Vaccines are used to protect against diseases, which are caused by pathogens. These pathogens are microbial organisms, such as bacteria and viruses, which affect animals, including humans. Vaccines are primarily derived from a pathogen by producing and administering either: a) an attenuated or avirulent version of the pathogen; b) the killed pathogen; c) extracted protective antigens or antigen mixes of the pathogen (homologous antigens); or d) a micro-organism expressing one or more protective antigens encoded by cloned genes originating in a microbial pathogen different from the vaccine strain (heterologous antigens).
Vaccines for both bacteria and viruses are engineered from microorganisms expressing one or more protective antigens, as described by K. Jones and M. Sheppard in Designer Vaccines, CRC Press (1997). Vaccines are intended to produce an immune response in the recipient consisting of at least one of an antibody mediated or T cell mediated immune response, thereby preventing future infection by a pathogen, or fighting a current pathogenic infection. In particular, vaccines against facultative intracellular pathogens, those growing inside the cells of the infected host, need to induce a strong and appropriate cell mediated immune response. In contrast, vaccines against obligate extracellular pathogens need to induce an appropriate antibody mediated immune response. Often, regardless of the pathogen, an appropriate combined antibody and cellular mediated immune response leads to sufficient protection or relief from infection. In order to achieve this protection or relief from infection, vaccines may express one or more homologous antigens, heterologous antigens, or a combination of both.
Vaccines may be administered to vertebrates both to prevent and treat infection by pathogens. Thus, vaccines are frequently administered to prevent the spread of a disease caused by a pathogen. In particular, herd animals, such as cows, goats, sheep and swine, are often vaccinated to prevent the spread of a disease among members of the herd. Further, because certain diseases may travel between vertebrates, including travel between various animals and between animals and humans, vaccines are used to prevent the spread of disease between various species, usually by administration to the infected animal and other uninfected animals in the immediate vicinity. However, other animals in the area which are less likely to contract the disease may also be vaccinated as a prophylactic measure. For example, an infected cow and its as yet uninfected herd may be vaccinated to treat a disease and prevent its further spread. As a prophylactic measure, other animals which are likely to contract the disease from the infected cow, such as neighboring cows, sheep or humans, may be vaccinated as well.
It has been found that vaccines derived from an attenuated or avirulent version of a pathogen are highly effective in preventing or fighting disease caused by that pathogen. In particular, it is known that such attenuated or avirulent pathogens can be modified to express heterologous antigens (antigens which are derived from a pathogen of a different species). In order to express heterologous antigens in a desired attenuated or avirulent pathogen, a gene encoding an antigen capable of providing protection against the pathogen is identified from the deoxyribonucleic acid of a heterologous species. The desired gene is isolated and then inserted into a plasmid capable of replication and expression in the attenuated or avirulent pathogen. The plasmid is then introduced into the attenuated or avirulent pathogen, and causes expression of the heterologous antigen upon administration to a subject vertebrate.
An example of such expression of an heterologous antigen is the bacterial vaccine Salmonella, which expresses a Streptococcus spaA protein. See U.S. Pat. No. 4,888,170. This vaccine comprises an avirulent derivative of a pathogenic microbe of the genus Salmonella, which in turn expresses a recombinant gene derived from a pathogen of the species Streptococcus mutans, thereby producing an antigen capable of inducing an immune response in a vertebrate against the pathogen.
A further example of heterologous expression is Vibrio cholera vaccines. A number of live attenuated strains of Vibrio cholera have been developed to vaccinate humans against cholera. See Kaper, J. B., et al., New and improved vaccines against cholera in New Generation Vaccines (eds. M M Levin et al.) Marcel Deker, Inc., NY, 1997. Some of these strains over-express heterologous antigens. See Butterton, J. R. and S. B. Calderwood, Attenuated Vibrio cholera as a live vector for expression of foreign antigens in New Generation Vaccines (eds. M M Levin et al.) Marcel Deker, Inc., NY, 1997. The immunity induced by the attenuated vaccine strains is the result of inducing antibodies which have either antibacterial and/or antitoxic activities. Some strains have been attenuated by the deletion of a number of genes encoding toxigenic components, including the A subunit of the cholera toxin encoded by the ctxA gene. However, in order for a cholera vaccine strain to be fully protective, it is necessary that the ctxB gene encoding the B subunit (to which the A subunit binds) be expressed to allow for the production of antibodies that neutralize the cholera toxin. The ctxB gene has been over-expressed in Vibrio cholera for the purpose of producing large amounts of the antigen cholera toxin B (CTB). The over-expressed antigen CTB is collected, purified and used as a subunit vaccine which is the extracted CTB antigen. See Lebens M., et al., 1993, Biotechnology (NY) December; 11:1574-1578. However, although an over-expressed antigen has itself been used as a vaccine, an attenuated or avirulent pathogen of Vibrio cholera which over-expresses the ctxB gene, or any other homologous gene, has not been used as a live vaccine.
Another example of heterologous expression is in Mycobacterium spp. vaccines, used to prevent tuberculosis in humans. The Mycobacterium tuberculosis GroEL protein induces protective immunity when expressed by the groEL gene transfected into macrophages (Silva, C. L. and Lowrie, D. B., 1994, Immunology 84:244-248), indicating that GroEL protein is a protective antigen if presented to T cells by this type of antigen presenting cell (APC). Naked DNA vaccines using Mycobacterium genes coding for a variety of antigens (hsp70, 85kDa, 65kDa, 36kDa, 6kDa) are also able to induce protective immunity. See Lowrie, D. B. et al., 1997, Vaccine 15:834-838; Tascon, E. et al., 1996, Nat. Med. 2:888-892; and Lozes, E. et al., 1997, Vaccine 15:880-833. It is believed that the naked DNA vaccines work because they transfect APCs (Chattergon, M. et al., 1997, FASEB J. 11:753-763.) which in turn present the antigen appropriately to T cells, thereby inducing a protective cell mediated immunity. M. bovis BCG, a live, attenuated strain of Mycobacterium, is used to induce protective immunity against M. tuberculosis infection in humans. Fine, P M. 1988, Br. Med. Bull. 44:91.
