CN1365286A - Method for treating chronic HBV infection - Google Patents

Abstract

Methods and kits for treating chronic HBV infection are described. Compositions containing PreS2 + S antigen of HBV and a metabolizable oil adjuvant are utilized.

Description

The method of treatment chronic HBV infection

Invention field

The present invention relates to treat the immunotherapeutic agent of chronic HBV (" HBV ") infection and disease.

Background of invention

85% above child HBV infection becomes chronic.The acute HBV of 1-5% infects and can not cure among the adult, also becomes chronic infection.Chronic infection can cause chronic active hepatitis (case of 20-50%), liver cirrhosis or even liver failure (case of 10-20%), cause hepatocarcinoma (case of 1-3%) in some cases.Hepar damnification mainly is that the hepatocellular cellullar immunologic response that infects is caused.

Only at just the have an appointment chronic carrier of 1,000,000 HBV of the U.S..The whole world has 300,000, the chronic carrier more than 000.Many chronic carriers are (being Taiwan, Japan, South Asia) and Africa in the pan-Pacific area.

The mechanism that causes chronic HBV infection it be unclear that.The existence of hepatitis B e-antigen (" HBeAg " or " HBe ", preceding core polypeptide) is relevant with the activeness virus replication, is the index of infectious increase and seriousness.If positive prognosis is better though the patient is anti--HBe, acute and chronically infected recovery is all relevant at the antibody of hepatitis B surface antigen (" HBsAg " or " HBs ") with generation.Along with neutrality anti--appearance of HBs antibody, it is to infect the index of removing that HBsAg disappears.Yet the spontaneous seroconversion of antagonism HBs is extremely rare.Though annual 10% patient experience the has been arranged spontaneous seroconversion of HBeAg to anti--HBe becomes anti-HBs to occur over just every year among about 1% the patient by the HBs seroconversion.

Though the therapy of existing treatment Chronic HBV, most of scopes and effectiveness are all limited.Interferon therapy only causes anti-HBs seroconversion in 3-5% patient.In addition, interferon therapy is very expensive, may have serious adverse, and must every day subcutaneous injection.

The antiviral drugs of upgrading can reduce viral load as lamivudine, but only cause anti-HBs seroconversion in a few patients.In addition, they must life-time service-interruption can cause virus to reappear, and making to need lifelong treatment-and the drug resistance mutant may occur.

Also existing immunotherapy.Some immunotherapies are used based on antibody.When the anti-HBs antibody of independent use (" HBIG "), can have therapeutical effect, infection proves to HBV as liver transplantation.Protein Design Laboratory (Protein Design Labs) just efforts be made so that with the anti-HBs antibody of humanization makes the Ig perfusion therapy.

Other immunotherapies are used based on antigen.A kind ofly utilized the HBsAg of yeast expression and oil/aqueous emulsion to add RIBI  and QS-21 (SmithKline Beecham) based on antigenic immunotherapy.Carrying out chronically infected second phase test at present.Another kind utilizes PreS1+PreS2+S/ Alumen (Medeva).Also have another kind of (just carrying out 150 patients' immunotherapy test now) to use PreS2+S/ Alumen (Institut Pateur).Another kind utilizes the deutero-peptide of core protein based on antigenic immunotherapy, is used for inducing cytotoxic T lymphocyte responses (Cytel).

Still need to improve the potent immunotherapy of chronic HBV infection.

The invention summary

On the one hand, the present invention relates to treat the method for chronic HBV infection, comprise and use a kind of compositions, said composition contains reorganization PreS2+S protein and a kind of metabolizable oily adjuvant and optional antiviral drugs or the chemical compound of HBsAg.

Another aspect the present invention relates to a kind of immunization therapy medicine box, and it comprises the PreS2+S protein of HBsAg, a kind of metabolizable oily adjuvant and optional antiviral drugs or chemical compound.

The accompanying drawing summary

Fig. 1 has described the HBV genome with interested restriction site and has been used to prepare the joint of expression plasmid.

Fig. 2 schematically describes this clone's strategy.

Fig. 3 a-b, 4a-b and 5a-b have described three patients' the anti-HBe of HBe/ (a) and Serum ALT/HBV DNA (b) result, and second patient shows the short-term burst aggravation.

Detailed Description Of The Invention

Enforcement of the present invention can be adopted (unless otherwise indicated) virology, immunology, microbiology, molecular biosciences The conventional method of learning and the recombinant DNA technology of this area. These technology are in the literature existing to be discussed comprehensively, sees example Such as Sambrook etc., Molecular Cloning:A Laboratory Manual (second edition, 1989); DNA Cloning:A Practical Approach, volume I﹠II (D.Glover volume); Methods In Enzymology (S. Colowick and N.Kaplan compile, Academic Press, Inc.); Handbook of Experimental Immunology, (D.M.Weir and C.C.Blackwell compile volume I-IV, Blackwell Scientific Publications) and Fundamental Virology, second edition, volume I﹠II (B.N.Fields and D.M.Knipe compiles).

The present invention relates to the immunotherapy based on antigen, be used for the treatment of chronic HBV infection. " control as used herein Treat " comprise and improve and/or eliminate HBV and infect, and stimulate immune response to it, preferred anti--HBsAg or anti--HBe replys. People HBV is the Hepadnaviridae of having a liking for liver property dna virus that infects birds and Mammalian The member. Outer membrane associated antigen HBsAg can induce neutrality protection antibody response. HBsAg is by three kinds of relevant eggs White matter forms, and they have common amino acid sequence-S, preS2+S and preS1+PreS2+S.

