
Critical

1987-06-11
Publication of AU562548B2
publication
Critical
patent/AU562548B2/en

2003-01-07
Anticipated expiration
legal-status
Critical

Status
Ceased
legal-status
Critical
Current

Links

Espacenet

Global Dossier

Discuss

Classifications

C—CHEMISTRY; METALLURGY

C07—ORGANIC CHEMISTRY

C07K—PEPTIDES

C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses

C—CHEMISTRY; METALLURGY

C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING

C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA

C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor

C12N15/09—Recombinant DNA-technology

C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts

C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

C12N15/86—Viral vectors

A—HUMAN NECESSITIES

A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE

A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES

A61K39/00—Medicinal preparations containing antigens or antibodies

C—CHEMISTRY; METALLURGY

C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING

C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA

C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses

C12N2710/00011—Details

C12N2710/10011—Adenoviridae

C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses

C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

C—CHEMISTRY; METALLURGY

C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING

C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA

C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense

C12N2760/00011—Details

C12N2760/20011—Rhabdoviridae

C12N2760/20211—Vesiculovirus, e.g. vesicular stomatitis Indiana virus

C12N2760/20222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

C—CHEMISTRY; METALLURGY

C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING

C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA

C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense

C12N2770/00011—Details

C12N2770/32011—Picornaviridae

C12N2770/32611—Poliovirus

C12N2770/32622—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

C—CHEMISTRY; METALLURGY

C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING

C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA

C12N2795/00—Bacteriophages

C12N2795/00011—Details

C12N2795/10011—Details dsDNA Bacteriophages

C12N2795/10311—Siphoviridae

C12N2795/10341—Use of virus, viral particle or viral elements as a vector

C12N2795/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE

Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather

Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Description

VIRUSES WITH RECOMBINANT SURFACE PROTEINS This invention relates generally to the introduction of protein segments having particular biological functions into animals including human _ beings. More particularly, this invention relates to the use of viral carriers to introduce into organisms small peptide segments possessing certain functions normally found on larger protein molecules. BACKGROUND OF THE INVENTION Many biological functions including antigenic functions, hormonal functions, enzymatic functions, and cell-regulatory functions are provided by proteins. Proteins consist of long chains of a ino acids in a particular sequence. The above-mentioned functions are typically attributable to rather limited segments of the protein comprising short sequences of amino acids. The rest of the protein molecule often serves as a carrier for the functional segment or segments. The carrier segments protect the functional segments of the protein and present the functional segments to substrates in an orientation which promotes activity. In addition, certain properties of the functional segments of the protein are only able to take effect when the short sequences of amino-acids comprising the functional segments are connected to a longer protein chain. For example, immune response to a particular short amino-acid sequence generally requires that the short sequence be coupled- to an extended molecule. In principle, the carrier segments of the protein could be replaced with a variety of other carrier segments without altering the properties of the functional protein segment. Such substitutions may effect certain distinct advantages, as will become clear, in the utilization of functional protein segments for commercial or medical purposes, such as the production of useful vaccines.
In particular, it will be seen that viral

proteins are particularly suited to being exploited as carriers for small amino-acid sequences possessing useful functions. One particularly useful function of proteins, typically attributable to limited segments of – a protein, is the ability to induce an immune response. When injected, inhaled, ingested, or otherwise placed into a live animal, a foreign protein, i.e., one not . naturally present in the host animal, elicits an immune response. The immune response consists of many different concerted processes in the animal, including the production of antibodies, which attack the foreign protein and thereby protect the animal from infection by a carrier of the foreign protein. Importantly, an additional feature of the immune response is a form of biological memory such that a second exposure to the same foreign protein results in a quicker and much stronger immune response. This is the principle of vaccination which is an important part of modern medicine. i has been found that effective immune responses are induced by small segments of proteins when they are attached to large carrier segments even if the carrier segments are not naturally of the same protein. Vaccinations with such proteins having a functional segment from one protein attached to unnatural carrier segments results not only in protection against further injection of the hybrid protein but also against the original protein from which the functional segment was obtained. Typically, vaccines are produced in laboratories by preparing agents having substantially reduced pathogenicity with respect to disease-causing viruses that contain protein segments that induce an immune response. These agents are either strains of microorganisms which produce only mild diseases or else are chemically inactivated microorganisms. The vaccines are introduced into an animal to induce an immune
O PI

response in the injected animal; however, there have been problems with such vaccines. Many infectious agents are difficult or impossible to grow under controlled conditions, and those which are grown and – then inactivated present the possiblity of partial escape from the inactivation process which poses an appreciable risk to the vaccinated animal. With . weakened strains of infectious microorganisms, the risk of natural mutation to more dangerous forms is inherent, similarly potentially endangering the vaccinated animal. Moreover, all the techniques involved in the production of such vaccines are time consuming and expensive.
Accordingly, it is advantageous to use, as vaccines, immunogenic (immune-response-producing) protein segments obtained from infectious agents attached to unnatural carriers in place of the infectious agents themselves. In accordance with one aspect of the invention, viral proteins are particularly useful as carriers, and immunogenic protein segments are inserted into viral proteins in such a way that the viruses carry the segments so that they will be exposed to the immune system of a vaccinated animal without the immunogenic protein segments interfering with viral viability or reproduction. Several kinds of viruses can be used to carry immunogenic protein segments, each with distinct advantages. Among these are DNA-containing bacteriophages, nonpathogenic DNA-containing animal viruses and nonpathogenic enveloped RNA-containing influenza viruses.
DNA-containing bacteriophages, such as lambda phage, are viruses which infect bacteria. These viruses multiply to great numbers in bacteria, and they may be produced at small cost, are not pathogenic for animals or humans and can be introduced by ingestion, inhalation, or injection. Nonpathogenic animal viruses, such as the DNA-containing adenoviruses and the

enveloped RNA-containing influenza viruses, replicate in human or animal cells resulting in inapparent or inconsequential infections. These can thus be safely introduced by injection, ingestion or inhalation. – Proteins exposed on the surface of these viruses are preferred as foreign immunogenic protein segments. Surface proteins are capsid proteins in the • case of non-enveloped viruses and trans-membrane proteins in the case of enveloped viruses. When an immunogenic protein segment is incorporated in an exposed manner in a surface virus protein, the entire virus serves as an extended carrier. The virus carrier retains the ability to replicate while the incorporated foreign protein segment has the potential for inducing the specific immune response. The virus carrier also retains its biological functions contributing to protein stability.
Other types of viruses may also be used to advantage as carriers in accordance with the invention. Furthermore, short protein segments with functions other than the capacity to stimulate immune responses may be incorporated as viral surface protein segments by the methods of the invention.
The joining of protein segments with specific functions to protein carriers may be accomplished by taking advantage of recent advances in understanding the genetic code, molecular biological processes and the technology of recombinant DNA genetics. The amino-acid sequences of cellular proteins, as well as most viral proteins, are determined by genes which are segments of deoxyribonucleic acids (DNA) sequenced according to the genetic code. The particular sequence of a ino-acids is synthesized in accordance with the sequence of codons (triplets of nucleic acid subunits) in the DNA. Insertion of foreign DNA sequences into the DNA of a host organism, under certain appropriate conditions, results in the expression of the amiπo-acid sequence
OMPI

