Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1996-05-17
2002-04-02
Martinell, James (Department: 1633)
Chemistry: molecular biology and microbiology
Vector, per se
C536S023100, C536S023700
Reexamination Certificate
active
06365401
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The current invention relates to a class of microbial coding sequences that are specifically induced during infection of a host by a microbial pathogen and more particularly to a set of probes that may be used to identify and isolate microbial virulence genes. The products of these virulence genes will provide potential targets for the development of vaccines or antimicrobial agents.
2. Description of the State of the Art
Microbial pathogens, or disease-producing microorganisms, can infect a host by one of several mechanisms. For example, they may enter through a break in the skin, they may be introduced by vector transmission, or they may interact with a mucosal surface. Disease ensues following infection of a host, when the potential of the pathogen to disrupt normal bodily functions is fully expressed. Each disease-producing microorganism possesses a collection of virulence factors, that enhance their pathogenicity and allow them to invade host or human tissues and disrupt normal bodily functions. Infectious diseases have been major killers over the last several thousand years, and while vaccines and antimicrobial agents have played an important role in the dramatic decrease in the incidence of infectious diseases, infectious diseases are still the number one cause of death world-wide.
Vaccines
Attempts to vaccinate are almost as old as man's attempt to rid himself of disease. However, during the last 200 years, since the time Edward Jenner deliberately and systematically inoculated a population with cowpox to avoid a smallpox epidemic, vaccination, at least in parts of the world, has controlled the following nine major diseases: smallpox, diphtheria, tetanus, yellow fever, pertussis, poliomyelitis, measles, mumps and rubella. In the case of smallpox, the disease has been totally eradicated. The impact of vaccination on the health of the world's population is hard to exaggerate. With the exception of safer water, no other modality, not even antibiotics, has had such a major effect on mortality reduction and population growth.
Following the first exposure of a host to an antigen, the immune response is often slow to yield antibody and the amount of antibody produced is small, i.e., the primary response. Upon secondary challenge with the same antigen the response is more rapid and of greater magnitude, i.e., the secondary response. Achieving an immune state equal to the accelerated secondary response following reinfection with a pathogenic microorganism is the goal that is sought to be induced by vaccines. Vaccines are basically suspensions of viral, bacterial, or other pathogenic agents or their antigens which can be administered prophylactically to induce immunity.
In general, active vaccines can be divided into two general classes: subunit vaccines and whole organism vaccines. Subunit vaccines are prepared from components of the whole organism and are usually developed in order to avoid the use of live organisms that may cause disease, or to avoid the toxic components present in whole organism vaccines.
The use of purified capsular polysaccharide material of
H. influenza
type b as a vaccine against the meningitis caused by this organism in humans is an example of a vaccine based upon an antigenic component. See Parks et al.,
J. Inf. Dis.
, 136 (Suppl.):551 (1977), Anderson et al.,
J. Inf. Dis.
, 136 (Suppl.):563 (1977); and Mäkela et al.,
J. Inf Dis.,
136 (Suppl.):543 (1977). Classically, subunit vaccines have been prepared by chemical inactivation of partially purified toxins, and hence have been called toxoids. Formaldehyde or glutaraldehyde have been the chemicals of choice to detoxify bacterial toxins. Both diphtheria and tetanus toxins have been successfully inactivated with formaldehyde resulting in a safe and effective toxoid vaccine which has been used for over 40 years to control diphtheria and tetanus. See, Pappenheimer, A. M., Diphtheria. In:
Bacterial Vaccines
(R. Germanier, ed.), Academic Press, Orlando, Fla., pp. 1-36 (1984); Bizzini, B., Tetanus. Id. at 37-68. In contrast to subunit vaccines, whole organism vaccines make use of the entire organism for vaccination. The organism may be used killed or alive (usually attenuated) depending upon the requirements necessary to elicit protective immunity. The following discussion will focus on live but attenuated microorganisms (live vaccines).
In the case of intracellular pathogens, it is generally agreed that live vaccines induce a highly effective type of immune response. Ideally, these attenuated microorganisms maintain the full integrity of cell-surface constituents necessary for specific antibody induction yet are unable to cause disease, because they fail to produce virulence factors, grow too slowly, or do not grow at all in the host. Additionally, these attenuated strains should have no probability of reverting to a virulent wild-type strain. Traditionally, live vaccines have been obtained by either isolating an antigenically related virus from another species, by selecting attenuation through passage and adaptation in a nontargeted species or in tissue cultures, or by selection of temperature-sensitive variants.
In contrast to these somewhat haphazard approaches of selecting for live vaccines, modern developmental approaches introduce specific mutations into the genome of the pathogen which affect the ability of that pathogen to induce disease, that is, specific mutations are introduced into genes involved in virulence. Defined genetic manipulation is the current approach being taken in an attempt to develop live vaccines for various diseases caused by pathogenic microorganisms.
U.S. Pat. No. 5,210,035, exemplifies this approach by describing the construction of vaccine strains from pathogenic microorganisms made non-virulent by the introduction of complete and non-reverting mutational blocks in the biosynthesis pathways, causing a requirement for metabolites not available in host tissues. Specifically, Stocker teaches that
S. typhi
may be attenuated by interrupting the pathway for biosynthesis of aromatic (aro) metabolites which renders Salmonella auxotrophic (i.e., nutritionally dependent) for p-aminobenzoic acid (PABA) and 2,3-dihydroxybenzoate, substances not available to bacteria in mammalian tissue. These aro
−
mutants are unable to synthesize chorismic acid (a precursor of the aromatic compounds PABA and 2,3-dihydroxybenzoate), and no other pathways in Salmonella exist that can overcome this deficiency. As a consequence of this auxotrophy, the aro
−
deleted bacteria are not capable of extensive proliferation within the host; however, they reside and grow intracellularly long enough to stimulate protective immune responses.
Unfortunately the development of vaccines based on chemical toxoids, discussed previously, is difficult since protective antigens and the genes encoding them must first be identified and then procedures must be developed to efficiently isolate the antigens. Similarly, modern approaches to the rational development of live vaccines has been hampered by the limited knowledge available concerning genes that are involved in virulence and thus the targets of mutagenesis.
Antimicrobial Agents
The medical literature up to about 1930 is full of vivid descriptions of gruesome infections by streptococci, staphylococci, and clostridia. The dawning of the age of antimicrobial therapy, with the introduction of the sulfonamides in the 1930s, allowed physicians finally to cure many of these fatal infections. From the outset, antibiotics were heralded as a panacea for everything from fungus-infected pear orchards to the common cold. Penicillin lozenges were popular as were nostrums such as antibiotic mouthwashes and throat sprays. By the 1950s, doctors jubilantly predicted an end to infectious diseases and, by the 1980s, half of all drug companies had stopped developing antibiotics, believing the battle won.
The stunning success of the pharmaceutical industry in the United Sates, Japan, the United Kingdom, Fran
Conner Christopher P.
Heithoff Douglas M.
Mahan Michael J.
Martinell James
Morrison & Foerster / LLP
The Regents of the University of California
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