Seroreactive regions on HPV 16-proteins E1 and E2

Details Seroreactive regions on HPV 16-proteins E1 and E2 Seroreactive regions on HPV 16-proteins E1 and E2

1995-06-06

2001-04-24

Salimi, Ali (Department: 1645)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S007940, C435S007950, C424S185100, C424S186100, C424S204100, C530S300000, C530S325000, C530S326000, C530S327000, C536S023720, C930S220000

Reexamination Certificate

active

06221577

ABSTRACT:

The invention relates to seroreactive regions on proteins E1 and E2 of human papillomavirus (HPV) 16.
The application also relates to a vaccine which contains such peptides which contain the seroreactive regions.
The invention likewise embraces compositions for diagnostic purposes which contain peptides with the seroreactive regions.
HPV 16 is one of the human papillomaviruses (Proc. Natl. Acad. Sci., USA 80, 3813-3815 (1983). The organization of the genome of HPV 16 has been described in Virology 145, 181-185 (1985).
Genomic sequences of HPV can be detected in most cases of preinvasive and invasive cervical tumors. HPV 16 has been identified world-wide as the virus type predominating in these tumors. The HPV 16 genome is detectable in more than 50% of cervical tumors, in which case it is often present integrated into the cellular DNA. Little is known about the immune response after infections with HPV 16 or other papillomaviruses.
Initial data: patients suffering from cervical tumors were compared with healthy individuals with regard to the presence of antibodies against viral proteins. These viral proteins were then linked as fusion products with various prokaryotic peptides at their N terminus and then used as antigens in Western blots.
The object of the present invention is the further identification of HPV 16 viral structures which can be used as tool in the prophylaxis, diagnosis and therapy of HPV 16-dependent tumorous diseases in humans. The identification of such structures is a prerequisite for the development of ELISAs which make it possible to test a large quantity of human sera for the presence of HPV 16.
The present invention therefore embraces seroreactive regions of the E1 protein of HPV 16, which have one of the following amino-acid sequences:
I.
NGWFYVEAVVEKKTGDAISDDENENDSDTGEDLVDFIVNDNDYLT
(SEQ ID NO: 1)

II.
NENDSDTGEDLVDFIVND
(SEQ ID NO: 2)

III.
MADPAGTNGEEGTGCNGWFYVEAVVEKKTGDAISDDENENDSDTGEDLVDFIVNDNDYLT
(SEQ ID NO: 3)

IV.
EDLVDFIVNDNDYLT
(SEQ ID NO: 4)

V.
EDLVDFIVNDNDYLTQAETETAHALFTAQEAKQH
(SEQ ID NO: 5)

VI.
NENDSDTGEDLVDFIVNDNDYLTQAETETAHALFTAQEAKQHRDAVQVLKRKYL
(SEQ ID NO: 6)

VII.
GSPLSDIS;
(SEQ ID NO: 7)
seroreactive regions of the E2 protein of HPV 16, which have one of the following amino-acid sequences:
I.
DKILTHYENDS
(SEQ ID NO: 8)