specified by the inserted, foreign DNA sequence. Recombinant DNA technology allows such manipulations to be conveniently carried out. Sequences of DNA encoding a particular protein or protein segment – may now be easily isolated and purified in large enough amounts to use biochemically. These sequences can then be cut in specific places, using enzymes known as ■• restriction endonucleaεes, and spliced together with other purified fragments using DNA ligases. These recombinant molecules can then be put into living organisms, such as bacteria or higher cells.
It would be desirable to utilize the recombinant techniques which have been developed to incorporate protein segments having specific functions in surface viral proteins to provide useful agents for commercial and medical processes.
It is an object of the present invention to provide a method for attaching useful protein segments to virus carriers. Another object of the invention is to provide viral carriers of immunogenic protein segments for inducing immune responses in animals. Specifically it is an object to produce new, safer vaccines. A further object is to provide improved vaccinations of mammals, including humans.
SUMMARY OF THE .INVENTION Foreign protein segments are incorporated as exposed segments of surface viral proteins in a manner which does not effect the reproductive viability of the virus. Viruses with recombinant or foreign protein segments are useful for introducing the function, e.g., immune response inducing, of the foreign protein segment into an animal, such as humans. The foreign protein segment is incorporated by inserting a DNA fragment with a nucleotide base sequence coding for the protein segment into the viral genome in a manner in which the inserted DNA fragment expresses itself as an exposed

-6- seg ent of a surface viral protein.
To incorporate the foreign DNA fragment, the viral genome, or a portion thereof, is inserted into a cloning vector which, in turn, is introduced into a host – microorganism to produce multiple copies of the recombinant cloning vector. The foreign DNA fragment is isolated and inserted into the recombinant cloning . vector at an appropriate location within the viral DNA genome portion. The foreign DNA segment-containing viral DNA genome portion is isolated from the cloning vector, and the complete viral genome is reconstructed. The viral genome containing the foreign DNA fragment is packaged as a complete virus; after infecting cells, it will generate progeny in which the foreign protein segment is expressed as a portion of one of its surface proteins.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In accordance with the present invention, functional protein segments are incorporated in viral protein carriers.
Herein the term “virus” shall include bacteria-infecting viruses including bacterial viruses or phages as well as animal-infecting viruses. The term ■recombinant protein” is used herein to refer to a protein which is the expression of a gene containing a foreign nucleotide base sequence, the recombinant protein including an amino-acid sequence which is foreign or unnatural to the protein of the virus.
In general, the method of the protein segment incorporation may be broken down into a series of seven discrete steps, which need not be performed strictly in the order given.
The first step is the selection of a virus having a protein appropriate for use as a carrier of the functional foreign protein segment. The selection of the particular virus depends, in part, on the ultimate use of the protein. “If, for example, a vaccine for

immunizing cattle is desired, an appropriate virus would be one capable of replicating in cattle without causing serious pathological effects. Moreover, it may be appropriate to use a strain of virus to which the cattle – in question have had little or no previous exposure to assure that strong, primary immune reactions develop. Other considerations of virus selection will become , apparent from the examples given below and from facts generally known about bacterial and animal viruses. Once a virus has been selected, a carrier protein of choice is based upon examination of the known molecular biology of that virus. Suitable carrier proteins are on the external surface of the virus, which are nonessential for viability and replication of the virus or which contain regions (where the functional protein segment will be incorporated) that are nonessential for viability and replication. Examples of such appropriate nonessential viral proteins are the D and E gene products of the bacteriophage , the neurominidase protein of orthomyxoviruses; and hexon and protein IX of the adenoviruses.
The second step of the method is to insert the DNA genome of the chosen virus, or a portion of the DNA genome containing the gene coding for the chosen viral carrier protein, into a cloning vector, such as a plasmid vector. The plasmid vector allows the DNA of the virus to be propagated in bacteria wherein a large number of copies of this DNA are produced for further manipulations. The virus genome portion is inserted into a plasmid by standard techniques. In brief, the plasmid, which is a circle of a particular DNA sequence, is cut at known sites with one or more restriction endonucleases. The same enzymes are used to cut the viral genome at specific sites chosen to contain the gene of interest between them, the sites of cutting being determined by the particular restriction enzymes

used. As is well known, cutting two different pieces of DNA with the same restriction enzyme leaves fragment ends which stick to each other by base pair hydrogen bonding and which may be covalently joined by the enzyme – T4-DNA ligase. If the sites of cutting necessitate the use of different restriction enzymes, the ends may be rendered compatible by treatment with the enzyme SI or , the Klenow fragment of DNA Pol I followed by the addition of short DNA segments called linkers. Once the compatible viral genome portion is joined to the plasmid, one of the plasmid genes is typically destroyed. The destroyed gene, in general, is one that codes for a drug resistance. Plasmids usually carry several such genes that encode for proteins which convey resistance to different drugs. This provides an easy method for screening for a successfully recombined plasmid. The plasmid is put or “transformed” into bacteria, and then colonies of bacteria are screened for various drug resistances. Multiple copies of the recombinant plasmid are obtained from lysing the cell colony.
The third step consists of isolating a DNA fragment having the nucleotide base sequence coding for the functional sequence of amino acids to be incorporated as the foreign protein segment of the viral protein. Such DNA fragments are obtained from DNA cloned in a vector or are otherwise prepared in pure form. The gene coding for the entire functional protein is isolated in its entirety by cutting with restriction enzymes. The desired DNA fragment is then isolated by separating various DNA fragments by standard techniques, generally by electrophoresis through an agarose or acrylamide gel, but also by other means, such as columns or gradients. Under certain conditions, the functional segment of the protein in question may not be known in advance. In such case, many fragments of the gene are

generated with restriction enzymes. The mixture of fragments is then used in place of a purified DNA fragment which would otherwise be isolated, and a recombinant virus having the correct DNA fragment is * obtained in the final step of the method when a functional screening is performed on the recombinant viruses as described below.
The DNA fragments coding for the functional protein segment are further prepared by joining them to appropriate linkers of various lengths. The type of linker is chosen to be compatible with the enzyme used to cut the viral gene in the fourth step. Linkers of various lengths are used in order to place at least some of the DNA fragments in the proper reading frame (so that three-nucleotide codons specifying amino acids are incorporated in the viral protein in phase with the codons of the viral DNA) and also to allow some flexibility in the recombinant protein, e.g., to assure that, in at least some of the recombinant proteins, the foreign segment is exposed in a manner which promotes its functioning. The fragments are joined to the linkers by first treating with an enzyme, such as S 1 or the Klenow fragment of DNA Pol I, followed by joining with T4 DNA ligase. Excess linkers are removed, typically with a column.
The fourth step is to cut the viral genome portion to prepare it for the insertion of the foreign DNA fragment coding for the functional protein segment, and then to attach the two together in a productive way. If nonessential portions of the viral protein are known, an enzyme for cutting in that region of the DNA is used; otherwise a variety of enzymes cutting in different places in the viral gene are utilized. If the enzymes cut more than once in the viral gene, a partial digestion is performed, and DNA molecules with single cuts existing in the region of choice are isolated by electrophoresis through a gel. Once the plasmid-bound

