Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Animal cell – per se – expressing immunoglobulin – antibody – or...
Reexamination Certificate
2000-03-29
2003-11-18
Park, Hankyel T. (Department: 1618)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Animal cell, per se, expressing immunoglobulin, antibody, or...
C435S069100, C435S325000, C536S023720, C424S208100
Reexamination Certificate
active
06649409
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a DNA vaccine against HIV, which is designed from a clinical primary isolate. One aspect of the invention relates to a method of producing a nucleotide sequence construct, in a prefered aspect based on a cassette system, the nucleotide sequence construct being used as a DNA vaccine. The method can, for example, lead to the disclosed synthetic BX08 HIV-1 envelope vaccine nucleotide sequence construct, designed to generate suitable DNA vaccines against HIV, specifically HIV-1. Furthermore, the invention can be used for the production of recombinant protein antigens.
BACKGROUND OF THE INVENTION
There is an urgent need for new vaccine strategies against HIV. One such new promising strategy is called genetic immunization or DNA vaccine (Webster et al 1997). Some of the advantages of a DNA vaccine against HIV is the induction of Th cell activation, induction of antibodies also against conformational dependent epitopes, and the induction of cellular immunity. So far, most DNA vaccine envelope genes tried, have been from tissue culture adapted virus strains (Boyer et al 1997) that often differs in several aspects from primary clinical isolates (such as early isolates) e.g. in co-receptor usage (Choe et al 1996, Dragic et al 1997).
One disadvantage in HIV envelope based DNA vaccines may be the intrinsic relatively low expression which is regulated by the Rev expression. This may prevent an optimal investigation of the vaccines in small animal models like mice where Rev is functioning suboptimally. Recently it has been shown using the tissue culture adapted HIV-1 MN strain, that an exchange of the HIV codon usage to that of highly expressed mammalian genes greatly improves the expression in mammalian cell lines and renders the HIV expression Rev independent (Haas et al 1996). Additionally, it is known that rare codons cause pausing of the ribosome, which leads to a failure in completing the nascent polypeptide chain and a uncoupling of transcription and translation. Pausing of the ribosome is thought to lead to exposure of the 3′ end of the mRNA to cellular ribonucleases. The world-wide spread of HIV-1 has presently resulted in 8,500 new infections daily and AIDS is now the number 1 cause of death among US males (and number 3 among US females) aged 25-40 years. The epidemic hot-spots now include Eastern Europe, India and South East Asia and southern Africa. The attempts to solve this world-wide problem involve education, prevention, treatment and vaccine development. Affordable protective vaccines represent the best solution to the world-wide problem of infection with HIV-1. Induction of virus neutralizing antibodies is one of the key components in vaccine development. Several recombinant envelope vaccines have been tested in humans and animals, but they seem unable to induce sufficient protection. In this respect DNA vaccination may provide a different and more natural mode of antigen presentation. It is hoped that the immune responses induced by such DNA vaccines could aid in limiting virus replication, slowing disease progression or preventing occurrence of disease. Unfortunately many HIV envelope vaccines induce only moderate levels of antibodies. This could in part be due to limitations in expression, influenced by regulation by the Rev protein and by a species-specific and biased HIV codon usage. Also the virus variability is considered a barrier for development of antibody based vaccines and thus a tool for more easy producing of closely related vaccine variants is needed.
It has been suggested to improve the immunogenicity and antigenicity of epitopes by certain mutations in the envelope gene. An elimination of certain immune dominant epitopes (like V3) could render less immune dominant but more relevant, conserved, or hidden epitopes more immunogenic (Bryder et al 1999). Also elimination of certain N-linked glycosylation sites could improve the exposure of relevant epitopes and increase the immunogenicity of those epitopes. Thus, it is possible that elimination of the glycosylation sites in V1 and V2 may in a more favourable way expose neutralising epitopes (Kwong et al 1998, Wyatt et al 1998). The HIV envelope contains putative internalization sequences in the intracellular part of gp41 (Sauter et al 1996). Thus it would be relevant to eliminate and/or mutate the internalization signals in a membrane bound HIV envelope vaccine gene to increase the amount of surface exposed vaccine derived HIV glycoproteins as gp150. Since the antibody response, that is measured and calculated in titers, is improved by adding the secreted gp120 as opposed to adding the membrane bound form (Vinner et al 1999), it could be advantageous to express the vaccine as a secreted gp120 or a secreted gp140. This would include important parts of gp41, such as the 2F5 neutralising linear epitope (Mascola et al 1997).
SUMMARY OF THE INVENTION
Our suggested solution to the problems described above is to design DNA envelope vaccines from a clinical primary isolate with Rev-independent high expression in mammals, that is-built as a cassette for easy variant vaccine production.
A method of producing a nucleotide sequence construct with codons from highly expressed mammalian proteins based on a cassette system coding for an early, primary HIV envelope is described. The method comprises the steps of direct cloning of an HIV gene, derived from a patient within the first 12 months of infection, thereby obtaining a first nucleotide sequence; designing a second nucleotide sequence utilising the most frequent codons from mammalian highly expressed proteins to encode the same amino acid sequence as the first nucleotide sequence; redesigning the second nucleotide sequence so that restriction enzyme sites surround the regions of the nucleotide sequence encoding functional regions of the amino acid sequence and so that selected restriction enzyme sites are removed, thereby obtaining a third nucleotide sequence encoding the same amino acid sequence as the first and the second nucleotide sequence; redesigning the third nucleotide sequence so that the terminals contain convenient restriction enzyme sites for cloning into an expression vehicle; producing snuts between restriction enzyme sites as well as terminal snuts and introducing snuts into an expression vehicle by ligation. The nucleotide sequence construct obtained by this method uses the mammalian highly expressed codons (
FIG. 1
) and renders the envelope gene expression Rev independent and allows easy cassette exchange of regions surrounded by restriction enzyme sites that are important for immunogenicity, function, and expression.
The method can, for example, lead to the disclosed synthetic, Rev-independent, clinical (such as early), primary HIV-1 envelope vaccine gene, built as a multi cassette. From the sequence of the envelope of the HIV-1 BX08 isolate (personal communication from Marc Girard, Institute Pasteur, Paris), the present inventors have designed a synthetic BX08 HIV-1 envelope vaccine nucleotide sequence construct.
With the great diversity of envelopes in HIV among different patients and within one patient, it would be of advantage to vaccinate with several envelope variants, all being highly expressed. To avoid synthesising several full length envelopes, it is much easier to exchange relevant parts of an envelope cassette to various strains in a multivalent vaccine.
Whether it is the disclosed synthetic BX08 nucleotide sequence construct, or any of the nucleotide sequence constructs obtained by the method, they are designed to generate suitable DNA vaccines against HIV, specifically HIV-1. In this case the mammal, preferably a human being, is inoculated with the nucleotide sequence construct in an expression vehicle and constitutes a host for the transcription and translation of the nucleotide sequence construct. The nucleotide sequence constructs of the present invention can furthermore be used for the production of recombinant protein antigens. In this case the nucleotide sequence construct is pla
Foley & Lardner
Park Hankyel T.
Statens Serum Institut
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