Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant or stably-transformed bacterium encoding one or...
Reexamination Certificate
2000-12-04
2004-04-20
Devi, S. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant or stably-transformed bacterium encoding one or...
C424S093400, C424S093200, C424S261100, C424S184100, C435S069300, C435S091420, C435S471000
Reexamination Certificate
active
06723323
ABSTRACT:
TECHNICAL SECTOR
The field of invention is that of Biotechnology and more specifically the generation of
Vibrio cholerae
vaccines and the methods of constructing them by using genetic engineering tools.
BACKGROUND OF THE INVENTION
A brief explanation on the terminology used through the text of the invention is listed below.
By ctx&PHgr; virus is meant the particle of protein-coated DNA, produced by certain
Vibrio cholerae
strains, which is capable of transducing its DNA, comprising cholera toxin genes, to other
Vibrio cholerae
strains.
By cholera toxin is meant the protein responsible for the clinical symptoms of cholerae when produced by the bacteria.
By ctx&PHgr;-encoded toxin genes are meant, in addition to cholera toxin genes, zot and ace genes, which code for the “zonula occludens toxin” and for the “accessory cholera enterotoxin”, respectively.
For non-toxigenic strains of
Vibrio cholerae
it most be understood any strain devoid of the genes coding for the above toxins, which are as well, useful as vaccines but still produce an undesired reactogenic syndrome.
The term safe vaccine or safe strain refers to such strain lacking the residual reactogenicity of non-toxigenic strains of
Vibrio cholerae.
By hemagglutinin/protease is meant the protein manifesting dual function, being one of them the ability to agglutinate erythrocytes from certain species and the other the property to degrade proteins such as mucin.
The term celA refers to the nucleotide sequence coding for the endoglucanase A protein. This protein naturally occurs in
Clostridium thermocellum
strains and has a &bgr;(1-4) glucan-glucane hydrolytic activity able to degrade cellulose and its derivatives.
By Thymidylate synthase is meant the protein capable of catalyzing the reductive methylation of deoxyuracil monophosphate (dUMP) by N
5
-N
10
-methylene-tetrahydrofolate to yield 2,5 deoxythymidyne phosphate (dTMP) and dihydrofolate.
Substantially pure DNA is DNA that is free from both of the coding sequences immediately contiguous by the 5′ or the 3′ end of thyA coding sequence, in the naturally occurring genome of the microorganism from which the DNA of the invention is derived. The term thereof includes, for example, a recombinant DNA which is incorporated on a vector strain, cell line or plasmid, or which exists as a separate molecule (e.g., cDNA, restriction or PCR fragment). It also includes recombinant DNA molecules which are part of a hybrid gene encoding additional sequences.
Homologous sequences refers to DNA or protein sequences which share similar or identical residues being nucleotides or amino acids, respectively, in identical positions of two or more given strings. The greater the number of identical/similar residues in certain position, the greater the percent of identity/similarity between two them.
Clinical cholera is an acute diarrheal disease that results from an oral infection with the bacterium
Vibrio cholerae
. After more than 100 years of research on cholera there remains the need for an effective and safe vaccine. Humankind has witnessed seven pandemics of cholera; the former six were caused by strains of the classical biotype and the current seventh pandemic is characterized by the predominance of Vibrios belonging to El Tor Biotype. Recently, beginning in January of 1991, this pandemic has extended to South America causing greater than 25 000 cases and over 2000 deaths in Peru, Ecuador, Colombia, and Chile. By November 1992, a new serogroup emerged in India and Bangladesh, the O139, showing a great epidemic potential that became a new cause of concern throughout the developing world. These recent experiences reinforce the need for effective cholera vaccines against disease caused by
V. cholerae
of serogroups O1 (El Tor) and O139.