viral DNA is cleaved, it is joined to the earlier prepared foreign DNA fragment from step three with T4 DNA ligase to produce a plasmid which contains the remainder of the original plasmid, at least a portion of – the viral genome and the the foreign DNA fragment within the viral genome portion. This modified plasmid is introduced by transformation into cells, typically of . the same culture that was transformed in step 2, to produce multiple copies of the modified plasmid. Multiple copies of the plasmid are obtained from the culture lysate.
The fifth step of the method is to reconstruct the viral genome with the desired foreign DNA fragment inserted. The viral DNA genome portion with the incorporated foreign DNA fragment is released from the plasmid by digestion with the same restriction enzyme or enzymes used for preparing the viral DNA fragment for insertion into the plasmid in step two. If only a portion of the virus genome was inserted (rather than the entire viral genome) , the virus is reconstructed by joining with other fragments in a series of well known steps, each using the enzyme T4 DNA ligase followed by purifying accurately joined fragments.
In a sixth step, the viral genome is packaged to produce an intact infectious virus with a complete protein coat. The packaging procedure depends upon the virus involved. Bacteriophage DNA, such as lambda phage, is packaged into viruses jji vitro by well known reactions using purified phage extracts. Animal virus genomes are placed into cells, typically by a technique known as DNA-calcium phosphate coprecipitate transfection. Precipitates made by mixing DNA, calcium chloride and phosphate buffer are known to be taken up by animal cells. Once in the cells, the viral DNA produces specific RNAs, and the RNA’s direct the synthesis of proteins from which the intact viruses are ultimately formed.

If packaged in vitro from phage extracts, the packaged viruses are transformed into a host cell culture in which the packaged virus strain reproduces. If packaged by transfection, viruses are reproduced in – the transfected culture. By growth in culture, substantial copies of recombinant viruses are obtained. The packaging and reproduction of recombinant . viruses constitutes a selection for viral genomes which have been reconstructed in such a way that the resulting viruses are reproductively viable. It is to be appreciated that virus genomes, which produce the protein coat of the complete virus and which are reproductively viable as complete viruses, may represent only a portion of the recombinant DNA products of the process. Recombinant genomes missing necessary genome segments or having necessary genome segments in the wrong reading frame do not form the protein coat or do not reproduce if packaged. After packaging and reproduction of correctly recombined virus genomes, incorrectly recombined genomes are either lost or represent an insignificant portion of the recombinant viruses. Packaged, reproductively viable viruses can be seen by standard methods, such as plaque assays, and positive plaque assays are indicative of successful genome recombination. Some of the successfully recombined genomes contain the foreign DNA fragment, and some of these express the function of the foreign protein segment.
The final step of the invention consists of screening individual plaques of viruses from step six (each representing an independent recombinant) for the functional incorporated protein segment. The particular screening procedure depends on the type of function desired from the recombinant virus. Standard assays exist for many hormonal and enzymatic functions which may be desired. In the case of assaying for the ability to induce an immune response, the plagues are screened

with antiserum raised against the original protein. Viruses are obtained and purified from duplicates of plaques sensitive to the antiserum, and the viruses are injected into host animals, e.g., rabbits. Antibodies – produced by the animal against the injected virus are, finally, tested for the ability to cross-react with the original protein. The induction of antibody production . to the original protein by the virus is conclusive proof that the virus incorporates at least an immune response-inducing segment of the original protein in a manner that the segment is exposed to the immune system of the host animal.
Since the virus incorporating the exposed recombinant protein segment is known to induce an immune response, it is useful as a vaccine provided that it remains substantially non-pathogenic and provided that the immune-response the virus induces results in effective neutralization of the infectious agent which naturally carries the immune-response-inducing protein segment. Because the virus into which the foreign protein is incorporated is in itself non-pathogenic, it is generally true that the virus with the recombinant protein segment is non-pathogenic as well, but this must be ascertained in each case. Whether the virus having the recombinant protein segment induces an immune response that counteracts the infectious agent must also be determined in each case, and an effective dose is determined for those viruses (vaccines) which do induce immunity to the infectious agent. Viruses having recombinant protein segments demonstrating effectiveness as vaccines are grown in appropriate cell cultures, and the viruses are recovered from lysates of such cultures. The method of administering the vaccine will vary according to the infectious agent being vaccinated against but is well known in the art; vaccines produced according to the invention may be administered along with a
OMPI

pharmaceutically acceptable diluent by injection, by ingestion through the mouth, nose, eye, ear or other body orifice, or by inhalation. The virus is admixed with an appropriate carrier suitable for the intended – method of introduction. For example, viruses having recombinant protein segments may be admixed with an aerosol and administered to animals through the air for • inhalation. An effective amount of the virus is administered as is well known in the art. Generally, viruses which contain a surface protein segment that induces an immune response to infectious agent are administered in an amount of about 10 9-1010 particles per kilogram of body weight of the animal.
The usefulness of viruses having recombinant protein segments is not limited to inducing immunological responses, although an immediate practical use of such viruses is as artificial vaccines. Viruses might, for example, incorporate a protein segment which has an enzymatic or hormonal function. By inducing a controlled, non-pathagenic infection in an animal, a continuous supply of a needed hormonal or enzymatic function might be made available. For example, a virus incorporating a segment of a gonadal hormone might be useful in long-term control of fertility in an animal. For purposes of further illustrating the invention the following examples are set forth. These examples are not intended to limit the scope of the invention.
EXAMPLE .1 This example is of the construction of a recombinant phage as a carrier of an antigenic site for vesticular stomatis virus (VSV) G protein, which is responsible for attachment of the virus to the host cells in the initial phase of infection. Antibodies to the G protein cause virus neutralization, i.e., abolish infectivity. Amino-acid sequences containing antigenic sites of the G protein are candidates for VSV vaccines.