Usedly among the present invention contain PreS2+S antigen and a kind of metabolizability oil adjuvant that antigenic compositions contains HBV.In one embodiment, the PreS2+S antigen generation of in Chinese hamster ovary cell, recombinating.In another preference, this metabolizability oil adjuvant is adjuvant MF59 hereinafter described.

In the method for the invention, above-mentioned composition is applied to individuality.Optional, use this contain antigenic compositions before, simultaneously or use a kind of antiviral drugs afterwards.Before containing antigenic compositions, use time of antiviral drugs can be from 1 month to 24 months.The hepatocyte that has jumpbogroup to infect among the Chronic HBV patient.Because hepar damnification is mainly owing to cause infected hepatocellular cellullar immunologic response, advised before other treatment, prolonging antiviral therapy, to reduce infected hepatocellular quantity (Genome, J.Clin.Invest.102 (5): 867-868, in JIUYUE, 1988).In addition, reduce the level of cyclicity HBsAg with antiviral drugs and can pass through to reduce the free antigen amount, reduce immunization therapy and begin the sedimentary probability of back antigen-antibody complex.

Can use the foregoing of effective dose to the individuality that has chronic HBV infection.Term used herein " effective dose " refers to realize the desired purpose of treatment, and does not have the required amount of adverse side effect (as poisoning, stimulation or anaphylaxis).Though individual needs may be different, the optimum range of determining the preparation effective dose is in those skilled in the art's limit of power.Human dosage also is not difficult to derive (Katocs etc., the 27th chapter are in Remington ' s Pharmaceutical Sciences, and 18 editions, Gennaro compiles, Mack PublishingCo.Easton, PA, 1990) from zooscopy.Usually, provide the required dosage (can regulate) of preparation of effective dose to change with several factors by those skilled in the art, comprise the character of age, health, physiological situation, body weight, disease that the receiver takes a disease or unusual kind or degree, therapeutic frequency, the character of treatment (if desired) simultaneously, required effect and scope (Nies etc., chapter 3 is in Goodman﹠amp; Gilman ' s The Pharmaceutical Basis ofTherapeutics, the 9th edition, volumes such as Hardman, McGraw-Hill, New York, NY, 1996).The dosage range of considering is about 5-100 microgram.

Also should consider to need repeatedly to use said composition.Time between the administration is decided by administration quantity.For example, if be administered twice, for the first time can be at the 0th month, for the second time respectively the 1st, 2 or June.In addition, administration in January at interval.Time between the multiple dosing is not difficult to be determined by those skilled in the art.Administration is 8 times in one embodiment.

Term used herein " administration " includes but not limited to that transdermal, gastrointestinal tract are outer, subcutaneous, intramuscular, oral cavity and local delivery.The common requirement of any administration is effectively and transmits conveniently.In a preference, intramuscular is used said composition.

The purpose of open the inventive method is to improve chronic HBV infection.This improvement can be by for example using the minimizing of HBV DNA in the nucleic acid test determination blood; Measure the minimizing of serum alanine aminotransferase (ALT) with routine test; Or increase with the short-term burst that transaminase is measured in routine test.Preferred treatment of the present invention will cause resisting-decline/disappearance appearance of HBe and HBsAg the time, and anti-HBs more preferably appears simultaneously with decline/disappearance of HBs.All these available routine immunization test determination.For the present invention, normally the ALT level is decided to be the about 6-34IU/L of women, the about 6-43IU/L of male.

Term used herein " metabolizability oil adjuvant " refers to contain individual nontoxic to what used, and can be by the metabolic oil adjuvant of individuality, preferably about 6-30 the carbon atom of this oil includes but not limited to alkane, alkene, alkynes and corresponding acid and alcohol, ether or ester thereof, and composition thereof.This oil can be any can be by the oil of the vegetable oil of host animal organism metabolism, fish oil, animal oil or synthetic preparation, the body metabolism of the host animal that they can be applied, nontoxic to individuality.Host animal is mammal normally, preferably the people.The clear and definite eliminating of mineral oil and similar poisonous oil distillate oil is outside the present invention.

The exemplary source of vegetable oil comprises nut, seed and corn.The example that Oleum Arachidis hypogaeae semen, Oleum Glycines, Oleum Cocois, these modal oil of olive oil are macadamia nut oils.Seed oil comprises safflower oil, Oleum Gossypii semen, Oleum Helianthi, Semen Sesami wet goods.In cereals, Semen Maydis oil is the easiest to be obtained, but also available other corn oil are as Semen Tritici aestivi, Herba bromi japonici, rye (Secale cereale L.), rice, Herba Eragrostidis pilosae, black Semen Tritici aestivi etc.

The technology of acquisition vegetable oil has obtained good exploitation and has known.These and other similarly oily components can find in for example Merck Index and the raw material about food, nutrition and food technology.

Though the glycerol of 6-10 carbon and 1, the fatty acid ester of 2-propylene glycol is natural in seed oil not to be existed, and they can be by the suitable parent material preparation from nut and seed oil of hydrolysis, separation and esterification.These products can NEOBEE  (PVO International INc., Chemical Specialties Division, 416Division Street, Boongon, trade name NJ) and other manufacturers buy.

Also can in adjuvant of the present invention and immunogenic composition, use oil from any animal origin.The animal oil ﹠ fat is solid-state under physiological temp usually, because they exist with the triglyceride form, and than the saturation height of fish or vegetable oil.Yet, can obtain fatty acid by saponification triglyceride (it provides free fatty) partially or completely from Animal fat.The fat of mammal milk and oil are metabolizable, therefore can be used for enforcement of the present invention.It is well known in the art obtaining the required separation of pure oil, purification, saponification and additive method from animal origin.