Antigen vaccines developed against Brucellosis provide examples of homologous antigen expression, wherein the antigen is derived from the same species as the attenuated pathogen. Brucellosis is an infectious bacterial disease which can be transmitted to human beings by animals. It is caused by any of a variety of species of pathogenic aerobic bacteria of the genus Brucella. In animals, Brucellosis can result in abortion and infertility. In humans, it causes fever, malaise and headaches. This disease has been extensively studied, resulting in the development of numerous vaccines.
It is known that existing vaccine strains of Brucella, such as B. abortus strains 19 and RB51, and B. melitensis strain REV1, can both protect against the Brucella species from which they were derived and cross protect against infection by other species, such as B. abortus, B. melitensis, B. ovis, B. suis, B. canis and B. neotomae. See Winter, A. J. et al., 1996, Am. J. Vet. Res., 57:677; P. Nicoletti in Animal Brucellosis, CRC Press (1990), pp. 284-296; J. M. Blasco in Animal Brucellosis, CRC Press (1990), pp. 368-370; and G. C. Alton in Animal Brucellosis, CRC Press (1990), pp. 395-400. New B. melitensis strain VTRM1 and B. suis strain VTRS1 also cross protect against various Brucella species. See Winter, A. J. et al., Am. J. Vet. Res., 57:677.
In the past, one of the most commonly used vaccines to prevent bovine Brucellosis was B. abortus strain 19, as described by P. Nicoletti in Animal Brucellosis, CRC Press (1990), pages 284-296. This particular strain of B. abortus provided immunity in cattle with a range of protection from 65 to 75% depending upon a number of variables, such as the age of the cattle at vaccination, the dose administered, the route of administration and prevalence of Brucellosis in the vaccinated herd.
B. Abortus strain RB51, a new attenuated live Brucella vaccine (marketed as RB-51.RTM.), is a stable vaccine approved for use in the United States. See Schurig, G. G. et al, 1991, Vet. Microbiol. 26:359; and Colby, L., 1997, M.Sc. Thesis, Virginia Tech, Blacksburg, Va. Attenuation of strain RB51 is indicated by studies carried out in mice, goats and cattle. See Schurig, G. G., 1991, Vet. Microbiol. 28:171; Palmer R. M. et al., 1997, Am.J. Vet Res. 58:472; Roop, R. M. et al., 1995, Res. Vet. Science, 51:359; and Zambrano, A. J. et al., 1995, Archivos de Medicina Veterinaria XXVIII, No. extraordinario:119-121. In comparison to the protection provided by strain 19, strain RB51 has been shown in single vaccination protocols to be similarly protective in cattle. See Cheville, N. F. et al., 1993, Amer. J. Vet Research 53:1881; and Cheville, N. F. et al., 1996, Amer. J. Vet Research, 57:1153. Further, oral administration of strain RB51 in mice and cattle has indicated protective immunity. See Stevens, M. G. et al., 1996, Infect. Immun. 64:534. In particular, the mouse model indicates that the protective immunity to Brucellosis induced by strain RB51 is solely T cell mediated because a passive transfer of RB51-induced antibodies does not protect against the disease, whereas adoptive T cell transfer does. See Bagchi, T., 1990, M.Sc. Thesis, Virginia Tech, Blacksburg, Va.; Jimenez deBagues, M. P. et al., 1994, Infect. Immun. 62:4990. It is believed that vaccination with RB-51.RTM. confers protection by inducing production of interferon gamma able to activate macrophages and specific cytotoxic T cells in the subject which are able to kill Brucella infected macrophages.
Although RB-51.RTM., derived from B. abortus strain 2308, is the best current vaccine against Brucellosis in animals, it is still not 100% effective. None of the current Brucellosis vaccines are totally effective. Therefore, research continues on promising strains, such as B. abortus strain RB51. For example, expression of heterologous antigens by B. abortus strain RB51 has been described by S. Cravero, et al. 1995, Proceedings 4th Intl. Vet. Immunol. Symposium, July, Davis, Ca., Abstract # 276; and S. Cravero et al., 1996, Conference of Research Workers in Animal Diseases, Nov., Chicago, Abstract # 150. Over-expression of a homologous antigen by Brucella has been described as a research tool for the purpose of complementing specific deletion mutants for the study of HtrA protein in B. abortus (P. H. Elzer, Inf. Immun., 1994, 62:4131), and for the study of physiological functions as discussed by R. Wright at an Oral Presentation of the Brucella Research Conference on Nov. 9, 1997 in Chicago, Ill.
However, over-expression of homologous antigens of Brucella or other pathogens, with or without concomitant expression of a heterologous antigen, has not been studied for use in vaccines. Over-expression of homologous antigens previously has been used primarily as a research tool, as described above. An attenuated or avirulent pathogen modified to over-express an homologous antigen has not been used as a live vaccine. However, we have found that a vaccine which is an attenuated or avirulent pathogen which over-expresses one or more homologous antigens, as described herein, will provide greater protection against a pathogenic disease than vaccines of attenuated pathogens which express wild type levels of the same homologous antigens.
Therefore, the invention is directed to a vaccine, a means of producing the vaccine, and its use for prophylaxis and treatment of a pathogenic disease wherein the vaccine is an attenuated or avirulent pathogen which over-expresses at least one homologous antigen, thereby providing greater protection against and treatment of the disease caused by the unattenuated pathogen in the subject vertebrate.