The purpose of this construction is to introduce the antigenic site of VSV into the D or E subunits of the head coat protein of bacteriophage lambda (which multiplies in Escherichia coli) in such a – way that the VSV antigenic site is exposed to the outside of the phage coat and accessible to the immune system of a vaccinated animal. The insertion is made in , such a way as not to interfere with the assembly of the lambda phage, nor with its infectivity. The constructed lambda phage thus contains the foreign or recombinant protein segment in its protein coat. In this recombinant, the carrier lambda phage contributes the viral stability, ability to reproduce abundantly in simple media and carrier function for immunogenicity; the incorporated protein segment contributing the specific function, i.e., the antigenic site for VSV neutralization.
The construction of the recombinant protein is carried out by iτ± vitro recombination between the DNA of phage lambda and DNA containing the G gene of VSV. The introduction of the DNA segment containing a G antigenic site into lambda phage DNA yields the advantage that the recombinant protein is generated in the regular phage multiplication so that all lambda phage that is produced carries it.
The choice of lambda phage, a bacteriophage that is non-pathogenic to animals, as an appropriate carrier virus for the G gene fragment constitutes step 1 of the general method. Step 2 involves the insertion of portions of lambda phage DNA into a plasmid which can be easily grown in a cell culture. In lambda phage, the two main proteins constituting the head coat are specified by genes D and E in the 0.11 to 0.15 kb segment of the phage DNA genome from its left end. For simplifying insertion of the VSV sequences, the lambda DNA is fragmented using restriction endonucleases Bam HI and

Kpn I. This isolates a fragment between 0.113 and 0.360kb of the phage DNA, including the D and E genes. This fragment is joined to plasmid pBR322 (containing a Kpn I site) which has been cut with Kpn I and Bam HI – enzymes. The PBR 322 plasmid with the inserted viral DNA fragment is transformed into a culture of E coli, and the recombinant plasmid is reproduced therein as the «• E coli is cultured in a suitable synthetic or broth medium as is well-known in the art. The recombinant plasmid imparts tetracycline-sensitivity and a plicillin-resistance to the transformed E coli providing an easy method of selecting E coli cultures infected with recombinant plaε ids. Upon lysing positively tested cultures of E coli, multiple copies of recombinant pBR322 are released.
The third step is the isolation of the DNA sequence encoding the functional segment of the G VSV protein. The selection of the G VSV gene fragment is based on the known amino-acid sequence, which implicates several base sequences in the determination of antigenic sites. The largest Alu I fragment of the G VSV gene is isolated and then cut with Sau 3a enzyme. The smaller fragment contains one of the antigenic sites, the larger segment two other sites. The latter two sites are further resolved by Hind III digestion.
In the fourth step, the isolated G VSV gene fragments are inserted into the plasmid-bound virus genome fragment after the plasmid-bound fragment is prepared by restriction enzyme digestion. The plasmid is cut by partial digestion with one of a variety of restriction enzymes, which cut lambda phage in the regions of genes D and E, and plasmid DNA with a single cut in those regions are isolated. These single-cut plasmid fragments are recombined with various G VSV gene fragments with appropriate linkers at their ends.
Linkers of various lengths are used in order to insure that at least some of the G VSV gene fragments are
OMPI * ι. wipo

placed in the proper reading frame and to allow some flexibility in the recombinant protein. To obtain multiple copies of the recombined plasmids, containing both the viral genome fragments and the G VSV fragment, – the plasmids are reproduced by transformation in E coli as per step 2 hereinabove, and multiple copies of the recombined plasmid is obtained from E coli lysate.
Step 5 consists of reconstituting the intact viral genome. The lambda phage DNA fragment is released from the recombinant plasmid by digestion with KPn I and Bam HI enzymes, and the released fragment is reconnected in two steps, first to the left end Bam HI fragment of phage lambda DNA and then to the right end KPn I fragment. The sixth step, in the case of bacteriophage lambda, involves packaging iji vitro the reconstituted lambda phage DNA into lambda capsids. The packaging of the phage is carried out in vitro with purified phage extracts according to the method of Sternberg, Tiemeier and Enquist. The recombinant phage is reproduced by infecting cultures of E coli therewith, and the lysing of the E coli releases multiple copies of the recombinant phage.
Finally, recombinant phages are screened by diluting the phage in saline and injecting in rabbits 10 9-1010 virus particles per kilogram of body weight. After 8 days, blood is drawn from the rabbits, and their blood serum is tested by radioimmunoassay for reactivity with G VSV antigen. Reactivity with G VSV antigen demonstrates the production of G VSV antibody by the rabbits, and accordingly, the incorporation of G VSV protein in the phage.
EXAMPLE ,2
This is an example utilizing a animal virus carrier appropriate for a human vaccine against another virus which is, in itself, pathogenic. Polio virus
– vaccines are of two types as presently used. The Salk
ftjREA
OMPI yΛ, WIPO

vaccine consists of chemically crippled polio virus with the inherent risk of a few polio viruses remaining intact in the vaccine and infecting the patient. The Sabin vaccine uses live virus of attenuated strains – which have the inherent risk of reverting to pathological form. To produce a safer vaccine, key immunogenic peptide segments can be inserted into a , truly safe virus, which can then be used to infect patients without the risks inherent in other vaccines. A suitable nonpathogenic virus suitable is adenovirus type 2 (Ad 2) , or vaccine strains of other types. According to step one of the method, an appropriate protein in Ad 2 capsids is identified. One of the proteins known to be exposed on the surface of adenoviruses is the hexon protein; another is “protein IV” or “fiber”. Moreover, the later protein is of various lengths in different strains of adenovirus, and thus some strains must contain regions which can be removed, or substituted for, without reducing infectivity.
The entirety of the Ad 2 genome has been inserted into plasmids in various laboratories. For convenience of further manipulation, the region encoding fiber or hexon is isolated and inserted into another plasmid according to step 2 of the method. The use of fiber will now be pursued. Fiber is known to extend from map units 87 to 91.5 kb. Importantly the fiber-coding region does not overlap messages for other proteins as do other Ad 2 genes. The Hind III “F” fragment (one of the products of the full digestion of Ad 2 DNA with the enzyme Hind III) extends from 89.5 to 97.3 map units. This fragment is isolated first. Ad 2 DNA genome is cut with Hind III, and the products separated on a 1% agarose gel. The fragment of appropriate size is removed by the technique of electroelution.
The plasmid pBR322 is prepared by Hind III

digestion and treatment with CIP to prevent religation. The F fragment is then inserted in the plasmid by treatment with T4 DNA ligase. A partial Hind III digestion is performed, and molecules cut only once are – purified from a gel. This material is Sma I digested. Sma I cuts at 91 map units releasing the DNA fragment extending from 91 to 91.3 map units. The fragment from , 89.5 to 91 units is still attached to the plasmid. Since Sma I is a “blunt end cutter”, the DNA can be directly joined to Hind III linkers. After further Hind III cutting, the plasmid is closed with T4 DNA ligase. The fragment, 89 to 91 map unit fragment, now carried in the plasmid, is entirely within the coding region of Ad 2 fiber (87 to 91.5). The plasmid is transformed into E coli. Ampicillin-resistant, tetracycline-sensitive E coli strains, transformed with recombinant plasmids, are selected, cultured and lysed in order to obtain multiple copies of the recombinant plasmid.
The third step is the isolation of a DNA fragment with a nucleotide base sequence coding for the desired functional protein segment to be attached to the carrier. In this case, the desired function is the ability to stimulate the immune system of a vaccinated human against polio virus. Such a protein segment will be found on the outside of the polio virus. After the virus is fully assembled, a capsid protein, VPO, is cleaved to form two protein segments VP4 and VP2. Since it is accessible to cleavage enzymes, it follows that the amino acids at the VP4-VP2 junctions are on the outermost portions of the virus particle and thus good candidates for immunogenic regions of capsid proteins.
Polio is an RNA* rather than a DNA virus. This creates a problem for the technology as described. However, full length DNA copies of the polio RNA genome have now been made by a process known as reverse transcription.
– DNA coding for the functional protein segment
JfREAt
OMPI