But many fishes contain the metabolism oil of being not difficult to reclaim.For example, cod liver oil, shark liver oil and whale oil such as spermaceti are the examples of several fish oil that can use in this article.Can synthesize many side chain oil, so-called terpene by 5 carbon isoprene unit biochemical methods.Shark liver oil contains paniculate unsaturated terpene, is called zamene, and 2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosa carbon hexane is particularly preferred in this article.Squalene (saturated analogues of zamene) also is particularly preferred oil.Fish oil (comprising zamene and Squalene) is not difficult to obtain from merchandise resources, or available methods known in the art obtain.

The oil ingredient of these adjuvants and immunogenic composition will be with about 0.5%-20% volume, preferably is not more than approximately 15%, and particularly the amount of about 1%-12% exists.Most preferably adopt about 1%-4% oil.

EP 0 399 843 B1 have described the metabolizability that this paper considered oil adjuvant, are incorporated herein for your guidance.Preferred adjuvant is than in the patent and " MF59; the design and the evaluation of the wisdom of vaccine for man and powerful adjuvant " the 10th chapter such as Ott, pp.277-296, Vaccine Design:The Subunit and Adjuvant Approach, Michael F.Powell and Mark J.Newman compile, Plenum Press, New York, the MF59 that 1995 (being incorporated herein for your guidance) are described.Particularly; the following preparation of MF59: the MTP-PE of the zamene of 4.3%w/v, the Tween of 0.5%w/v 80 , 0.5%Span 85 and 400 mcg/ml (the different glutamyl of N-acetyl group muramyl-L-alanyl-D--L-alanine-2-[1,2-two palmityls-Xi-glyceride-3-3 (hydroxyl phosphorus acyloxy)] acetamide).The mechanism of action of MF59 seemingly produces strong CD4 ⁺T cell response.

This adjuvant can also contain emulsifying and/or immunostimulant.In pharmaceutical science, use the emulsifying and the suspending agent of many quantity usually.These comprise that natural deutero-material is as natural gum, phytoprotein, based on polymer such as the alginate and the cellulose etc. of sugar.Some oxygen polymer or polymer that has hydroxyl or other hydrophilic substituents on carbon skeleton has surface activity, as the list or the multi-functional compounds of povidone, polyvinyl alcohol and glycerol ether.The long-chain fatty acid derived compounds forms and can be used for the 3rd group of emulsifying and suspending agent of the present invention.If nontoxic, available aforementioned any surfactant.

Exemplary immunostimulant comprises the synthetic property adjuvant that can improve host immune power, as levamisole and inosine pranobex.Levamisole is the laevoisomer of tetramisole, and it strengthens body fluid and cellular immunity by T-cell dependency mechanism.Inosine pranobex, a kind of complex that contains the purine precursor of inosine, ribosidoadenine and guanosine can promote the T cell mitogen.Tuftsin (Tuftsin), the sequence homology in a kind of 4 amino acid whose peptides (Thr-Lys-Pro-Arg) and immunoglobulin (Ig) heavy chain mainly stimulates macrophage.

Term used herein " individuality " refers to animal, normally mammal, preferably people.

Term " about " used herein refers to ± 10%.

Term used herein " one " and " being somebody's turn to do " refer to single or multiple.

" chronic HBV infection " used herein is often referred to the HBV infection that continues to surpass acute phase.Chronic infection may be that the chronic disease that obtains in child or manhood causes.The chronic infection that disease causes usually can be by HBSAg existence and ALT level unusual, but and with or identify without the HBeAg of detection level.The normal common scope of ALT level is 10-32U/L (women 9-24U/L, be adult's twice baby's normal range) (webpage: On Hepatitis C International, " laboratory blood testing " 7/10/98 modification).Yet chronic infection also comprises the asymptomatic carrier with normal ALT; Begin to use antiviral drugs,, but make virus be reduced to the following patient of detection level as interferon therapy; With the patient after the liver transplantation, they are continuing antiviral therapy or are accepting the Ig perfusion.The HBsAg that continues above 6 months after the actute infection is chronically infected characteristics.In chronically infected patient, recorded antiviral t cell response defective (Boni etc., J.Clin.Invest., 102 (5): 968-975, in JIUYUE, 1998).

Term used herein " antiviral agent " or " antiviral compound " include but not limited to interferon and nucleoside analog, comprise lamivudine.Lamivudine has been used to reduce the hepar damnification that Chronic HBV causes, but can each patient's every day oral 25-300 milligram dosage, up to 1 year.Yet the dosage of suitable antiviral drugs will specifically depend on as medicament and patient.

Shown that the vaccine that contains reorganization HBV PreS2+S antigen (10 microgram) and metabolizability oil-in-water adjuvant MF59 can strengthen its immunogenicity in the adult (Poland etc., 37 ICAAC, p.216, JIUYUE in 1997 28 days-October 1).After using a vaccinating agent, 89% test individuality has produced the anti-HBs titre of serum protectiveness.100% test individuality is protected behind double injection.In addition, the geometric average antibody titre of PreS2+S/MF59 vaccine is than containing the control vaccine high 100 times (Poland etc. see on) of aluminum as adjuvant.The inventor predicts that available similar compositions treats chronic HBV infection as immunotherapeutic agent.

Embodiment 1

Prepared the compositions that contains reorganization PreS2+S and oil-in-water adjuvant MF59.Be prepared as follows reorganization PreS2+S.Hyclone provides whole culture fluid and supplement.

