Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
1993-11-08
2003-05-06
Smith, Lynette R. F. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S200100, C435S252330, C435S238000, C435S849000, C435S252100
Reexamination Certificate
active
06558678
ABSTRACT:
The present invention relates to the preparation and use of formalin-killed colonization-factor-antigen (CFA)-expressing
E. coli
organisms for vaccination against enteric infection/diarrhea caused by enterotoxigenic
E. coli
bacteria in humans.
Specially, the invention relates to a method of producing a vaccine composition against enteric infection caused by enterotoxigenic
E. coli
bacteria in humans, and a method of preventing an enteric infection caused by enterotoxigenic
E. coli
bacteria in humans.
BACKGROUND
Diarrhea caused by enterotoxinogenic
Escherichia coli
(ETEC) is an important health problem, particularly in developing countries and in travellers to these areas. In hospital- and clinic-based studies of acute diarrhea in developing countries ETEC has been identified in 10-50% of the cases, the average being ca 20% in children less than 5 years, and slightly higher in older age groups. Likewise, ETEC has been identified as the causative agent in at least one third to one half of cases of acute diarrhea among persons travelling from industrialized to developing countries.
The illness caused by ETEC ranges from mild diarrhea without dehydration to cholera-like disease. In the first 5 years of life many children in developing countries suffer from 1-2 episodes of diarrhea caused by ETEC each year. Although in the majority of ETEC infections symptoms are relatively mild, ETEC accounts for more than one billion diarrheal episodes and one million deaths annually among children in developing countries. Thus, any interventions that could reduce ETEC mortality and morbidity even partially might be of great public health significance.
No vaccine for use in humans against ETEC diarrhea is yet available. However, in a large field trial of a newly developed oral cholera vaccine it was found that the B subunit component of this vaccine, which cross-reacts immunologically with the heat-labile enterotoxin (LT) of ETEC, afforded significant protection not only against cholera but also against diarrhea caused by LT-producing ETEC. The protection against ETEC infection was particularly pronounced against illness associated with severe, life-threatening dehydration which was reduced by 86% by the vaccine during the first three months after immunization.
MECHANISMS OF DISEASE AND IMMUNITY. To cause disease, ETEC must be able to colonize the small intestine, and to elaborate LT and/or a heat-stable enterotoxin (ST, STa).
E. coli
LT is similar to cholera toxin in structure and function, consisting of a toxin-active A subunit attached to five B subunits that mediate binding to cell membrane receptors. The ST molecule is a small polypeptide of only 19 amino acid residues, that stimulates guanylate cyclase activity in intestinal cells. Different from LT which is a strong immunogen, ST is non-immunogenic unless experimentally conjugated to a larger carrier protein. ST-only and LT/ST-producing strains are important causes of diarrhea in endemic areas, whereas LT-only strains frequently cause disease in travellers to developing countries. The proportion of ETEC strains with different enterotoxin profiles varies from country to country.
In many ETEC strains adhesion to intestinal mucosa is mediated by antigenically distinct fimbriae. In strains pathogenic for man three main adhesins have been identified; they are referred to as colonization factor antigens CFA/I, CFA/II, and CFA/IV (formerly called PCF8775). CFA/I is a single homogenous fimbrial antigen whereas CFA/II comprises the
coli
surface (CS) antigens CS1, CS2 and CS3, and CFA/IV the CS4, CS5 and C6 antigens. Although the prevalence of these different colonization factors varies geographically, CFA/I, CFA/II or CFA/IV are usually found on one-half to three-quarters of ETEC isolated from cases with clinically significant diarrhea. However, additional adhesins are also likely to be identified.
The highest ETEC infection rates in endemic areas are seen in young children. The findings of a decreased attack rate with increasing age and of a higher proportion of asymptomatic cases in adults than in children suggest that naturally acquired protective immunity may develop. Similarly, a degree of resistance against ETEC diarrhea develops among travellers during prolonged residence in high-risk countries. Experimental studies in animals and human volunteers have also shown that ETEC infection may give rise to substantial immunity against rechallenge with the homologous organisms.
There is evidence that both antibacterial and antitoxic immunity contributes to protection against ETEC diarrhea. Antibacterial immunity against ETEC may to a large extent be ascribed to immunity against the different colonization factor antigens (CFA), even though antibodies against O-antigen may play a role as well for protection against ETEC of homologous O-gruoups. In animals, anti-CFA antibodies have protected against challenge with ETEC expressing the homologous CFAS. similarly, in both animals and human volunteers oral or intraintestinal immunization with ETEC strains expressing CFA/I, CFA/II or CFA/IV has induced protective immunity against subsequent challenge with
E. coli
carrying the homologous CFA/CS-factor.
Naturally induced antitoxic ETEC immunity is only directed against LT since native ST is not immunogenic. The anti-LT immune response is mainly against the B subunit portion of the molecule, which cross-reacts immunologically with the B subunits of cholera toxin. This explains why, as mentioned, oral immunization with cholera B subunit could induce protection against ETEC diarrhea [1]; interestingly protection was induced not only against LT-only but also against LT/ST ETEC strains [1]; a finding also supported by findings in animals after immunization with either cholera or LT B subunits (unpublished data).
It is also known that in humans clinical ETEC disease evokes significant antitoxic as well as antibacterial immune responses in the intestine resulting in specific IgA antibody titer increases in intestinal lavage fluid against LT and homologous CFA and O antigens [2]. Both anti-enterotoxin and anti-colonization factor antibodies can independent of each other protect against experimental ETEC infection and, when being present together in the intestine, these antibody specificities have been found to cooperate synergistically in protecting against ETEC disease [3].
In contrast to the well-established protective function of anti-LT immunity against ETEC disease, the significance of anti-ST immunity for protection remains undefined. Although ST in its natural state is not immunogenic it may give rise to ST-neutralizing antibodies when being used coupled to a carrier protein. This suggests that also vaccine-induced anti-ST immunity may be an attainable goal. However, different ST-carrier conjugates tested to date, derived either by chemical coupling or recombinant DNA techniques, have all retained significant, though sometimes reduced toxic activity. Therefore, several synthetic modified ST-peptides have been prepared recently in an attempt to identify nontoxic ST-related epitopes. Synthetic oligonucleotides encoding for similar peptides have also been made and fused to the gene for cholera B-subunit and when inserted into Vibrio cholerae these chimeric genes encode production of high concentrations of completely nontoxic ST-B subunit fusion protein. Immunization of experimental animals with such chemically derived or genetically engineered nontoxic peptide-B-subunit conjugates has evoked anti-ST antibody responses but so far with only weak neutralizing activity.
CANDIDATE VACCINES. Based on this knowledge about the key protective antigens of ETEC bacteria and of the main immune mechanisms operating against ETEC infections, it may be concluded that an effective ETEC vaccine should be given orally and ideally evoke both anti-colonization and antitoxic immune responses in the intestine. Thus, the vaccine should contain a combination of bacterial cell- and toxin-derived antigens. Different live or inactivat
Holmgren Jan
Svennerholm Ann-Mari
Darby & Darby
Portner Ginny Allen
Smith Lynette R. F.
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