Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1995-05-22
2001-08-21
Allen, Marianne P. (Department: 1631)
Chemistry: molecular biology and microbiology
Vector, per se
C435S069100, C435S069300, C435S069800, C435S069900, C435S471000, C435S476000, C435S483000, C435S940000, C536S023100, C536S023720, C530S826000
Reexamination Certificate
active
06277631
ABSTRACT:
The invention relates to recombinant proteins with the immunoreactivity of hepatitis B virus e antigen (HBeAg), to a process for the preparation thereof in yeasts and to the use thereof in immunoassays and in vaccines.
Immunoassays nowadays play an important part in the diagnosis of hepatitis B virus (HBV) infection. Thus, the acute phase of an infection is characterized by immunological detection of hepatitis B virus surface antigen (HBsAg). Determination of other HBV parameters allows confirmation of the diagnosis or differential diagnosis. Thus, it is assumed that HBsAg- and HBeAg-positive samples are acutely infectious, whereas the appearance of antibodies against HBeAg (anti-HBeAg-antibodies) marks the start of the period of patient convalescence.
Radioimmunoassays and enzyme-linked immunoassays have become used world-wide for determining HBeAg and anti-HBeAg-antibodies and have substantially displaced other less sensitive techniques such as agglutination methods. These assays operate on the “sandwich principle”. A solid phase, for example the wells of a microtiter plate or beads which are coated with human or mouse anti-HBeAg antibodies, is incubated with the patient's sample. If the sample contains HBeAg, it binds to the solid-phase antibodies. Unbound constituents are removed in a washing step. The HBeAg bound to the solid phase is labeled during a second incubation with an anti-HBeAg antibody which is coupled to an enzyme or radioisotope. After another washing step, detection is carried out by the conversion of a substrate or by measuring the radio-activity of this ternary complex.
The same reagents and the same assay scheme can be used to detect anti-HBeAg-antibodies when a defined amount of HBeAg (the so-called neutralization reaction) is also added to the sample. If the test material, usually a serum sample, contains no anti-HBeAg-antibodies, a certain signal is generated thereby. However, if anti-HBeAg antibodies are present in the sample, they bind to the HBeAg of the neutralization reagent and prevent its binding to the solid phase and thus also the formation of the signal. This type of assay design thus combines aspects of the sandwich and of the competitive assay principle.
Assay systems for determining HBeAg and anti-HBeAg-antibodies in accordance with the principles described above can be obtained from several manufacturers and are also described many times in the literature (for example Abbott HBe(rDNA), Wiesbaden; Behring
R
Enzygnost-HBe, Marburg; Sorin Biomedica EBK EIA, Düsseldorf).
To check that the assay is carried out correctly, all assay systems for determining HBeAg use a so-called positive control which contains a defined amount of HBeAg and thus must give rise to a defined signal if the assay has been carried out correctly. If this signal is not reached, the assay run is worthless because of the obvious error in carrying it out, and it must be repeated. For determination of anti-HBeAg antibodies, in fact HBeAg is in principle necessary in the neutralization reagent, as explained above, for carrying out the assay.
Some of the assay systems established to date use HBeAg which has had to be obtained from the blood of HBV-infected people, because it has not been possible to establish cell culture systems for growing the virus. The disadvantage of this material is the difficulty of obtaining large quantities of high-titer HBeAg-positive serum from the infected people.
In addition, manipulation of HBeAg-positive serum is, because of its infectious risk, possible only with elaborate and costly safety precautions.
Currently, the only protection from hepatitis B infection is, besides general hygienic measures, regarded as being vaccination.
The only immunogen used in vaccines currently commercially available is HBsAg, although there have been indications for some years in the literature that it might be possible to achieve or improve vaccination protection by using HBcAg and/or HBeAg components, singly, as mixture or as fusion with another immunogen. It would therefore likewise be important for immunization purposes to generate by genetic engineering methods an HBeAg which has no infectious potential and which additionally ought to have advantages, compared with the materials known to date, owing to optimal immunoreactivity without further denaturation measures, and should be possible to prepare in sufficient quantities straightforwardly and at low cost.
The first papers which showed that denaturation of HBcAg of human origin in, for example, SDS causes it to lose a large part of the HBc immunoreactivity and instead gain HBe immunoreactivity appeared in 1979 and 1980 (for example Takahashi et al., J. Immunol. 122 (1979), 275-279). However, application of this method to HBcAg of human origin has no advantages over HBeAg of human origin because the problems of acquisition and infectiosity remain.
Once it became possible to express HBcAg by genetic engineering methods in
E. coli
, the denaturation technique was also carried out with rHBcAg. The disadvantage in this case too is that there is still a certain HBcAG immunoreactivity remaining in these preparations.
EP-A 075 395 then described a truncated recombinant HBcAg (up to amino acid 144) from
E. coli
, which had HBeAg immunoreactivity in addition to HBcAg immunoreactivity. However, it was again necessary to eliminate the remaining HBcAg immunoreactivity by denaturation measures.
Once Takahashi et al. (loc. cit.) were able to show that the C-terminal amino-acid sequence of HBeAg corresponds to HBcAg apart from amino acids being missing from position 150 onward, the truncated HBcAg was also expressed as fusion protein in
E. coli
. Even with this material it was necessary to eliminate the remaining HBcAg immunoreactivity by denaturation (Mimms et al., Vir. Hepatitis and Liver Disease (1988), 248-251).
Thus, in the early 1980s it was assumed that HBeAg represented a denaturation product and/or a breakdown product of HBcAg. However, it has emerged that a DNA sequence with an open reading frame of 29 amino acids is located in front of the translation start signal of HBcAg (pre C sequence, amino acids −29 to −1).
It is assumed on the basis of current knowledge that in the region of the HBcAg gene (pre C plus C sequence) two different mRNAs are read or one mRNA species codes for two different translation products. HBcAg-specific mRNA contains an open reading frame with codons +1 to +183, and translation results in HBcAg which comprises amino acids +1 to +183.
By contrast, HBeAg-specific mRNA contains an open reading frame with codons −29 to +183. Translation results in formation of a precursor molecule which contains amino acids −29 to +183. The first 19 amino acids of this precursor protein function as signal sequence and lead to translocation of the precursor protein into the endoplasmic reticulum (ER). During further processing there is also proteolytic elimination of the C-terminal amino acids from position 150 onward so that, finally, HBeAg is secreted into the bloodstream.
Despite being substantially identical in amino-acid sequence, HBcAg and HBeAg have completely different immunological, structural and functional properties. Since, moreover, transcription starts at different starting points and leads to different mRNAs, it must be assumed that HBcAg and HBeAg are, ultimately, encoded by different genes, although with a certain overlap.
The paper by Kim et al. (The 1990 International Symposium on Viral Hepatitis and Liver Disease, Houston, Tex., Apr. 4-8, 1990, poster abstract No. 62) may be mentioned here as an example of the importance of the signal sequence for the formation of HBeAg. It shows that only HBcAg-reactive material is obtained in the cytosol of yeasts when the intention is to express HBeAg without the signal sequence directly in yeasts, and describes that the HBeAg must be fused to the alpha-factor signal sequence if the intention is to obtain material which has predominantly HBeAg reactivity.
Secretion by means of the signal seq
Broker Michael
Noah Michael
Allen Marianne P.
Dade Behring Marburg GmbH
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Zeman Mary K
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