The present invention relates to methods for inducing an immunological response in vertebrates, including non-avian vertebrates, using synthetic recombinant avipox virus. More particularly, the invention relates to a method for inducing an immunological response in a vertebrate, particularly a mammal, to a vertebrate pathogen by inoculating the vertebrate with a synthetic recombinant avipox virus containing DNA which encodes for and expresses the antigenic determinants of said pathogen, and to vaccines comprising such a modified avipox virus. Further, the invention relates to modified avipox virus, to methods for making and using the same, and to certain DNA sequences produced or involved as intermediates in the production of modified avipox virus and to methods for making such sequences.
Avipox or avipoxvirus is a genus of closely related pox viruses which infect fowl. The genus avipox includes the species fowlpox, canary pox, junco pox, pigeon pox, quail pox, sparrow pox, starling pox, and turkey pox. The species fowlpox infects chickens, and is not to be confused with the human disease called chickenpox. The genus avipox shares many characteristics with other pox viruses and is a member of the same subfamily, poxviruses of vertebrates, as vaccinia. Pox viruses, including vaccinia and avipox, replicate within eukaryotic host cells. These viruses are distinguished by their large size, complexity, and by the cytoplasmic site of replication. However, vaccinia and avipox are different genera and are dissimilar in their respective molecular weights, their antigenic determinants, and their host species, as reported in Intervirology Vol. 17, pages 42-44, Fourth Report of the International Conmmittee on Taxonomy of Viruses (1982).
The avipox viruses do not productively infect non-avian vertebrates such as mammals, including humans. Further, avipox does not propaqaze when inoculated into mammalian (including human) cell cultures. In such mammalian cell cultures inoculated with avipox the cells will die because of a cytotoxic effect, but show no evidence of productive viral infection.
The inoculation of a non-avian vertebrate such as a mammal with live avipox results in the formation of a lesion at the inoculation site which resembles a vaccinia inoculation. However, no productive viral infection results. Nevertheless, it has now been found that a mammal so inoculated responds immunologically to the avipox virus. This is an unexpected result.
Vaccines composed of killed pathogen or purified antigenic components of such pathogens must be injected in larger quantities than live virus vaccines to produce an effective immune response. This is because live virus inoculation is a much more efficient method of vaccination. A relatively small inoculum can produce an effective immune response because the antigen of interest is amplified during replication of the virus. From a medical standpoint, live virus vaccines provide immunity that is more effective and longer lasting than does inoculation with a killed pathogen or purified antigen vaccine. Thus, vaccines composed of killed pathogen or purified antigenic components of such pathogens require production of larger quantities of vaccine material than is needed with live virus.
It is clear from the foregoing discussion that there are medical and economic advantages to the use of live virus vaccines. One such live virus vaccine comprises vaccinia virus. This virus is known in the prior art to be a useful one in which to insert DNA representing the genetic sequences of antigens of mammalian pathogens by recombinant DNA methods.
Thus, methods have been developed in the prior art that permit the creation of recombinant vaccinia viruses by the insertion of DNA from any source (e.g. viral, prokaryotic, eukaryotic, synthetic) into a nonessential region of the vaccinia genome, including DNA sequences coding for the antigenic determinants of a pathogenic organism. Certain recombinant vaccinia viruses created by these methods have been used to induce specific immunity in mammals to a variety of mammalian pathogens, all as described in U. S. Pat. No. 4,603,112, incorporated herein by reference.
Unmodified vaccinia virus has a long history of relatively safe and effective use for inoculation against smallpox. However, before the eradication of smallpox, when unmodified vaccinia was widely administered, there was a modest but real risk of complications in the form of generalized vaccinia infection, especially by those suffering from eczema or immunosuppression. Another rare but possible complication that can result from vaccinia inoculation is post vaccination encephalitis. Most of these reactions resulted from inoculating individuals with skin diseases such as eczema or with impaired immune systems, or individuals in households with others who had eczema or impaired immunological responses. Vaccinia is a live virus, and is normally harmless to a healthy individual. However, it can be transmitted between individuals for several weeks after inoculation. If an individual with an impairment of the normal immune response is infected either by inoculation or by contagious transmission from a recently inoculated individual, the consequences can be serious.