By inserting the S+PreS2 gene, made up recombiant plasmid pKSVAdPreS-BglII (Fig. 1-2) with two joints.Make up the pKSV-10 that the cloning vehicle that uses in this recombinant expression system is Pharmacia.The long 7.2kb of pKSV-10 carrier, cloning site is BglII.As Valenzuela etc., Nature, 280:815-819, on August 30th, 1979 (being incorporated herein for your guidance) is described, separates to obtain viral DNA.Partly digest the EcoRI/BglII fragment of about 1880 bp of virus genom DNA with HhaI, separate the EcoRI/HhaI fragment that obtains 1230bp.Designed joint and be connected, formed the BglII/BglII fragment, be used to be cloned into pKSV-10 with the EcoRI/HhaI fragment.Then pKSV-10/BglII is connected with Pre S BglII/BglII fragment, obtains DNA plasmid vector pKSVAdPreS-BglII.Transformed competence colibacillus cell HB101 then.Screening obtains transformant, prepares in a large number.

The dhfr sequence is inserted pSV7d made up cloning vehicle pSV7d/AML-dhfr (123:436-442 1989, is incorporated herein for your guidance for Stibbe etc., Virology).With pKSVAPres-BglII and pAML/dhfr cotransfection DG44 (dhgr-) Chinese hamster ovary celI (77:4216-4220 1980, obtains with passage number about 100 in JIUYUE, 1985 from the Dr.Leslie Rall of Chiron for Urlaub etc., PNAS USA).Then, in 24 hole microtitration plates, cultivate 48 clones.With Abbott ELISA method screening and cloning, select the high yield of amplification called after PKSVAdPres 16 to clone with methotrexate, finally obtain cloning PKSVAdPreS 16-12-80-30 (being abbreviated as HEP 30 cells).Amplification HEP 30 cells, preparation master/work (Master/Working) cell bank, called after HBV B30 cell.

The cell of clone HBV B30 master cell bank increases in the DME/F12 culture fluid of FBS that has replenished 6% dialysis and 50 μ M methotrexate (MTX).The amplification back makes cell adapted MTX-DMEM/F12 culture fluid by several steps (FBS concentration from 6% to 2% progressively descends in 3 liters of rotary flasks).After the adaptation, cell inoculation in the stirring perfusion container of 3L, is used DMEM/F12, the 2%FBS perfusion.

Then some perfusion cells sub-clone in the presence of 50 μ M MTX is incubated in the DMEM/F12 10%FBS culture fluid, in the presence of 2%FBS and 2 μ M MTX, goes down to posterity, make it be adapted to HBV and produce culture fluid by jolting flask repeatedly.In the 14th generation, the productivity of test sub-clone is with parent's culture small test tube frozen (be deposited in Chiron cell culture preservation center, be numbered CMCC11336).In these cells some are melted in the DM122 culture fluid, in the DM122 culture fluid that has replenished 200mM L-glutaminate, 1mM (1: 500) MTX, increase then.When cell density reaches 4.0-8.0 * 10 ⁵During living cells/milliliter, with cell at DM122, contain among 10% human albumin (w/v)+7.5% (v/v) DMSO and be concentrated to 1.0 * 10 ⁹, be distributed to 1 milliliter/test tube, produce new HBV Master cell bank (MBVMK001).With 1 ℃/minute these cell freezings are arrived-96 ℃, be stored at then≤-176 ℃.

Of (on seeing, being incorporated herein for your guidance) such as Ott, prepared MF59 as aseptic O/w emulsion.10 microgram PreS2+S in the 0.25 ml volumes antigen are mixed with 0.25 milliliter of MF59, and final dose is 0.5 milliliter.Hereinafter table 1 and 2 has been listed the composition of this vaccine.

Table 1

The HBV antigen composition

Amount in the every final dose of composition

0.02 milligram of PreS2+S antigen

Sodium chloride, 3.80 milligrams of USP

Sodium citrate, dihydrate, 2.15 milligrams of USP

Citric acid, monohydrate, 0.04 milligram of USP

(Tween 80 for polysorbate80 ^TM) 0.01 milligram

The water for injection capacity

PH scope 6.0-7.0

Table 2

The MF59 adjunvant composition

Amount in the every final dose of composition

9.75 milligrams of zamene

(Tween 80 for polysorbate80 ^TM) 1.18 milligrams

(Span 85 for three oleic acid sorbitol esters ^TM) 1.18 milligrams

Sodium Citrate, usp, Dihydrate Powder, 2.15 milligrams of USP

Citric acid monohydrate, 0.04 milligram of USP

The water for injection capacity

pH6.4

Embodiment 2

In the triangular muscle with 500 microlitre embodiment, 1 described compositions intramuscular injection to 13 patient, these patients determine to suffer from Chronic HBV by continuing to exist more than HBsAg6 month.8 patient HBe positives.Whole 13 patients serum ALT raise (being higher than 1.2 * ULN, i.e. upper limits of normal), do not have Decompensated hepatic disease evidence.The 0th, 1,2 and administration in 6 months.By measuring HBV DNA, Serum ALT levels reduction, the HBeAg level reduces, and administration for the second time anti--HBe antibody occurs and determines effective after one month.Measured HBV DNA (Chiron and Chen etc., J.Virol.Methods, 53 (1): 131-7, May nineteen ninety-five, for your guidance) with the bDNA method in this complete introducing.HBeAg, anti--HBe, HBsAg and anti-HBs level have been measured with following chemiluminescence immunoassay test.Other sees U.S. Patent number 5,395,752, is incorporated herein for your guidance.(s/co) reported the result with ODU.