can be isolated from the DNA, produced by reverse transcription, by a double digest with the restriction enzymes Nru I and Bam HI. A 0.5 kb fragment is purified by electroelution from bisacrylamide gel. The fragment – is then further digested into short fragments with Fnn 441 or Mnl I, each of which cuts the fragment in three places.
The small DNA fragments produced, some of which code for the functional protein segments, are prepared for insertion in the phage-bound Ad 2 DNA fragment by blunt ending with Klena or Pol I and then by joining to Bam HI linkers with T4 DNA ligase. Linkers of various sizes are used in order to assure that some fragments will be joined in the proper reading frame in the next step.
The fourth step of the general method involves the joining of the carrier coding DNA to the functional protein segment coding DNA. The plasmid carrying the Ad 2 fragment is digested very lightly with the enzyme Mbo I. Mbo I is a very frequent cutter of the DNA and is used because the best place to incorporate the foreign protein segment is not known in advance. By cutting very lightly, generally, each plasmid-bound Ad 2 fragment is cut one time.. Mbo I and Bam HI give compatible ends, and the polio fragments are attached to the Mbo I-cut Ad2 DNA with T4 DNA ligase. Such a joining will not be recut by the enzyme Bam HI. Again, the recombinant plasmid is transformed into E coli in order to obtain multiple copies thereof. The Ad 2 genome portions containing the inserted pseudo-polio fragments are excised from the plasmid by digesting the plasmid with Hind III.
In the fifth step, the full Ad2 genome, containing the inserted pseudo-polio fragments, is reconstructed in two steps. First, a partial Sma I digest of intact Ad 2 DNA yields the combined G-K Sma I fragment (the right arm) , which can be purified. Since
* REXD
OMPI ^ ATlO

S a I is a blunt end cutter, Hind III linkers are directly attached thereto with T4 DNA ligase at the side cut by Sma I. The left side is the end of the viral DNA and is uncut. This plasmid DNA is Hind III cut, and the – polio DNA-containing Ad 2 viral genome is isolated from the plasmid fragment by electrophoresis. The viral genome fragment is attached to the prepared Ad 2 arm , with T4 DNA ligase, half of the molecules being of the correct orientation. The other required Ad 2 arm is prepared by partial Hind III digestion and isolation of the combined G-E-C-H-D-A-B Hind III fragment. This is then attached to the recombinant polio DNA-containing Ad 2 fragment on the free side generating Ad 2 genomes, one-half having a configuration with potential virus-producing capabilities.
In the sixth step, the recombinant Ad 2 genomes are transfected into Hela cells by phosphate calcium coprecipitation, and the transfected Hela cells are cultured in DME medium with 10% horse serum. Only viruses having all of the genes necessary for virus viability and reproduction, in correct reading frame, generate the viral protein coat and cause lytic infection of the Hela cells to produce progeny viruses. Some of these reconstructed viruses also incorporate polio virus DNA fragments which are expressed in the fiber protein of the Ad 2 virus.
Which of the recombinant viruses incorporate polio virus proteins in an exposed manner is determined by injecting rabbits with various viral fractions to determine whether the rabbits produce antibodies to polio virus. Rabbits are injected with 10 9-1010 particles of recombinant”Ad 2 virus, diluted in saline, per kilogram of body weight. After 8 days, blood is drawn. Reactivity of the blood serum with polio virus is determined by radioimmunoassay. Recombinent Ad 2 virus, incorporating polio virus protein as established by antibody induction in rabbits, has potential as a
JJRE OMPI

safer human polio vaccine.
While the invention has been described in terms of certain preferred embodiments, modifications obvious to one with ordinary skill in the art may be made – without departing from the scope of the invention. For example, while the invention has been described in terms of inserting DNA nucleotide sequences into viral . genomes, an RNA nucleotide sequence would be inserted in viruses which have an RNA genome, working through DNA intermediates, and such recombinant RNA viruses are within the scope of the invention.
Various features of the invention are emphasized in the following claims.

Claims (9)

1. A method of modifying a virus to give the – virus a new biological function comprising inserting a foreign nucleotide base sequence into the viral genome at a location where said foreign nucleotide base 5. seguence will express itself as an exposed segment of surface viral protein.

2. A method according to Claim 1 including determining a surface viral protein which is not critical to the reproductive viability of said virus or 0 a portion of a surface protein which is not critical to the reproductive viability of said virus and inserting said foreign nucleotide base sequence into the viral genome at a location where said foreign nucleotide base sequence expresses itself as an exposed segment of said 5 noncritical surface protein or of said noncritical portion of a surface protein.

3. A method according to Claim 1 including selecting a virus for modification which is non-pathogenic in an animal to which said modified virus 0 is to be administered.

4. A method according to Claim 1 wherein said foreign nucleotide base sequence is inserted into said genome by splicing into a cloning vector a portion of said viral genome containing at least a fragment of the 5 gene for said surface protein, transfecting an organism with said spliced cloning vector to obtain multiple copies of said spliced cloning vector, cleaving said spliced cloning vector at a location within said surface protein gene fragment, l’inking said foreign nucleotide 0 base sequence to the cleaved ends of said surface protein gene fragment, isolating said spliced portion of said viral genome portion containing said foreign nucleotide base sequence from said cloning vector, and joining said isolated genome portion with additional viral genome portions necessary to create a functional viral genome.

5. A method according to Claim 4 including packaging said viral genome which contains said foreign – nucleotide base sequence as a complete modified virus.

6. A method according to Claim 1 including obtaining said foreign nucleotide base sequence by
. determining a segment of a protein that induces an immunological response in an animal and isolating a nucleotide base sequence which codes for said protein segment, and then inserting said isolated foreign nucleotide base sequence into said virus at such location that said modified virus induces an immunological response when administered to such an animal.

7. A method according to Claim 6 including obtaining said virus genome from a virus that is non-pathogenic to said animal, isolating said nucleotide base sequence from an agent which is infectious to said animal, inserting said isolated nucleotide base sequence into said viral genome at a location whereat said nucleotide base sequence is expressed as an exposed protein segment of said modified virus so that the reproductive viability of said modified virus is maintained, and administering said modified virus to said animal to induce an immune response of a magnitude sufficient to counteract future exposure to said infectious agent.

8. A reproductively viable virus having a foreign nucleotide base sequence inserted in its genome, which foreign nucleotide base sequence expresses itself as a biologically active segment of a surface protein of said virus.

9. A virus according to Claim 8 selected from the group consisting of bacteriophages, adenoviruses and enveloped RNA-containing influenza viruses.
-10. A virus according to Claim 8 wherein said
OMPI ψ IPO protein segment is incorporated in a surface protein at a location where said protein segment does not adversely affect viability of the virus.