Thus, it can be appreciated that a method which confers on the art the advantages of live virus inoculation but which reduces or eliminates the previously discussed problems would be a highly desirable advance over the current state of technology. This is even more important today with the advent of the disease known as acquired immune deficiency syndrome (AIDS). Victims of this disease suffer from severe immunological dysfunction and could easily be harmed by an otherwise safe live virus preparation if they came in contact with such virus either directly or via contact with a person recently immunized with a vaccine comprising such a live virus.
It is an object of the present invention to provide a vaccine which is capable of immunizing vertebrates against a pathogenic organism, which has the advantages of a live virus vaccine, and which has few or none of the disadvantages of either a live virus vaccine or a killed virus vaccine as enumerated above, particularly when used to immunize non-avian vertebrates.
It is a further object of this invention to provide synthetic recombinant avipox viruses for use in such vaccines.
It is a further object of this invention to provide a method for inducing an immunological response in avian and non-avian vertebrates to an antigen by inoculating the vertebrate with a synthetic recombinant avipox virus which, in the case of non-avian vertebrates such as mammals, cannot productively replicate in the animal with the production of infectious virus. In this case, the virus is self-limiting, reducing the possibility of spreading to non-vaccinated hosts.
It is a still further object of the invention to provide a method for inducing an immunological response in a vertebrate to an antigen, which method comprises inoculating the vertebrate with a vaccine including synthetic recombinant avipox virus which comprises and expresses the antigenic determinant of a pathogen for said vertebrate.
It is another object of the invention to provide a method for expressing a gene product in a vertebrate by inoculating the vertebrate with a recombinant virus containing DNA which encodes for and expresses the gene product without productive replication of the virus in the vertebrate.
It is yet another object of the invention to provide a method for inducing an immunological response in a vertebrate to an antigen by inoculating the vertebrate with a recombinant virus containing DNA which encodes for and expresses the antigen without productive replication of the virus in the vertebrate.
In one aspect the present invention relates to a method for inducing an immunological response in a vertebrate to a pathogen by inoculating the vertebrate with a synthetic recombinant avipox virus modified by the presence, in a nonessential region of the avipox genome, of DNA from any source which encodes for and expresses an antigen of the pathogen.
In a further aspect, the present invention is directed to a method for expressing a gene product or inducing an immunological response to an antigen in a vertebrate with a recombinant virus which does not productively replicate in the cells of the vertebrate but which does express the gene product or the antigen in those cells.
The virus can be a poxvirus, e.g., an avipox virus, such as a fowlpox virus or canarypox virus. As discussed below, a condition for expression of inserted DNA is a promoter in a proper relationship to the inserted DNA.
Thus, the invention comprehends a recombinant virus, for instance a recombinant poxvirus, e.g., avipox virus such as fowlpox virus or canarypox virus, or vaccinia virus, which contains a promoter operably linked to the inserted DNA for expression of the gene product or antigen. The promoter can be a vaccinia promoter, an avipox promoter, an entomopox promoter.
Accordingly, in certain embodiments, the invention provides a recombinant poxvirus synthetically modified by the presence of DNA not naturally occurring in the poxvirus operably linked to a promoter, e.g., a recombinant poxvirus containing the DNA and an entomopox promoter for expressing the DNA, or a recombinant vaccinia virus containing the DNA and an avipox promoter for expressing the DNA, or a recombinant avipox virus containing the DNA and an avipox promoter for expressing the DNA or a non-avipox promoter such as a vaccinia promoter, e.g., HH, 11K or Pi, or an entomopox promoter, for expressing the DNA.
The methods can comprise inoculating the vertebrate with the recombinant virus, e.g., by introducing the virus into the vertebrate subcutaneously, intradermally, intramuscularly, orally or in ovum.
The antigen can be an antigen of a vertebrate pathogen, e.g., a mammalian pathogen or an avian pathogen, such as a rabies G antigen, gp51,30 envelope antigen of bovine leukemia virus, FeLV envelope antigen of feline leukemia virus, glycoprotein D antigen of herpes simplex virus, avian influenza hemagglutinin antigen, a fusion protein antigen of the Newcastle disease virus, an RAV-1 envelope antigen of rous associated virus, nucleoprotein antigen of avian influenza virus, a matrix antigen of the infectious bronchitis virus and a peplomer antigen of the infectious brochitis virus.