In order to measure HBeAg, with 100 microlitre serum people anti--the emulsion 100 microlitre couplings of HBe have the mixing of newborn magnetic-particle (600 mcg/ml), cultivated 18 minutes for 37 ℃.Add the link coupled people of 100 microlitres and DMAE (dimethyl acridinium ester, 300 30,000,000 relative light unit, " RLU " every milliliter) and resist-HBe, cultivated 18 minutes for 37 ℃.Wash this granule 3 times with buffer, measure chemiluminescence then.Used buffer has following ingredients: 50mM TRIS, pH8.0; 500mM potassium chloride; 0.09% Hydrazoic acid,sodium salt; 1mM EDTA; 0.05%Tween20; And 1.75%BSA, sulfydryl modification filters with 0.22 micron filter unit of MILLIPAK-60.

In order to measure HBsAg, with above-mentioned buffer 1/100 dilution 100 microlitre serum, itself and 100 microlitres are coupled to the mouse anti-HBs (specificity combines anti-" a " neutralizing epitope of HBsAg) of paramagnetic particle (being respectively 280 mcg/ml and 150 mcg/ml for antibody concentration) and (resist " a " with mouse anti-HBs, 75.0 nanograms/milliliter) link coupled (with above-mentioned buffer dilution) 50 microlitre DMAE mix, cultivated 18 minutes for 37 ℃.With buffer washing granule 3 times, measure chemiluminescence then.

Anti-in order to measure-HBe, 50 microlitre serum and 100 microlitres reorganization HBeAG SOD fusion rotein (50 nanograms/milliliter) (is for example seen Choo etc., Science, 1989,244,359-362 is incorporated herein for your guidance) and 100 microlitres be coupled to the anti-HBe of the people of latex magnetic-particle (600 mcg/ml) and mix, cultivated 36 minutes for 37 ℃.Adding resists-HBe with the link coupled 100 microlitre people of DMAE (300 * 10E, 6 RLU/ milliliters), cultivates 18 minutes for 37 ℃.With buffer washing granule 3 times, measure chemiluminescence then.Used buffer has following component: 100mM borate buffer, pH8.9; 0.09% Hydrazoic acid,sodium salt; 5mM EDTA; 0.05%TWEEN 20; With 0.2% gelatin (fish), filter with MILLIPAK-60,0.22 micron filter unit.

Measure anti--HBs (Abbott) with the standard enzyme immunologic test.

In 8 male patients of initial HBe, 4 remarkable decline and anti--HBe production of antibodies of having induced the HBeAg level after 3-4 injection are with the decline of HBV DNA and Serum ALT.Fig. 3 a-b, Fig. 4 a-b and Fig. 5 a-b have described 3 patients' data.Also in several patients, induce the significant level of anti--HBs antibody, comprised the patient's (see and for example show 3-5, be anti--the HBs hurdle) who does not have HBe at first.Two among 8 patients show that Serum ALT significantly increase in research process.The both is cured.

After further following up a case by regular visits to, the more than half generation resists-HBsAg among 13 patients.4 patients' seroconversion becomes anti-HBe, and 3 become HBV DNA feminine gender among 4 seroconversion patients.

Embodiment 3

500 microlitre embodiment, 1 described compositions and 100 milligrams of lamivudines were applied to patient at 0 o'clock.The described 500 microlitre compositionss in addition of embodiment and other 100 milligrams of lamivudines were used in the time of 1 month.Measure anti-HBs titre, and the existence that detects the HBs after 1 month of administration for the second time, determine effect.If detectable HBs still exists, carry out administration for the third time.Repeat this process, up to no longer detecting HBs.

Embodiment 4

100 milligrams of lamivudines were begun to be applied to patient at 0 o'clock, and every day the potion successive administration.With 500 microlitre embodiment, 1 described compositions the 1st, 2,3 and intramuscular injection in July give patient.Measure HBeAg and anti-HBe titre, and after the compositions of using embodiment 1 January detect HBV DNA and determine effect.If still there is detectable HBe, further use the compositions of embodiment 1.If after administration again, still can detect the existence of HBe January, carry out another time administration.Repeat this process, up to detecting less than HBe.

Embodiment 5

500 microlitre embodiment, 1 described compositions was applied to patient at 0 o'clock.Be applied to 100 milligrams of lamivudines of this patient during January.Measure anti-HBs titre, and after the compositions of using embodiment 1, detect the existence of HBs and HBe January, to determine effect.If still there is detectable HBs, use the compositions of embodiment 1 for the second time.If still can detect the existence of HBs or HBe, carry out administration for the third time.Repeat this process, up to detecting less than HBs.

Embodiment 6

Used 100 milligrams of lamivudines every day 6 months.500 microlitre embodiment, 1 described compositions was applied to patient at the 7th, 8,9 and 13 month.If have detectable HBs or HBe, further administration.

Embodiment 7

Material:

Chiron  HBV/MF59: antigen: the reorganization HBV PreS2+S (20 micrograms/dosage) of Chinese hamster ovary celI generation and purification.Adjuvant: MF59, the O/w emulsion of micro-liquefaction.Provide antigen and MF59 with the tubule that separates, when injection, mix.0.5 milliliter of every dose of cumulative volume of intramuscular injection.Timetable is included in 0-, 1-, 2-and the 6-month uses 4 times (table 6).

Lamivudine (Epivir , Glaxo Wellcome): patient must treat 1 month before beginning Chiron  HBV/MF59 treatment at least.Lamivudine after this (i.e. back 1 month of the 4th injection) continues 7 months with the dosage level (100 milligrams-300 milligrams of every days) before the ChironHBV/MF59 treatment.