AU11596/83A
1982-01-11
1983-01-07
Virus with recombinant surface proteins

Ceased

AU562548B2
(en)

Applications Claiming Priority (2)

Application Number
Priority Date
Filing Date
Title

US338416

1982-01-11

US06/338,416

US4593002A
(en)

1982-01-11
1982-01-11
Viruses with recombinant surface proteins

Publications (2)

Publication Number
Publication Date

AU1159683A

AU1159683A
(en)

1983-07-28

AU562548B2
true

AU562548B2
(en)

1987-06-11

Family
ID=23324738
Family Applications (1)

Application Number
Title
Priority Date
Filing Date

AU11596/83A
Ceased

AU562548B2
(en)

1982-01-11
1983-01-07
Virus with recombinant surface proteins

Country Status (16)

Country
Link

US
(1)

US4593002A
(en)

EP
(1)

EP0098299B1
(en)

JP
(1)

JP2593295B2
(en)

AU
(1)

AU562548B2
(en)

CA
(1)

CA1211060A
(en)

DE
(1)

DE3367851D1
(en)

ES
(1)

ES518870A0
(en)

FI
(1)

FI833174A
(en)

GB
(1)

GB2125065B
(en)

GR
(1)

GR77173B
(en)

IT
(1)

IT1164858B
(en)

NO
(1)

NO833205L
(en)

NZ
(1)

NZ202954A
(en)

PT
(1)

PT76079B
(en)

WO
(1)

WO1983002393A1
(en)

ZA
(1)

ZA8358B
(en)

Cited By (2)

* Cited by examiner, † Cited by third party

Publication number
Priority date
Publication date
Assignee
Title

WO1994024268A1
(en)

1993-04-14
1994-10-27
Arthur Webster Pty. Ltd.
Recombinant avian adenovirus vector

AU676042B2
(en)

1993-04-14
1997-02-27
Commonwealth Scientific And Industrial Research Organisation
Recombinant avian adenovirus vector

Families Citing this family (95)

* Cited by examiner, † Cited by third party

Publication number
Priority date
Publication date
Assignee
Title

EP0117767A1
(en)

1983-01-07
1984-09-05
Mgi Pharma, Inc.
Production of parvovirus subunit vaccines

IL70704A0
(en)

1983-01-19
1984-04-30
Amgen
Methods and materials for development of parvovirus vaccines

US5338674A
(en)

1983-02-25
1994-08-16
Wright Stephen E
Process for producing a vaccine for a pathogenic RNA virus and product thereof

NZ209308A
(en)

1983-08-30
1991-08-27
Genentech Inc
Vaccine against hsv involving a truncated membrane-free derivative of a membrane-bound protein

US7264817B1
(en)

1983-08-30
2007-09-04
Genentech, Inc.
Immunogenic composition based on a truncated derivative of a membrane bound protein and process for making it

US5288641A
(en)

1984-06-04
1994-02-22
Arch Development Corporation
Herpes Simplex virus as a vector

CA1282721C
(en)

1984-06-04
1991-04-09
Bernard Roizman
Herpes simplex virus as a vector

US4722840A
(en)

1984-09-12
1988-02-02
Chiron Corporation
Hybrid particle immunogens

IL76751A
(en)

1984-11-01
1991-04-15
American Home Prod
Method and oral vaccines against infectious organisms using live,recombinant adenovirus for the production of antibodies

US5643579A
(en)

1984-11-01
1997-07-01
American Home Products Corporation
Oral vaccines

FR2573436B1
(en)

1984-11-20
1989-02-17
Pasteur Institut

RECOMBINANT DNA COMPRISING A NUCLEOTIDE SEQUENCE ENCODING A DETERMINED POLYPEPTIDE UNDER THE CONTROL OF AN ADENOVIRUS PROMOTER, VECTORS CONTAINING THIS RECOMBINANT DNA, EUKARYOT CELLS TRANSFORMED BY THIS RECOMBINANT DNA, THE CONSTITUTION OF VACCINES

US6696420B1
(en)

1984-11-20
2004-02-24
Institut Pasteur
Adenoviral vector with a deletion in the E1A coding region expressing a hetorologous protein

IL78775A
(en)

1985-05-15
1992-06-21
Biotech Australia Pty Ltd
Oral vaccines

IL79880A0
(en)

1985-08-29
1986-11-30
Inst Medical W & E Hall
Recombinant virus

US5763269A
(en)

1985-09-06
1998-06-09
Syntro Corporation
Recombinant infectious bovine rhinotrocheitis virus

US5310668A
(en)

1986-06-20
1994-05-10
Merck & Co., Inc.
Varicella-zoster virus as a live recombinant vaccine

DE3632038A1
(en)

1986-09-20
1988-03-31
Behringwerke Ag

TISSUE-SPECIFIC VIRAL VECTORS BASED ON THE LPV AND THEIR USE

NZ219515A
(en)

1987-02-10
1989-09-27
Wellcome Found
Fusion proteins comprising influenza virus ha and a nonnatural antigenic epitope

ATE114723T1
(en)

1987-03-02
1994-12-15
Enzon Lab Inc

ORGANISM AS CARRIER FOR ”SINGLE CHAIN ANTIBODY DOMAIN (SCAD)”.

AU628294B2
(en)

1987-07-27
1992-09-17
Syntro Corporation
Attenuated herpes viruses, herpes viruses which include foreign dna encoding an amino acid sequence and vaccines containing same

FR2619012B1
(en)

1987-08-07
1989-12-22
Pasteur Institut

VACCINES IN WHICH THE CHARACTERISTIC EPITOPE IS INCORPORATED IN A PROTEIN OF PICORNAVIRUS, ESPECIALLY POLIOVIRUS

US5182211A
(en)

1987-08-07
1993-01-26
Institut Pasteur
Plasmid vectors encoding a protein of a picornavirus

US5316931A
(en)

1988-02-26
1994-05-31
Biosource Genetics Corp.
Plant viral vectors having heterologous subgenomic promoters for systemic expression of foreign genes

US6054566A
(en)

1988-02-26
2000-04-25
Biosource Technologies, Inc.
Recombinant animal viral nucleic acids

US20030150019A1
(en)

1988-02-26
2003-08-07
Large Scale Biology Corporation
Monopartite RNA virus transformation vectors

US6284492B1
(en)

1988-02-26
2001-09-04
Large Scale Biology Corporation
Recombinant animal viral nucleic acids

US5037743A
(en)

1988-08-05
1991-08-06
Zymogenetics, Inc.
BAR1 secretion signal

US5223409A
(en)

1988-09-02
1993-06-29
Protein Engineering Corp.
Directed evolution of novel binding proteins

ATE221119T1
(en)

1988-12-13
2002-08-15
Harvard College

PROTOTYPICAL FELV ISOLATES FOR USE AS DISEASE SAMPLE OR VACCINE

US5691170A
(en)

1989-04-18
1997-11-25
Therion Biologics
Generation of hybrid genes and proteins by virus-mediated recombination

US7413537B2
(en)

1989-09-01
2008-08-19
Dyax Corp.
Directed evolution of disulfide-bonded micro-proteins

ATE194384T1
(en)

