Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1998-05-13
2001-02-20
Duffy, Patricia A. (Department: 1643)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S093100, C424S093200, C424S093400, C424S093480, C424S831000, C435S879000
Reexamination Certificate
active
06190669
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to attenuated Salmonella mutants which constitutively express the Vi antigen, as well as vaccines against typhoid fever containing the same, live vector vaccines containing the same, and DNA-mediated vaccines containing the same.
BACKGROUND OF THE INVENTION
I. The Vi Antigen
The Vi antigen, a capsular polysaccharide, was first described by Felix et al,
Lancet,
227:186-191 (1934). This capsular polysaccharide is present in Salmonella, such as
S. typhi, S. paratyphi
C, and
S. dublin,
as well as in
Citrobacter freundii.
Structurally, the Vi antigen is a linear polymer of &bgr;-4,2-deoxy-2N-acetylgalacturonic acid with variable O-acetylation (Daniels et al,
Infect. Immun.,
57:3159-3164 (1989)). Its presence in
S. typhi
has been correlated, in vitro, with a significant decrease in lysis by serum, complement activation and phagocytosis (Looney et al,
J. Lab. Clin. Med.,
108:506-516 (1986)). Thus, the Vi antigen may act as a shield protecting
S. typhi
against the immune system.
A. The viaB Chromosomal Region and the Regulation of Expression of the Vi Antigen
Three widely separated chromosomal loci, viaA, viaB, and ompB are thought to be necessary for expression of the Vi antigen (Johnson et al,
J. Bacteriol.,
90:302-308 (1965); and Snellings et al,
J. Bacteriol.,
145:1010-1017 (1981)). Of these, the viaB locus is always found in Vi antigen-positive strains, and is thought to contain the genes encoding the enzymes necessary for the synthesis of Vi (Hashimoto et al,
J. Bacteriol.,
175:4456-4465 (1993); and Virlogeux et al,
Microbiol.,
141:3039-3047 (1995)). The viaB locus consists of 11 open reading frames (ORF) (FIG.
1
A), of which the vipA and vipB genes encode the enzymes that synthesize the nucleotide sugar of the Vi polysaccharide, and the five vex genes (vexA-E) are thought to be responsible for translocation of the Vi antigen (Hashimoto et al (1993), supra). The first ORF of the viaB region, i.e., vipR (FIG.
1
A), is a positive transcriptional regulator for its own expression, as well as for the expression of vipA, vipB, orf4, vipC, and perhaps others genes downstream of vipR (Hashimoto et al,
J. Bacteriol.,
178:1430-1436 (1996)). Furthermore, the promoter upstream of vipR also controls the transcription of (at least) vipA and vipB (encoding structural units of the Vi antigen), forming an operon within the viaB region. Another chromosomal region, the ompB operon, comprising the ompR-envZ genes, plays a role in the expression of the Vi antigen as a transcriptional regulator of viaB (Pickard et al,
Infect. Immun.,
62:3984-3993 (1994)). The ompR-envZ region forms part of the adaptive response of
E. coli
to conditions of high osmolarity. In
S. typhi,
the Vi antigen is osmotically regulated and ompR is necessary for its expression (Pickard et al, supra).
B. Relationship Between Exposure of the Vi Antigen and Immunoprotection
The Vi capsular polysaccharide of
S. typhi
is a virulence factor and a protective antigen in humans (Felix et al (1934), supra). Purified Vi polysaccharide is a licensed parenteral typhoid vaccine that elicits a moderate degree of protective immunity following inoculation with a single dose, and protection is mediated by serum IgG antibodies (Acharya et al,
New England Journal of Medicine,
317:1101-1104 (1987); and Klugman et al,
Lancet,
2:1165-1169 (1987)). In contrast, while attenuated
S. typhi
strain Ty21a, a licensed live oral vaccine, does not express the Vi antigen, nor does it elicit serum Vi antibody; nevertheless, Ty21a confers at least moderate levels of protection (Wahdan et al,
J. Infect. Dis.,
145:292-296 (1982); and Levine et al,
Lancet,
1:1049-1052 (1987a)). It is believed that cell-mediated immune mechanisms mediate protection in this situation. Several new attenuated strains of
S. typhi
that express the Vi antigen in vitro have failed to elicit serum Vi antibodies when administered as oral vaccines, even though they elicit high titers of O and H antibodies (Tacket et al,
J. Infect. Dis.,
163:901-904 (1991); Tacket et al,
Vaccine,
10:443-446 (1992a); and Tacket et al,
Infect. Immun.,
65:452-456 (1997)). The likely explanation for this phenomenon is that the expression of the Vi antigen is highly regulated in relation to osmotic stimuli (Pickard et al, supra). The supposition is that Vi antigen expression is interrupted, except when the bacteria are extracellular in the blood or other body fluids (e.g., bile).