Thus, the vertebrate can be a mammal or a bird, e.g., dog, cat, mouse, rabbit, cattle, sheep, pigs, chicken.
In another aspect, the present invention is directed to synthetic recombinant avipox virus modified by the insertion therein of DNA from any source, and particularly from a non-avipox source, into a nonessential region of the avipox genome. Synthetically modified avipox virus recombinants carrying exogenous (i.e. non-avipox) genes encoding for and expressing an antigen, which recombinants elicit the production by a vertebrate host of immunological responses to the antigen, and therefore to the exogenous pathogen, are used according to the invention to create novel vaccines which avoid the drawbacks of conventional vaccines employing killed or attenuated live organisms, particularly when used to inoculate non-avian vertebrates.
It must be noted again that avipcx viruses can only productively replicate in or be passaged through avian species or avian cell lines. The recombinant avipox viruses harvested from avian host cells, when inoculated into a non-avian vertebrate such as a mammal in a manner analogous to the inoculation of mammals by vaccinia virus, produce an inoculation lesion without productive replication of the avipox virus. Despite the failure of the avipox virus to productively replicate in such an inoculated non-avian vertebrate, sufficient expression of the virus occurs so that the inoculated animal responds immunologically to the antigenic determinants of the recombinant avipox virus and also to the antigenic determinants encoded in exogenous genes therein.
When used to inoculate avian species, such a synthetically recombinant avipox virus not only produces an immunological response to antigens encoded by exogenous DNA from any source which may be present therein, but also results in productive replication of the virus in the host with the evocation of an expected immunological response to the avipox vector per se.
Several investigators have proposed creating recombinant fowlpox, specifically viruses for use as veterinary vaccines for the protection of fowl livestock. Boyle and Coupar, J. Gen. Virol. 67, 1591-1600 (1986), and Binns et al., Isr. J. Vet. Med. 42, 124-127 (1986). Neither proposals nor actual reports directed to the use of recombinaont avipox viruses as a method to induce specific immunity in mammals have been uncovered.
Sticki and Mayer, Fortschr. Med. 97(40), pages 1781-1788 (1979) describe the injection of avipox, specifically fowlpox, virus into humans. However, these studies relate only to the use of ordinary fowlpox to enhance nonspecific immunity in patients suffering from the after effects of cancer chemotherapy. No recombinant DNA techniques are employed. There is no teaching of an avipox into which DNA coding for antigens of vertebrate pathogens had been inserted, or of a method for inducing specific immmunity in vertebrates. Instead, the prior art depended upon a general and nonspecific tonic effect on the human host.
A more complete discussion of the basis of genetic recombination may help in understanding how the modified recombinant viruses of the present invention are created.
Genetic recombination is in general the exchange of homologous sections of deoxyribonucleic acidl (DNA) between two strands of DNA. (In certain viruses ribonucleic acid [RNA] may replace DNA). Homologous sections of nucleic acid are sections of nucleic acid (RNA or DNA) which have the same sequence of nucleotide bases.
Genetic recombination may take place naturally during the replication or manufacture of new viral genomes within the infected host cell. Thus, genetic recombination between viral genes may occur during the viral replication cycle that takes place in a host cell which is co-infected with two or more different viruses or other genetic constructs. A section of DNA from a first genome is used interchangeably in constructing the section of the genome of a second co-infecting virus in which the DNA is homologous with that of the first viral genome.
However, recombination can also take place between sections of DNA in different genomes that are not perfectly homologous. If one such section is from a first genome homologous with a section of another genome except for the presence within the first section of, for example, a genetic marker or a gene coding for an antigenic determinant inserted into a portion of the homologous DNA, recombination can still take place and the products of that recombination are then detectable by the presence of that genetic marker or gene.
Successful expression of the inserted DNA genetic sequence by the modified infectious virus requires two conditions.