1989-09-12
2000-07-15
Hoffmann La Roche

TNF-BINDING PROTEINS

US5427908A
(en)

1990-05-01
1995-06-27
Affymax Technologies N.V.
Recombinant library screening methods

US5723286A
(en)

1990-06-20
1998-03-03
Affymax Technologies N.V.
Peptide library and screening systems

US20030064480A1
(en)

1990-06-28
2003-04-03
Leander Lauffer
Fusion proteins with immunoglobulin portions, the preparation and use thereof

US6916605B1
(en)

1990-07-10
2005-07-12
Medical Research Council
Methods for producing members of specific binding pairs

US7063943B1
(en)

1990-07-10
2006-06-20
Cambridge Antibody Technology
Methods for producing members of specific binding pairs

US6172197B1
(en)

1991-07-10
2001-01-09
Medical Research Council
Methods for producing members of specific binding pairs

GB9015198D0
(en)

1990-07-10
1990-08-29
Brien Caroline J O
Binding substance

WO1992006191A1
(en)

1990-09-28
1992-04-16
Protein Engineering Corporation
Proteinaceous anti-dental plaque agents

CA2095633C
(en)

1990-12-03
2003-02-04
Lisa J. Garrard
Enrichment method for variant proteins with altered binding properties

US5989886A
(en)

1991-01-02
1999-11-23
The Johns Hopkins University
Method for the inhibition and prevention of viral replication using fusions of a virus protein and a destructive enzyme

ES2287206T3
(en)

1991-03-01
2007-12-16
Dyax Corporation

PROCESS FOR THE DEVELOPMENT OF MINI-PROTEINS OF UNION.

GB9108386D0
(en)

1991-04-19
1991-06-05
Agricultural Genetics Co
Modified plant viruses as vectors

WO1993000103A1
(en)

1991-06-21
1993-01-07
The Wistar Institute Of Anatomy And Biology
Chimeric envelope proteins for viral targeting

WO1993002202A1
(en)

1991-07-19
1993-02-04
Syngene, Inc.
Compositions and methods for reproducing positive diagnostic indications

US5270170A
(en)

1991-10-16
1993-12-14
Affymax Technologies N.V.
Peptide library and screening method

US5733731A
(en)

1991-10-16
1998-03-31
Affymax Technologies N.V.
Peptide library and screening method

WO1993011236A1
(en)

1991-12-02
1993-06-10
Medical Research Council
Production of anti-self antibodies from antibody segment repertoires and displayed on phage

US6511845B1
(en)

1992-08-07
2003-01-28
Alan R. Davis
Methods for producing an immune response against HIV-1

US6057093A
(en)

1992-09-28
2000-05-02
Chiron Corporation
Methods and compositions for controlling translation of HCV proteins

US5633234A
(en)

1993-01-22
1997-05-27
The Johns Hopkins University
Lysosomal targeting of immunogens

US6296852B1
(en)

1993-04-14
2001-10-02
Commonwealth Scientific And Industrial Research Organisation
Recombinant avian adenovirus vector

US6010861A
(en)

1994-08-03
2000-01-04
Dgi Biotechnologies, Llc
Target specific screens and their use for discovering small organic molecular pharmacophores

US5559099A
(en)

1994-09-08
1996-09-24
Genvec, Inc.
Penton base protein and methods of using same

US5962311A
(en)

1994-09-08
1999-10-05
Genvec, Inc.
Short-shafted adenoviral fiber and its use

US5846782A
(en)

1995-11-28
1998-12-08
Genvec, Inc.
Targeting adenovirus with use of constrained peptide motifs

US6465253B1
(en)

1994-09-08
2002-10-15
Genvec, Inc.
Vectors and methods for gene transfer to cells

US5965541A
(en)

1995-11-28
1999-10-12
Genvec, Inc.
Vectors and methods for gene transfer to cells

WO1996012027A1
(en)

1994-10-18
1996-04-25
Scottish Crop Research Institute
Method of producing a chimeric protein

US5770442A
(en)

1995-02-21
1998-06-23
Cornell Research Foundation, Inc.
Chimeric adenoviral fiber protein and methods of using same

US6127525A
(en)

1995-02-21
2000-10-03
Cornell Research Foundation, Inc.
Chimeric adenoviral coat protein and methods of using same

US6783980B2
(en)

1995-06-15
2004-08-31
Crucell Holland B.V.
Packaging systems for human recombinant adenovirus to be used in gene therapy

ES2231813T5
(en)

1995-06-15
2012-12-17
Crucell Holland B.V.

Packaging systems for human recombinant adenovirus used in gene therapy

US6083693A
(en)

1996-06-14
2000-07-04
Curagen Corporation
Identification and comparison of protein-protein interactions that occur in populations

AU4070499A
(en)

1998-04-30
1999-11-16
Cornell Research Foundation Inc.
Adenoviral vectors with tandem fiber proteins

US20040043489A1
(en)

1998-07-08
2004-03-04
Menzo Havenga
Gene delivery vectors provided with a tissue tropism for dendritic cells and methods of use

US20030017138A1
(en)

1998-07-08
2003-01-23
Menzo Havenga
Chimeric adenoviruses

US6455314B1
(en)

1998-09-11
2002-09-24
Genvec, Inc.
Alternatively targeted adenovirus

US6929946B1
(en)

1998-11-20
2005-08-16
Crucell Holland B.V.
Gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells

US6913922B1
(en)

1999-05-18
2005-07-05
Crucell Holland B.V.
Serotype of adenovirus and uses thereof

US20050232900A1
(en)

1999-05-18
2005-10-20
Crucell Holland B.V.
Serotype of adenovirus and uses thereof

US6492169B1
(en)

1999-05-18
2002-12-10
Crucell Holland, B.V.
Complementing cell lines

EP1157999A1
(en)

2000-05-24
2001-11-28
Introgene B.V.
Methods and means for enhancing skin transplantation using gene delivery vehicles having tropism for primary fibroblasts, as well as other uses thereof

JP2003534806A
(en)

2000-05-31
2003-11-25
ジェンベク、インコーポレイティッド

Methods and compositions for targeting adenovirus vectors

US20040033605A1
(en)

2000-09-20
2004-02-19
Menzo Havenga
Gene delivery vectors provided with a tissue tropism for dendritic cells

US7235233B2
(en)

2000-09-26
2007-06-26
Crucell Holland B.V.
Serotype 5 adenoviral vectors with chimeric fibers for gene delivery in skeletal muscle cells or myoblasts

ATE519783T1
(en)

2000-12-22
2011-08-15
Grad Carole Legal Representative Of Kaplan Howard

ßPHAGE DISPLAYß LIBRARY OF HUMAN VH FRAGMENTS

US20030113714A1
(en)

2001-09-28
2003-06-19
Belcher Angela M.
Biological control of nanoparticles

US20030148380A1
(en)

2001-06-05
2003-08-07
Belcher Angela M.
Molecular recognition of materials

US20050164515A9
(en)

2001-06-05
2005-07-28
Belcher Angela M.
Biological control of nanoparticle nucleation, shape and crystal phase

US20030073104A1
(en)

2001-10-02
2003-04-17
Belcher Angela M.
Nanoscaling ordering of hybrid materials using genetically engineered mesoscale virus

MXPA04008891A
(en)

2002-04-25
2004-11-26
Crucell Holland Bv
Means and methods for the production of adenovirus vectors.