It was postulated in the present invention that if Vi antigen expression is made constitutive, this would result in the stimulation of serum IgG Vi antibodies, thereby enhancing the overall protection against typhoid fever. It was also postulated in the present invention, that constitutive expression would improve the immune responses to foreign antigens expressed by attenuated Salmonella live vector vaccines and DNA-mediated vaccines.
II. Target Populations of Typhoid Fever
Typhoid fever is exceedingly uncommon in modern industrialized countries where populations have access to treated, bacteriologically-monitored water supplies, and sanitation that removes human fecal waste. In contrast, in less-developed countries, among populations lacking such amenities, typhoid fever is often endemic, and from the public health perspective, typically constitutes the most important enteric disease problem of school age children (Levine et al,
Pediatr. Infect. Dis. J.,
8:374-381 (1989a)). Systematic clinical, epidemiologic and bacteriologic surveillance for typhoid fever in relation to field trials of candidate vaccines has established, with great accuracy, the incidence of typhoid fever in many populations (Levine et al,
Lancet,
336:891-894 (1990); Black et al,
Vaccine,
8:81-84 (1990); Levine et al . (1987a), supra; Ferreccio et al,
J. Infect. Dis.,
159:766-769 (1989); and Simunjuntak et al,
Lancet,
338:1055-1059 (1991)). The incidence rates revealed were much higher than predicted, based on unsystematic surveillance. Systematic surveys have also demonstrated a surprising frequency of clinically mild yet, bacteremic typhoid infection among infants and toddlers in endemic areas (Ferreccio et al,
J. Pediatr.,
104:899-901 (1984)).
Besides school age children in less-developed countries, two other populations at increased risk of typhoid fever are travelers and clinical microbiologists. Among U.S. travelers, the risk is highest in countries along the Pacific coast of South America, and in the Indian sub-continent (Ryan et al,
Rev. Infect. Dis.,
11:1-8 (1989); and Mathieu et al,
Arch. Intern. Med.,
154:1713-1718 (1994)). In addition, clinical microbiologists, including those in industrialized countries, have increased exposure to
Salmonella typhi
in the work environment, and therefore constitute a high risk group (Blaser et al,
J. Clin. Microbiol.,
13:855-858 (1981)).
III. Multi-Resistant
S. typhi
Strains
Since, circa 1990, sporadic cases and localized outbreaks of typhoid fever have begun to appear in the Middle East, Northeast Africa and South and Southeast Asia. These outbreaks are caused by strains of
S. typhi
encoding plasmid-mediated resistance to trimethoprim/sulfamethoxazole, as well as resistance to chloramphenicol (Gupta et al,
Pediatr. Infect. Dis.,
13:124-140 (1994); and Rowe et al,
Lancet,
336:1065-1066 (1990)). Therapy against such strains requires the use of quinolone antibiotics, such as oral ciprofloxacin, or third generation cephalosporins, such as parenteral ceftriaxone. These antibiotics are costly for developing countries. In contrast with previous antibiotic-resistant strains that caused sporadic cases or extended epidemics, as in Mexico from 1972-1973 (Olarte et al,
Antimicrob. Agents Chemother.,
4:597-601 (1973)); and in Peru from 1979-1981 (Goldstein et al,
J. Infect. Dis.,
2:261-266 (1986)), and that eventually disappeared to be replaced once again by sensitive strains, the multiple antibiotic-resistant
S. typhi
strains that appeared in the Middle East and the Indian sub-continent circa 1990 ar
Levine Myron M.
Noriega Fernando R.
Sztein Marcelo B.
Duffy Patricia A.
Sughrue, Mion, Zinn Macpeak & Seas. PLLC
University of Maryland Baltimore
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