First, the insertion must be into a nonessential region of the virus in order that the modified virus remain viable. Neither fowlpox nor the other avipox viruses have as yet demonstrated nonessential regions analogous to those described for the vaccinia virus. Accordingly, for the present invention nonessential regions of fowlpox were discovered by cleaving the fowlpox genome into fragments, then separating the fragments by size and inserting these fragments into plasmid constructs for amplification. (Plasmids are small circular DNA molecules found as extra chromosomal elements in many bacteria including E. coli. Methods for inserting DNA sequences such as the genes for antigenic determinants or other genetic markers into plasmids are well known to the art and described in detail in Man iatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory New York [1982]). This was followed by insertion or genetic markers and/or genes coding for antigens into the cloned fowlpox fragments. Those fragments which directed successful recombination, as proved by successful recovery of the genetic marker or antigens, were those which comprised DNA inserted into a nonessential region of the fowlpox genome.
The second condition for expression of inserted DNA is the presence of a promoter in the proper relationship to the inserted DNA. The promoter must be placed so that it is located upstream from the DNA sequence to be expressed. Because avipox viruses are not well characterized and avipox promoters have not previously been identified in the art, known promoters from other pox viruses are usefully inserted upstream of the DNA to be expressed as part of the present invention. Fowlpox promoters also can be successfully used to carry out the methods and make the products of the invention. According to the present invention, fowlpox promoters, vaccinia promoters and entomopox promoters have been found to promote transcription in recombinant pox virus.
Boyle and Coupar, J. gen. Virol. 67, 1591, (1986) have published speculation that vaccinia promoters xe2x80x9cmight be expected to operate in (fowlpox) virus.xe2x80x9d The authors located and cloned a fowlpox TK gene (Boyle et al., Virology 156, 355-365 [1987]) and inserted it into a vaccinia virus. This TK gene was expressed, presumably because of recognition of the fowlpox TK promoter sequence by vaccinia polymerase functions. However, despite their speculation, the authors did not insert any vaccinia promoter into a fowlpox virus nor observe any expression of a foreign DNA sequence present in a fowlpox genome. It was not known before the present invention that promoters from other pox viruses, such as vaccinia promoters, would in fact promote a gene in an avipox genome.
Fowlpox and canarypox viruses have been particularly used according to the present invention as preferred avipox species to be modified by recombination in incorporating exogenous DNA thereinto.
Fowlpox is a species of avipox which infects chickens in particular, but does not infect mammals. The fowlpox strain designated herein as FP-5 is a commercial fowlpox virus vaccine strain of chicken embryo origin available from American Scientific Laboratories (Division of Schering Corp.) Madison, Wis., United States Veterinary License No. 165, Serial No. 30321.
The fowlpox strain designated herein as FP-1 is a Duvette strain modified to be used as a vaccine in one-day old chickens. The strain is a commercial fowlpox virus vaccine strain designated O DCEP 25/CEP67/2309 October 1980 and is available from Institute Merieux, Inc.
Canarypox is another species of avipox. Analogously to fowlpox, canarypox particularly infects canaries, but does not infect mammals. The canarypox strain designated herein as CP is a commercial canarypox vaccine strain designated LF2 CEP 524 24 10 75 and is available from Institute Merieux, Inc.
The DNA genetic sequences inserted into these avipox viruses by genetic recombination according to the present invention include the Lac Z gene, of prokaryotic origin; the rabies glycoprotein (G) gene, an antigen of a non-avian (specifically mammalian) pathogen; the turkey influenza hemagglutinin gene, the antigen of a pathogenic avian virus other than an avipox virus; the gp51,30 envelope gene of the bovine leukemia virus, a mammalian virus; the fusion protein gene of the Newcastle disease virus (Texas strain), an avian virus; the FeLV envelops gene of the feline leukemia virus, a mammalian virus; the RAV-1 env gene of the rous associated virus which is an avian virus/poultry disease; the nucleoprotein (NP) gene of the Chicken/Pennsylvania/1/83 influenza virus, an avian virus; the matrix gene and peplomer gene of the infectious bronchitis virus (strain Mass 41), an avian virus; and the glycoprotein D gene (gD) of herpes simplex virus, a mammalian virus.
Isolation of the Lac Z gene is described by Casadaban et al., Methods in Enzymology 100, 293-308 (1983). The structure of the rabies G gene is disclosed, for example, by Anilionis et al., Nature 294, 275-278 (1981).