Efficacy determinations: when this research finishes, measure the patient's ratio with following parameters: HBsAg disappearance or minimizing, HBeAg disappears and anti-HBs or anti-HBe seroconversion.Treating the variation of back HBV dna level and Serum ALT/AST in advance will and summarize afterwards during Chiron  HBV/MF59 treatment with Chiron  HBV/MF59.For present disclosure, resulting efficacy data is limited to selected time point, does not carry out rounded analysis.

Method:

The compositions (a kind of oil-in-water adjuvant) that will contain reorganization PreS2+S antigen and MF59, i.e. Chiron  HBV/MF59) (table 2) is applied to suffers from chronic HBV infection, accepts the patient of lamivudine.Patient 0,1,2, June intramuscular accept 4 Chiron  HBV/MF59 injections.Patient continues to use lamivudine therapy in 6 months of Chiron  HBV/MF59 injection, and accepts lamivudine 1 month again after the 4th dose, the lamivudine of stopping using then.

Qualified patient comprises chronic HBV infection patient (positive definite with the male history of screening detection HBsAg by at least 6 months serum HBsAgs), and it has the compensatory hepatic disease, begins the candidate of lamivudine therapy when just accepting lamivudine or screening.At least accepted lamivudine therapy one month before using first dose of Chiron  HBV/MF59 in when screening with the patient of lamivudine therapy.These patients continue to accept lamivudine (100-300 milligram/day) with the preceding dosage of its research.The patient who does not accept lamivudine therapy during screening begins before the Chiron  HBV/MF59, treats at least one month at least.These patients accept lamivudine with 100 milligrams/day approval dosage.Participate in having got rid of among the patient according to medical history, health check-up, clinical laboratory's assessment or previous liver biopsy evidence show suffer from the compensatory difference or late period hepatic disease all patients.

Possible patient 1-8 before becoming a full member of research has experienced initial screening in week, during taked their case history, physical examination and blood and urine sample.Serology and virusology parameter (qualitative and quantitative assay) data: HBsAg, HBeAg, anti-HBs and anti-HBe that following HBV infects when screening, have been obtained.Other tests comprise: liver function detects (LFT): ALT, AST, alkali phosphatase, bilirubin (total and directly), prothrombin time, gross protein and albumin; Full blood count (CBC); Protein, hemanalysis and feces with urine.With polymerase chain reaction (PCR) quantitative assay HBV dna level.If prove on evidence before screening or when screening (according to the HBV dna level that improves) the lamivudine drug resistance mutant is arranged, get rid of this class patient.Other clinical laboratory's tests comprise during the screening investigation: HCV antigen/antibody combination, anti-HIV antibody and hepatitis anti-.Arbitrary male patient excludes from the participant with these antibody.

Except the standard that comprises and get rid of, it is qualified that possible the qualified patient who does not accept lamivudine therapy in this research when investigation screening must meet following extra standard ability: Serum ALT is higher 1.2 times than upper limits of normal, is higher than the 0.7MEq/ milliliter with bDNA test determination HBV DNA.The blood sample of collecting during the screening investigation has obtained these experiment values.

Meet all patients that comprise standard and do not have any exclusion standard (comprise clinical experiment test result) and return to accept its intramuscular injection first.Whether observed 30 minutes after the per injection, observing it has local and general reaction symptom.They fill in the diary card, describe back 7 days part (as injection site pain, heating, erythema, sclerosis etc.) of injection and whole body (as tired, uncomfortable, generate heat, feel cold, myalgia, arthralgia, nauseating, headache, erythra) react.Patient keeps keeping a diary card, record medical care problem and the medicine of obeying during 7 days.If any time, patient experiences any unusual, serious or severe adverse events that may be relevant with treatment in experiment, allow their the clinic prescription on individual diagnosis that comes right away.Allowed patient see the doctor in preceding 14 days at second and third and four injections (as being respectively 1 month, 2 months and 6 months), before accepting injection next time, to carry out clinical experiment test (LFT, urinalysis, creatine) inspection.

For the first time inject Chiron  HBV/MF59 and stop to use lamivudine after seven months.Patient goes back to hospital and does clinical and laboratory is followed up a case by regular visits in the time of 7.5,8,9,10.5 months.

In experimentation, begin to monitor all adverse events (AE) to final visit from Chiron  HBV/MF59.Whole prescription drugs used in the whole experiment have been write down.

Number of patients: plan and actual the recruitment are 24 people.All patients accept 4 Chiron  HBV/MF59 injections.

Table 6

24 (100%) immune #2 of day immune #1:# immunity occur in injection: average 31.7 medians of fate 28.0 standard errors 11.3 minimum 21 maximum 66 numbers, 24 immune #3 after the immunity inoculation for the first time: average 60.7 medians of fate 56.0 standard errors 12.4 minimum 51 maximum 94 numbers, 24 immune #3 after the immunity inoculation for the first time: average 29.0 medians of fate 28.0 standard errors 3.2 minimum 23 maximum 39 numbers, 24 immune #4 after the immunity inoculation for the second time: average 180.0 medians of fate 180.5 standard errors 9.1 minimum 168 maximum 198 numbers, 24 immune #4 after the immunity inoculation for the first time: average 148.3 medians of fate 148.0 standard errors 11.4 minimum 119 maximum 168 numbers, 24 immune #4 after the immunity inoculation for the second time: average 119.3 medians of fate 120.0 standard errors 11.5 minimums after the immunity inoculation for the third time

Table 7

Patient's statistics and patient characteristics feature patient number (altogether 24 patients) HBsAg during screening: positive 24 (100%) anti-HBs: negative 20 (83%) positive 4 (17%) HBeAg: negative 9 (38%) positive 14 (58%) other 1 (4%) anti-HBe: negative 15 (65%) positive 8 (33%) other 1 (4%) third livers: negative 24 (100%) hepatitis δ infect: negative 24 (100%) HIV infect average 69.5 medians of anergy 24 (100%) ALT (U/L) 49.0 standard errors 64.7 minimum 17 maximum 276 numbers 24

