Over the past few years the editors of the Morbidity and Mortality Weekly Report have chronicled the unexpected rise in tuberculosis cases. It has been estimated that one billion people are infected with M. tuberculosis worldwide, with 7.5 million active cases of tuberculosis. Even in the United States, tuberculosis continues to be a major problem especially among the homeless, Native Americans, African-Americans, immigrants, and the elderly. HIV-infected individuals represent the newest group to be affected by tuberculosis. Of the 88 million new cases of tuberculosis expected in this decade, approximately 10% will be attributable to HIV infection.
The emergence of multi-drug resistant strains of M. tuberculosis has complicated matters further and even raises the possibility of a new tuberculosis epidemic. In the U.S. about 14% of M. tuberculosis isolates are resistant to at least one drug, and approximately 3% are resistant to at least two drugs. M. tuberculosis strains have even been isolated that are resistant to all seven drugs in the repertoire of drugs commonly used to combat tuberculosis. Resistant strains make treatment of tuberculosis extremely difficult: for example, infection with M. tuberculosis strains resistant to isoniazid and rifampin leads to mortality rates of approximately 90% among HIV-infected individuals. The mean time to death after diagnosis in this population is 4-16 weeks. One study reported that, of nine immunocompetent health care workers and prison guards infected with drug-resistant M. tuberculosis, five died. The expected mortality rate for infection with drug-sensitive M. tuberculosis is 0%.
The unrelenting persistence of mycobacterial disease worldwide, the emergence of a new, highly susceptible population, and the recent appearance of drug-resistant strains point to the need for new and better prophylactic and therapeutic treatments of mycobacterial diseases.
Infection with M. tuberculosis can take on many manifestations. The growth in the body of M. tuberculosis and the pathology that it induces is largely dependent on the type and vigor of the immune response. From mouse genetic studies it is known that innate properties of the macrophage play a large role in containing disease, Skamene, Ref Infect. Dis. 11:S394-S399, 1989. Initial control of M. tuberculosis may also be influenced by reactive T xcex3xcex4 cells. However, the major immune response responsible for containment of M. tuberculosis is via helper T cells (Th1) and to a lesser extent cytotoxic T cells, Kaufmann, Current Opinion in Immunology 3:465-470, 1991. Evidence suggests that there is very little role for the humoral response. The ratio of responding Th1 to Th2 cells has been proposed to be involved in the phenomenon of suppression.
Th1 cells are thought to convey protection by responding to M. tuberculosis T cell epitopes and secreting cytokines, particularly INF-xcex3, that stimulate macrophages to kill M. tuberculosis. While such an immune response normally clears infections by many facultative intracellular pathogens, such as Salmonella, Listeria, or Francisella, it is only able to contain the growth of other pathogens such as M. tuberculosis and Toxoplasma. Hence, it is likely that M. tuberculosis has the ability to suppress a clearing immune response, and mycobacterial components such as lipoarabinomannan are thought to be potential agents of this suppression. Dormant M. tuberculosis can remain in the body for long periods of time and can emerge to cause disease when the immune system wanes due to age or other effects such as infection with HIV-1.
Historically it has been thought that one needs replicating mycobacteria in order to effect a protective immunization. An hypothesis explaining the molecular basis for the effectiveness of replicating mycobacteria in inducing protective immunity has been proposed by Orme and co-workers, Orme et al., Journal of Immunology 148:189-196, 1992. These scientists suggest that antigens are pinocytosed from the mycobacterial-laden phagosome and used in antigen presentation. This hypothesis also explains the basis for secreted proteins effecting a protective immune response.
Antigens that stimulate T cells from mice infected with M. tuberculosis or from PPD-positive humans are found in both the whole mycobacterial cells and also in the culture supernatants, Orme et al., Journal of Immunology 148:189-196, 1992; Daugelat et al., J. Infect. Dis. 166:186-190, 1992; Barnes et al., J. Immunol. 143:2656-2662, 1989; Collins et al., Infect. Immun. 56:1260-1266, 1988; Lamb et al., Rev. Infect. Dis. 11:S443-S447, 1989; and Hubbard et al., Clin. exp. Immunol. 87: 94-98, 1992. Recently Pal and Horwitz, Infect. Immun. 60:4781-4792, 1992, induced partial protection in guinea pigs by vaccinating with M. tuberculosis supernatant fluids. Similar results were found by Andersen using a murine model of tuberculosis, Andersen, Infection and Immunity 62:2536, 1994. Other studies include Hubbard et al., Clin. exp. Immunol. 87: 94-98, 1992, and Boesen et al., Infection and Immunity 63:1491-1497, 1995. Although these works are far from definitive, they do strengthen the notion that protective epitopes can be found among secreted proteins and that a non-living vaccine can protect against tuberculosis.
For the purposes of vaccine development one needs to find epitopes that confer protection but do not contribute to pathology. An ideal vaccine would contain a cocktail of T-cell epitopes that preferentially stimulate Th1 cells and are bound by different MHC haplotypes. Although such vaccines have never been made, there is at least one example of a synthetic T-cell epitope inducing protection against an intracellular pathogen, Jardim et al., J. Exp. Med. 172:645-648, 1990.