US20050064508A1
(en)

2003-09-22
2005-03-24
Semzyme
Peptide mediated synthesis of metallic and magnetic materials

US7329486B2
(en)

2003-03-31
2008-02-12
The Board Of Regents Of The University Of Texas System
High-throughput assay for virus entry and drug screening

EP2139993A2
(en)

2007-04-27
2010-01-06
Dow Global Technologies Inc.
Improved production and in vivo assembly of soluble recombinant icosahedral virus-like particles

US10736928B2
(en)

2016-01-20
2020-08-11
Richard Brian Murphy, JR.
Pegylated recombinant bacteriophage

CA3044074A1
(en)

2016-11-16
2018-05-24
Immunomic Therapeutics, Inc.
Nucleic acids for treatment of allergies

WO2018195527A1
(en)

2017-04-22
2018-10-25
Immunomic Therapeutics, Inc.
Improved lamp constructs

JP7222915B2
(en)

2017-05-02
2023-02-15
イミュノミックセラピューティックス，インコーポレイテッド

Improved LAMP constructs containing cancer antigens

EP3793595A1
(en)

2018-05-15
2021-03-24
Immunomic Therapeutics, Inc.
Improved lamp constructs comprising allergens

JP7097465B2
(en)

2018-06-19
2022-07-07
アルモ・バイオサイエンシーズ・インコーポレイテッド

Composition of IL-10 agent to be used in combination with chimeric antigen receptor cell therapy and its usage

WO2021077051A1
(en)

2019-10-18
2021-04-22
Immunomic Therapeutics, Inc
Improved lamp constructs comprising cancer antigens

WO2021146487A2
(en)

2020-01-14
2021-07-22
Synthekine, Inc.
Il2 orthologs and methods of use

WO2023201201A1
(en)

2022-04-10
2023-10-19
Immunomic Therapeutics, Inc.
Bicistronic lamp constructs comprising immune response enhancing genes and methods of use thereof

Family Cites Families (8)

* Cited by examiner, † Cited by third party

Publication number
Priority date
Publication date
Assignee
Title

DE2712615A1
(en)

1977-03-18
1978-09-21
Max Planck Gesellschaft

PROCESS FOR MANUFACTURING FILAMENTOES PHAGEN AS A VECTOR FOR SYNTHETIC RECOMBINATES

JPS53133684A
(en)

1977-04-26
1978-11-21
Noda Sangyo Kagaku Kenkyusho
Novel bacteriophage and fabricating same

DE2860386D1
(en)

1978-01-05
1981-03-19
Bayer Ag
Molding compositions consisting essentially of unsaturated polyesters, method of preparation and application

JPS5558096A
(en)

1978-10-25
1980-04-30
Noda Sangyo Kagaku Kenkyusho
Method of making novel recombined dna

JPS5561798A
(en)

1978-10-30
1980-05-09
Noda Sangyo Kagaku Kenkyusho
Preparation of novel recombination dna

FR2442271A1
(en)

1978-11-27
1980-06-20
Pasteur Institut

VECTOR FOR THE INSERTION OF A PROKARYOTE OR EUKARYOTE GENE, AND EXCRETION OF THE EXPRESSED PROTEIN

FR2480779B2
(en)

1979-08-30
1986-07-18
Anvar

VECTOR CONTAINING A NUCLEOTIDE SEQUENCE OF THE SURFACE ANTIGEN OF HEPATITIS B VIRUS AND METHOD FOR MANUFACTURING AN IMMUNOGENIC MOLECULE USING THE SAME

US4442205A
(en)

1981-09-22
1984-04-10
The United States Of America As Represented By The Department Of Health And Human Services
Simian virus recombinant that directs the synthesis of hepatitis B surface antigen

1982

1982-01-11
US
US06/338,416
patent/US4593002A/en
not_active
Expired – Lifetime

1983

1983-01-05
ZA
ZA8358A
patent/ZA8358B/en
unknown

1983-01-06
NZ
NZ202954A
patent/NZ202954A/en
unknown

1983-01-07
GR
GR70220A
patent/GR77173B/el
unknown

1983-01-07
GB
GB08322556A
patent/GB2125065B/en
not_active
Expired

1983-01-07
WO
PCT/US1983/000015
patent/WO1983002393A1/en
active
IP Right Grant

1983-01-07
EP
EP83900590A
patent/EP0098299B1/en
not_active
Expired

1983-01-07
AU
AU11596/83A
patent/AU562548B2/en
not_active
Ceased

1983-01-07
CA
CA000419086A
patent/CA1211060A/en
not_active
Expired

1983-01-07
JP
JP58500629A
patent/JP2593295B2/en
not_active
Expired – Lifetime

1983-01-07
DE
DE8383900590T
patent/DE3367851D1/en
not_active
Expired

1983-01-10
PT
PT76079A
patent/PT76079B/en
unknown

1983-01-10
ES
ES518870A
patent/ES518870A0/en
active
Granted

1983-07-10
IT
IT47530/83A
patent/IT1164858B/en
active

1983-09-06
FI
FI833174A
patent/FI833174A/en
not_active
Application Discontinuation

1983-09-08
NO
NO833205A
patent/NO833205L/en
unknown

Cited By (2)

* Cited by examiner, † Cited by third party

Publication number
Priority date
Publication date
Assignee
Title

WO1994024268A1
(en)

1993-04-14
1994-10-27
Arthur Webster Pty. Ltd.
Recombinant avian adenovirus vector

AU676042B2
(en)

1993-04-14
1997-02-27
Commonwealth Scientific And Industrial Research Organisation
Recombinant avian adenovirus vector

Also Published As

Publication number
Publication date

ES8500322A1
(en)

1984-10-01

NO833205L
(en)

1983-09-08

GB2125065B
(en)

1985-05-15

EP0098299A4
(en)

1984-07-05

PT76079B
(en)

1985-11-11

EP0098299B1
(en)

1986-11-26

CA1211060A
(en)

1986-09-09

ES518870A0
(en)

1984-10-01

EP0098299A1
(en)

1984-01-18

AU1159683A
(en)

1983-07-28

WO1983002393A1
(en)

1983-07-21

US4593002A
(en)

1986-06-03

NZ202954A
(en)

1985-08-16

JP2593295B2
(en)

1997-03-26

IT8347530D0
(en)

1983-07-10

GR77173B
(en)

1984-09-10

JPS59500042A
(en)

1984-01-12

DE3367851D1
(en)

1987-01-15

PT76079A
(en)

1983-02-01

FI833174A0
(en)

1983-09-06

IT1164858B
(en)

1987-04-15

ZA8358B
(en)

1983-10-26

GB2125065A
(en)

1984-02-29

GB8322556D0
(en)

1983-09-21

FI833174A
(en)

1983-09-06

Contact information