The result sums up

Patient characteristics and statistics: 42 years old patient's mean age (27-63 year).20 people (83%) male among 24 patients.13 patients (54%) are asian ancestry, 9 (38%) individual be the Caucasian, 1 people is the Black American, a people is the quadroon.When recruiting, 14 (58%) the people HBeAg positives, and the anti-HBe positive of 8 (33%) people (detecting) with EIA and Abbott; A patient's data do not obtain, and another patient both is negative.The horizontal 70IU/L of average serum ALT (scope 17-276IU/L) (table 7) during recruitment.The average duration of using lamivudine was 7.7 months (scope 1-33 month) (table 7) before accepting to inject Chiron  HBV/MF59 for the first time.16 people have accepted the other treatment outside the lamivudine among 24 patients before participating in this test.These treatments comprise interferon (comprising interferon-a and CIFN form), famciclovir (famciclovir) and Hepatitis B virus vaccine (CV-1899) (table 9).

On January 1st, 2000, all 24 patients accepted whole 4 Chiron  HBV/MF59 injections.Follow the tracks of 22 patients and interrupted lamivudine at least 2 months.In these 22 patients, 5 patients' data collection to 10.5 month, and two patients have finished whole research (December).

Table 8

The lamivudine persistent period before lamivudine is used and recruited: month

Average 7.67

Median 5.35

Standard error 7.78

Minimum 0.9

Maximum 33.3

Number 24 uses the total duration of lamivudine: month

Average 14.46

Median 12.25

Standard error 7.72

Minimum 7.6

Maximum 40.3

Number 24

Table 9

Treating hepatitis treatment patient interferon 101,601,602 interferon-ALPHA 201 before the test except lamivudine, 203,204,208,401,402,403,404,501 interferon (CIFN (common sequences interferon)), 202 famciclovirs 102,602 Hepatitis B virus vaccines (CV-1899) 101,207

Clinical laboratory estimates: collected the historical information of Serum ALT levels before the research as far as possible widely.The T/A difference of each patient's gained data.Many patients changed the ALT level and extensively fluctuate along with the time, were included in that the ALT level significantly improves in the research of accepting before the lamivudine.In 24 patients, 9 symptoms that record the preceding hepatitis burst of research aggravation, the Serum ALT levels peak value is more than the 700IU/L.ALT horizontal peak median among these 9 patients is 1035IU/L, and scope is 782IU/L-3116IU/L.

Serum ALT levels is in most patients, in when screening or in initial 7 middle of the month of participating in research normal or too high slightly (less than 2 times of upper limits of normal).This is contemplated to, because all patients accept lamivudine during this period.In two patients (205 and 601), it is undesired that Serum ALT levels continues, and the highest ALT level that writes down when accepting lamivudine in the research is respectively 112IU/L and 119IU/L.Two patients detect HBV DNA with the bDNA test when accepting lamivudine, patient 601 shows that the HBV dna level significantly raises when accepting lamivudine, and prompting may produce the lamivudine drug resistance mutant.

Table 10 and 11 has been summed up the data of Serum ALT levels.Table 10 shows among 24 patients that have no progeny in the lamivudine 22 the highest ALT level, and its data are investigated just up to date and obtained.In 10 patients, the highest ALT value is lower than 50IU/L.6 patients' the highest ALT value is between 50-100IU/L, and 5 patients' peak is between 101-200IU/L, and a patient (502) has peak ALT value＞200IU/L, and the peak level is 1870IU/L.In 12 patients of the highest ALT level greater than 50IU/L, ALT value the highest behind the interruption lamivudine is gone into the highest history value suitable (table 11) that writes down before this research with recruitment.Except a patient (502), all patients are interrupting having the highest ALT level behind the lamivudine, its than the research of record before peak low (22 patient in 10) or quite (22 patient in 1).

Patient 502 shows the early stage viral infection recurrence of having no progeny in the lamivudine, alleviates in the time of 10.5 months.Behind the interruption lamivudine therapy one month (8th month), the HBV dna level is increased to the 511mEq/ milliliter, and the ALT level is increased to 63IU/L, shows early stage viral infection recurrence has taken place.Interrupt latter two month of lamivudine therapy (9th month), patient 502 ALT horizontal peak is 1870IU/L, yet the level of HBV DNA drops to the 118mEq/ milliliter.After 9th month, patient 502 ALT level is the stable 46IU/L that drops in the time of 10.5 months.

Table 10

Interrupt the highest ALT level of the highest Alt level behind the lamivudine (lamivudine cure the disease number patient number treat interrupt between the later stage) (N=22)≤50IU/L 10 101,102,201,202,203,207,403,404,50

5,60251IU/L-100IU/L 6 204，206，401，501，503，601101IU/L-200IU/L 5 205，402，504，603，604＞200IU/L 1 502

The highest ALT water before the research of the highest ALT level of having no progeny in table 11 lamivudine therapy greater than the patient of 50IU/L

Have no progeny in the gentle Lamivudine have no progeny in the horizontal lamivudine therapy of highest serum ALT before the horizontal patient number research of the highest ALT follow-up period (N=12) (IU/L) between the highest ALT level of serum (IU/L) 204 342 54,205 141 144,206 271 52,401 328 77,402 400 125,501 112 57,502 810 1,870,503 62 53,504 924 167,601 339 69,603 299 130,604 285 155