Because convalescence to cholera is followed by an state of immunity lasting at least 3 years, much of the efforts in
Vibrio cholerae
vaccinology have been made to produce live, attenuated cholera vaccines, that closely mimic the disease in its immunization-prbperties after oral administration, but do not result reactogenic to the individuals ingesting them. Vaccines of this type involve deletion mutations of all toxin genes encoded by the ctx&PHgr; Vibrio-phage. See patents of Kaper, J. et al.; WO 91,18979 and Mekalanos, J., WO 95,18633).
The first vaccine to be assayed against cholera dates from 1885-1892. It was a traditional vaccine that comprised administration by parenteral route of “attenuated” vibrios. It resulted limited in efficacy and unacceptably reactogenic (Finkelstein R. A., International Symposium on Cholera on the America Continent. Sao Paulo, SP, Brasil, 1992). Oral vaccination was tried first in 1892, using attenuated
Vibrio cholerae
strains. The results of this attempt were misinterpreted and the strategy immediately abandoned. Oral vaccination was later rescued in 1970-1980 at the Center for Vaccine Development of Maryland, USA, by using chemically mutagenized vibrios as immunizing agents. Reversion to virulence of these mutants impeded further spread of the strategy (Levine et al., Infect and Imm, No 2, 1984; Finkelstein et al., patent U.S. Pat. No. 4,328,209) and prompted the researchers to generate genetically defined non-toxigenic mutants unable to revert. Although these mutants have shown to confer solid immunological protection against disease (Kaper J. B. and Levine M. Patents U.S. Pat. No. 06,472,276 and U.S. Pat. No. 581,406), the essential drawback for their use is the high level of adverse reactions they produce in vaccinees (Levine et al., Infect. and Imm. Vol 56, No 1, 1988). According to these data the major issue to be overcome when producing an effective cholera vaccine is safety. Additionally, researches worldwide are currently concerned on the horizontal transfer of genetic information among bacteria, thus it is necessary to pay attention to this aspect when designing live bacterial vaccines, with the aim to ameliorate the environmental impact during vaccination. It is also necessary to achieve good levels of stability and immunogenicity.
A dead cholerae vaccine consisting of whole cells supplemented with the B subunit of cholera toxin is available (Holmgren et al., Current topics in Microbiology and Immunology, Vol. 146, 1989). Such vaccine is safe and effective but requires multiple doses to generate an immune response equivalent to that of a cholera infection and consequently is very expensive.
Another alternative for cholera vaccination is the recent licensed CVD103-HgR, a live cholera vaccine belonging to the classical biotype. It is safe, effective and cheap; however its protective efficacy against the current circulating El Tor and O139 vibrios is not as good as against Classical vibrios (See patent U.S. Pat. No. 5,399,494).
Other live vaccine candidates have been described in patent WO 95/18633. Such mutants represent all serotypes of the current pandemic, including the O139. They are safe, their production is cheap, and have been shown to be preliminary effective; however they are not as extensively tested as CVD103-HgR. All these candidates are protothrophic bacteria able to survive natural conditions of the environment. Additionally, although a procedure to obtain defined mutants is described in the document, the proposed candidates constitute non-motile spontaneous mutants. It has been proposed by the inventors that the non-motile nature of these vaccine candidates limits their ability to reach the enterocyte surface and avoid the elicitation of the reactogenic response characteristic of their parentals.
Finkelstein et. Al, J. Bacteriology, Vol. 173, No. 11, 3311-17, cloned and sequenced the gene coding for the Hemagglutinin/Protease (hap). The mutan: HAP-1 was additionally isolated by insertional inactivation of this gene with a kanamycin resistance cassette in the bacterial chromosome. Rather than for vaccine purposes, thisstrain was a fundamental research approach. Finkelstein et. Al, Infection and Immunity, Vol. 60, No. 2, 472-78 demonstrated that wild type and hap mutants were v
Calzada Rafael Alfredo Fando
Díaz Edgar Valle
Gómez Javier Campos
Gónzalez Boris Luís Rodríguez
Pérez Talena Yamilé Ledón
Centro Nacional de Investigaciones Cientificas
Devi S.
Lackenbach & Siegel LLP
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