II.
DKILTHYENDSTDLRDHI
(SEQ ID NO: 9)

III.
DLRDHIDYWKH
(SEQ ID NO: 10)

IV.
AIYYKAREMGFKHINHQVVPTLA
(SEQ ID NO: 11)

V.
AIYYKAREMGFKHINHQVVPTLAVSKNKAL
(SEQ ID NO: 12)

VI.
YYKAREMGFKHINHQVVPTLAVSKN
(SEQ ID NO: 13)

VII.
INHQVVPTLAVSKNKALQAI
(SEQ ID NO: 14)

VIII.
INHQVVPTLAVSKNKAL
(SEQ ID NO: 15)

IX.
TLAVSKNKALQAIELQLTLETIYNSQYSNEKWTLQDV
(SEQ ID NO: 16)

X.
QLTLETIYNSQYSNEKWTLQDVSLE
(SEQ ID NO: 17)

XI.
TLETIYNSQYSNEK
(SEQ ID NO: 18)

XII.
TSVFSSNEVSSPEII
(SEQ ID NO: 19)

XIII.
VFSSNEVSSPEIIRQHLANHPAATHTKAVALGTEET
(SEQ ID NO: 20)

XIV.
EIIRQHLANHPAATHTKAVALGTEETQTTIQRPRSEP
(SEQ ID NO: 21)

XV.
TEETQTTIQRPRSEPDTGN.
(SEQ ID NO: 22)
The invention furthermore embraces peptides with one or more of the seroreactive regions identified above, a vaccine which contains one or more of the peptides identified above, a composition for diagnostic purposes for the identification of specific antibodies against HPV E1 and/or E2 protein, which likewise contain the peptides identified above, and monoclonal antibodies which have an affinity for one or more of the seroreactive regions of the E1 or E2 protein of HPV 16, and a composition for diagnostic purposes which contains these monoclonal antibodies.
In order to identify seroreactive regions in proteins E1 and E2 of HPV, the experimental route described in Science 228, 1315-1317 (1985) was followed. Subgenomic HPV 16 DNA fragments which had been randomly generated by ultrasound treatment and partial DNAse I treatment were cloned into the phage vector fusel and then expressed as part of a phage coat protein. Seroreactive phage recombinants were identified using sera prepared against E1 and E2, and purified, and the seroreactive regions were characterized by sequencing the HPV 16 portion. Polyclonal rabbit sera against an HPV 16 E1 MS2 polymerase fusion protein and against the amino- and carboxyl-terminal part of HPV 16 E2 (separate, likewise MS2 fusion proteins) were prepared.
The filamentous phages embrace the three groups f1, fd and M13. It is common to them all that binding and uptake of the phages takes place via F pili of the bacteria, i.e. that only F
+
strains can be infected. The fd wild-type phage, from which the vector system used is derived, forms particles which are about 900×6 nm in size and which are composed in particular of about 2700 subunits of the main coat protein. In addition, in each case 5 molecules of the minor coat proteins pIII, pVI, pVII and pIX are located at both ends of the virions. The single-stranded, circular phage genome which, in the case of the fd wild-type, is 6408 bp in size, carries the information for a total of 10 different proteins.
In the fd derivatives fusel, fuse2 (Parmley and Smith, Gene, 7, 305-318 (1988)) and fusemm, a tetracycline-resistance gene is integrated, by insertion of a part of the Tn10 transposon, in the phage genome, which has been enlarged to about 9.2 kbp in this way. This means that the replicative DNA double-stranded phage genomes behave in the bacteria like selectable plasmids and can accordingly be prepared and used for clonings. Another modification from the wild-type is the presence of a reading frame mutation in the gene for the minor coat protein pIII in conjunction with an inserted restriction site for cloning expressable DNA fragments. The gene for pIII is composed of two almost completely independent domains (Crissmann and Smith, 1984): an N-terminal domain which mediates the binding of the phages to the bacterial cell receptor (F pili) and a C-terminal protein domain which is responsible for phage morphogenesis. The reading frame mutation, which is located directly behind the signal sequence of the protein, thus leads to inactivation of the gene and accordingly also prevents the formation of infectious particles. This is of importance for the replication of these phage mutants as plasmids because the fd genomes inactivated in the morphogenesis do not damage the host bacteria (Smith, in: Vectors, A Survey of Molecular Cloning Vectors and Their Uses, Butterworth Publishers, Stoneham, Mass. 61-85, 1987).
Insertion of suitable DNA fragments and restoration of gene III functions lead to the formation of infectious phage particles which carry additional amino-acid sequences on their coats. These sequences are accessible to various ligands, for example antibodies, in the natural state of the phages.
The fd expression system used in this invention is essentially based on setting up phage banks by cloning DNA foreign sequences into the gene III, and examining the latter with the aid of monoclonal or polyclonal sera for seroreactive recombinants. An amplification normally takes place on preparation of these expression banks. The extent of this replication of individual clones in turn depends on the nature and size of the inserted DNA sequence. This means that different clones differ in frequency, which may differ by up to several powers of ten. It is therefore possible to derive from the stated properties the following two features of the fd expression banks:
Amplification of the banks, which leads to repeated cloning of identical phage clones isolated by immunoscreening.
Possibility of enriching seroreactive phages by affinity chromatography (columns) because phages in the active state can be bound and eluted again.
The repeated isolation of identical recombinants was avoided by using separately set up banks, there being an extremely low probability of cloning a DNA fragment prepared identically and in parallel, or of the phage recombinant derived therefrom.
In this invention, a total of 11 different expression banks for HPV 16 DNA in fusel were set up. The number of primary, tetracycline-resistant and insert-harbouring recombinants was in this case between 2000 and 90000 per bank. Sin

Affiliated with

Also associated with

Rating

Seroreactive regions on HPV 16-proteins E1 and E2 does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Seroreactive regions on HPV 16-proteins E1 and E2, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Seroreactive regions on HPV 16-proteins E1 and E2 will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2514286