Quantitatively HBV DNA estimates: the quantitative HBV dna level data that obtain the limited quantity time point.Three patients (402,504 and 601) HBV DNA when accepting lamivudine significantly increases (＞100 times).The possible cause of this increase is to have occurred that lamivudine is had chemical sproof HBV escape mutants.To carry out the HBV genetic typing of these samples.Obtained 16 in the 8th and JIUYUE measured the early stage information of the patient of HBV dna level about virus recurrence after the lamivudine therapy discontinued.Among these 16 patients, seven people (401,403,404,501,503,504,505) the 8th or the HBV dna level of 9 months (have no progeny in the lamivudine one or two months) below bDNA test detection level.One of them patient (504) may produce escape mutants (the HBV dna level is 577MEq/ml during July) simultaneously at lamivudine therapy, but he became HBV DNA feminine gender afterwards between follow-up period.All the other 9 patients that measured HBV DNA are interrupting having detectable HBV DNA behind the lamivudine therapy.

Table 12

Before the research and the highest HBeAg level and anti-HBe level after the last injection

Top level research back top level patient before the research

The anti-HBe of the anti-HBe of HBeAg (IU/ml) (IU/ml) HBeAg (IU/ml) (IU/ml) 101 16.4 1.6 30.2 1.8102 0.2 9.9＜0.1 46.4201＜0.1 41.5＜0.1 47.0202＜0.1 36.9＜0.1 24.2203＜0.1 5.2＜0.1 5.7204 1.0 4.5 1.9 3.2205＜0.1 122.6＜0.1 137.3206 1.9 5.7 18.4 2.7207 9.7 2.9 18.9 2.4208 3.3 4.6 17.8 2.5401＜0.1 44.1＜0.1 68.8402 0.2 4.1 0.4 4.2403＜0.1 419.7＜0.1 355.5404 1.2 5.0＜0.1 4.5501＜0.1 261.9＜0.1 338.2502 1.4 4.3 13.0 2.8503 0.2 62.1 1.1 17.1504 1.9 3.8 3.8 2372.0505 3.1 4.4＜0.1 30.7601 14.7 1.8 17.6 1.8602 2.9 4.3＜0.1 5.2603＜0.1 274.2＜0.1 162.0604＜0.1 165.1＜0.1 205.5

Quantitatively HBeAg and anti-HBe estimate: estimated the finite data about the change of HBeAg and anti-HBe level amount.HBeAg and anti-HBe titre have been measured with inner EIA.The detection lower limit of inner HBeAg test equates with commodity EIA (Abbott).Among the male patient of HBeAg (＞0.1), 4 patients (102,404,505 and 602) are lower than detectable limit at last injection back HBeAg when 14 screenings.Two (102 and 505) anti-HBe titres among these 4 patients also significantly increase (greater than 4 times).

The foregoing description is for the present invention is described, rather than limits the present invention by any way.Those skilled in the art will recognize that each modification is all in the spirit and scope of the invention as the accessory claim illustrated.

All list of references is incorporated herein for your guidance.

Claims (21)

1. a method for the treatment of chronic HBV infection is characterized in that, described method comprises the compositions that is applied to individual effective dose, and described compositions contains PreS2+S antigen and metabolizability oil adjuvant.

2. the method for claim 1 is characterized in that, uses PreS2+S antigen with every dose of about 5-100 microgram.

3. method as claimed in claim 2 is characterized in that, uses PreS2+S antigen with every dose of about 20 micrograms.

4. the method for claim 1 is characterized in that, uses more than the PreS2+S antigen 1 time.

5. method as claimed in claim 4 is characterized in that, uses PreS2+S antigen 8 times.

6. method as claimed in claim 4 is characterized in that, uses PreS2+S antigen 4 times.

7. method as claimed in claim 4 is characterized in that, uses PreS2+S antigen and infects improvement up to HBV.

8. method as claimed in claim 4 is characterized in that, uses PreS2+S antigen and is eliminated up to the HBV infection.

9. method as claimed in claim 7 is characterized in that, determines the improvement that HBV infects by the reduction of measuring serum component concentration, and described component is selected from HBV DNA, alanine aminotransferase (ALT), HBsAg and HBeAg.

10. method as claimed in claim 7 is characterized in that, determines the improvement that HBV infects by the increase of measuring serum component concentration, and described component is selected from anti-HBs and anti-HBe.

11. method as claimed in claim 9 is characterized in that, drops to the improvement that normal level determines that HBV infects by measuring Serum ALT concentration.

12. the method for claim 1 is characterized in that, the generation of recombinating in Chinese hamster ovary cell of described PreS2+S antigen.

13. the method for claim 1 is characterized in that, described metabolizability oil adjuvant is MF59.

14. the method for claim 1 is characterized in that, this method also comprises uses antiviral drugs or chemical compound.

15. method as claimed in claim 14 is characterized in that, described antiviral drugs is a lamivudine.

16. method as claimed in claim 15 is characterized in that, uses the about 100-300 milligram of lamivudine every day.

17. method as claimed in claim 16 is characterized in that, uses about 100 milligrams of lamivudine every day.

18. method as claimed in claim 14 is characterized in that, described antiviral drugs was used one month before using PreS2+S antigen at least.

19. treat the medicine box that chronic hepatitis B infects for one kind, it is characterized in that this medicine box comprises a kind of antigenic compositions of reorganization PreS2+S that contains, described medicine box also contains antiviral drugs or chemical compound and contains the compositions of metabolizability oil adjuvant.

20. medicine box as claimed in claim 20 is characterized in that, described metabolizability oil adjuvant is MF59.

21., it is characterized in that reorganization PreS2+S antigen produces as claim 19 or 20 each described medicine boxs in Chinese hamster ovary cell.

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