It is an object of this invention to provide M. tuberculosis DNA sequences that encode bacterial peptides having an immunostimulatory activity. Such immunostimulatory peptides will be useful in the treatment, diagnosis, and prevention of tuberculosis.
The present invention provides inter alia, DNA sequences isolated from Mycobacterium tuberculosis. Peptides encoded by these DNA sequences stimulate the production of the macrophage-stimulating cytokine, gamma interferon (xe2x80x9cINF-xcex3xe2x80x9d), in mice. Critically, the production of INF-xcex3 by CD4 cells in mice correlates with maximum expression of protective immunity against tuberculosis, Orme et al., J. Immunology 151:518-525, 1993. Furthermore, in human patients with active xe2x80x9cminimalxe2x80x9d or xe2x80x9ccontainedxe2x80x9d tuberculosis, it appears that the containment of the disease may be attributable, at least in part, to the production of CD4 Th-1-like lymphocytes that release INF-xcex3, Boesen et al., Infection and Immunity 63:1491-1497, 1995.
Hence, the DNA sequences provided by this invention encode peptides that can of stimulate T-cells to produce INF-xcex3. That is, these peptides act as epitopes for CD4 T-cells in the immune system. Studies have demonstrated that peptides isolated from an infectious agent and which are shown to be T-cell epitopes can protect against the disease caused by that agent when administered as a vaccine, Mougneau et al., Science 268:536-566, 1995 and Jardim et al., J. Exp. Med 172:645-648, 1990. For example, T-cell epitopes from the parasite Leishmania major have been shown to be effective when administered as a vaccine, Jardim et al., J. Exp. Med. 172:645-648, 1990; Mougneau et al., Science 268:536-566, 1995; and Yang et al., J. Immunology 145:2281-2285, 1990. Therefore, the immunostimulatory peptides (T-cell epitopes) encoded by the DNA sequences according to the invention may be used, in purified form, as a vaccine against tuberculosis.
As noted, the nucleotide sequences of the present invention encode immunostimulatory peptides. In a number of instances, these nucleotide sequences are only a part of a larger open reading frame (ORF) of an M. tuberculosis operon. The present invention enables the cloning of the complete ORF using standard molecular biology techniques, based on the nucleotide sequences provided herein. Thus, the present invention encompasses both the nucleotide sequences disclosed herein and the complete M. tuberculosis ORFs to which they correspond. However, it is noted that since each of the nucleotide sequences disclosed herein encodes an immunostimulatory peptide, the use of larger peptides encoded by the complete ORFs is not necessary for the practice of the invention. Indeed, it is anticipated that, in some instances, proteins encoded by the corresponding ORFs may be less immunostimulatory than the peptides encoded by the nucleotide sequences provided herein.
According to one aspect of the present invention, immunostimulatory preparations are provided comprising at least one peptide encoded by the DNA sequences presented herein. Such a preparation may include the purified peptide or peptides and one or more pharmaceutically acceptable adjuvants, diluents, and/or excipients.
According to another aspect of the invention, vaccines are provided comprising one or more peptides encoded by nucleotide sequences provided herein. Such a vaccine may include one or more pharmaceutically acceptable excipients, adjuvants, and/or diluents.
According to another aspect of the present invention, antibodies are provided that are specific for immunostimulatory peptides encoded by a nucleotide sequence according to the present invention. Such antibodies may be used to detect the presence of M. tuberculosis antigens in medical specimens, such as blood or sputum. Thus, these antigens may be used to diagnose tuberculosis infections.
The present invention also encompasses the diagnostic use of purified peptides encoded by nucleotide sequences according to the present invention. Thus, the peptides may be used in a diagnostic assay to detect the presence of antibodies in a medical specimen, which antibodies bind to the M. tuberculosis peptide and indicate that the subject from which the specimen was removed was previously exposed to M. tuberculosis. 
The present invention also provides improved methods of performing the tuberculin skin test to diagnose exposure of an individual to M. tuberculosis. In this improved skin test, purified immunostimulatory peptides encoded by the nucleotide sequences of this invention are employed. Preferably, this skin test is performed with one set of the immunostimulatory peptides, while another set of the immunostimulatory peptides is used to formulate vaccine preparations. In this way, the tuberculin skin test will be useful in distinguishing between subjects infected with tuberculosis and subjects who have simply been vaccinated. In this manner, the present invention may overcome a serious limitation inherent in the present BCG vaccine/tuberculin skin test combination.
Other aspects of the present invention include the use of probes and primers derived from the nucleotide sequences disclosed herein to detect the presence of M. tuberculosis nucleic acids in medical specimens.
A further aspect of the present invention is the discovery that a significant proportion of the immunostimulatory peptides is homologous to proteins known to be located in bacterial cell-surface membranes. This discovery suggests that membrane-bound peptides, particularly those from M. tuberculosis, may be a new source of antigens for use in vaccine preparations.