Its incorporation into vaccinia and expression in this vector are discussed by Kieny et al., Nature 312, 163-166 (1984). The turkey influenza hemagglutinin gene is described by Kawaoka et al., Virology 158, 218-227 (1987). The bovine leukemia virus gp51,30 env gene has been described by Rice et al., Virology 138, 82-93 (1984). The fusion gene of the Newcastle disease virus (Texas strain) is available from Institute Merieux, Inc., as plasmid pNDV 108. The feline leukemia virus env gene has been described by Guilhot et al., Virology 161, 252-258 (1987). The rous associated virus type 1 is available from Institute Merieux, Inc., as two clones, penVRVIPT and mp19env (190). Chicken influenza NP gene is available from Yoshihiro Kawaoka of St. Jude Children""s Research Hospital as plasmid pNP 33. An infectious bronchitis virus cDNA clone of the IBV Mass 41 matrix gene and peplomer gene are available from Institute Merieux, Inc. as plasmid pIBVM63. The herpes simplex virus gD gene is described in Watson et al., Science 218, 381-384 (1982).
The recombinant avipox viruses described in more detail below incorporate one of three vaccinia promoters. The Pi promoter, from the Ava I H region of vaccinia, is described in Wachsman et al., J. of Inf. Dis. 155, 1188-1197 (1987). More in particular, this promoter is derived from the Ava I H(Xho I G) fragment of the L-variant WR vaccinia strain, in which the promoter directs transcription from right to left. The map location of the promoter is approximately 1.3 Kbp (kilobase pair) from the left end of Ava IH, approximately 12.5 Kbp from the left end of the vaccinia genome, and about 8.5 Kbp left of the Hind III C/N junction. The sequence of the promoter is:
(GGATCCC)-ACTGTAAAAATAGAAACTATAATCATATAATAGTGTAGGTTGGTAGTA GGGTACTCGTGATTAATTTTATTGTTAAACTTG-(AATTC),
wherein the symbols in parentheses are linker sequences.
The Hind III H promoter (also xe2x80x9cHHxe2x80x9d and xe2x80x9cH6xe2x80x9d herein) was defined by standard transcriptional mapping techniques. It has the sequence
ATTCTTTATTCTATACTTAAAAAATGAAAA TAAATACAAAGGTTCTTGAGGGTTGTGTTAAATTGAAAGCGAGAAATAATCATAAATT ATTTCATTATCGCGATATCCGT TAAGTTTGTATCGTAATG.
The sequence is identical with that described as being up-stream of open reading frame H6 by Rosel et al., J. Virol. 60, 436-449 (1986).
The 11K promoter is as described by Wittek, J. Virol. 49, 371-378 (1984) and Bertholet, C. et al., Proc. Natl. Acad. Sci. USA 82, 2096-2100 (1985).
The recombinant avipox viruses of the present invention are constructed in two steps known in the art and analogous to those disclosed in aforementioned U. S. Pat. No. 4,603,112 for creating synthetic recombinants of the vaccinia virus.
First, the DNA gene sequence to be inserted into the virus is placed into an E. coli plasmid construct into which DNA homologous to a section of nonessential DNA of the avipox virus has been inserted. Separately, the DNA gene sequence to be inserted is ligated to a promoter. The promoter-gene linkage is then inserted into the plasmid construct so that the promoter-gene linkage is flanked on both ends by DNA homologous to a nonessential region of avipox DNA. The resulting plasmid construct is then amplified by growth within E. coli bacteria. (Plasmid DNA is used to carry and amplify exogenous genetic material, and this method is well known in the art. For example, these plasmid techniques are described by Clewell, J. Bacteriol. 110, 667-676 (1972). The techniques of isolating the amplified plasmid from the E. coli host are also well known in the art and are described, for instance, by Clewell et al. in Proc. Natl. Acad. Sci. U.S.A. 62, 1159-1166 (1969).)
The amplified plasmid material isolated after growth within E. coli is then used for the second step. Namely, the plasmid containing the DNA gene sequence to be inserted is transfected into a cell culture, e.g. chick embryo fibroblasts, along with the avipox virus (such as fowlpox strain FP-1 or FP-5). Recombination between homologous fowlpox DNA in the plasmid and the viral genome respectively gives an avipox virus modified by the presence, in a nonessential region of its genome, of non-fowlpox DNA sequences.