WO1992022654A1 - Vaccine and treatment method of human immunodeficiency virus infection - Google Patents
Vaccine and treatment method of human immunodeficiency virus infection Download PDFInfo
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- WO1992022654A1 WO1992022654A1 PCT/US1992/004980 US9204980W WO9222654A1 WO 1992022654 A1 WO1992022654 A1 WO 1992022654A1 US 9204980 W US9204980 W US 9204980W WO 9222654 A1 WO9222654 A1 WO 9222654A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/21—Retroviridae, e.g. equine infectious anemia virus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55505—Inorganic adjuvants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14041—Use of virus, viral particle or viral elements as a vector
- C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- HIV-1 The Human Immunodeficiency Virus Type-1 (HIV-1) is a retrovirus which causes a systemic infection with a major pathology in the immune system and is the etiological agent responsible for Acquired Immune Deficiency Syndrome (AIDS) . Barre-Sinoussi, et al., Science. 220: 868-871 (1983); Popovic et al., Science, 224: 497-500 (1984). Clinical isolates of HIV-1 have also been referred to as Lym- phadenopathy-Associated Virus (Feorino, et al., Science. 225: 69-72 (1984) and AIDS-related Virus (Levy et al., Science 225: 840-842 (1984)).
- Lym- phadenopathy-Associated Virus Feorino, et al., Science. 225: 69-72 (1984)
- AIDS-related Virus Levy et al., Science 225: 840-842 (1984
- AIDS has become pandemic and the development of a vaccine has become a major priority for world public health.
- a high percentage of persons infected with HIV-1 show a progressive loss of immune function due to the depletion of T4 lymphocytes.
- T4 cells as well as certain nerve cells, have a molecule on their surface called CD4.
- HIV-1 recognizes the CD4 molecule through a receptor located on the envelope of the virus particles, enters these cells, and eventually replicates and kills the cell.
- An effective AIDS vaccine might be expected to elicit antibodies which would bind to the envelope of HIV-1 and prevent it from infecting T4 lymphocytes or other susceptible cells.
- Vaccines are generally given to healthy individuals before they are exposed to a disease organism as an immune prophylactic. However, it is also reasonable to consider using an effective AIDS vaccine in post-exposure immunization as i munotherapy against the disease. Salk, J., Nature, 127: 473-476 (1987). It is widely believed that the HIV-1 envelope
- HIV-1 envelope protein is initially synthesized as a 160,000 molecular weight glycoprotein (gpl60) .
- the gpl60 precursor is then cleaved into a 120,000 molecular weight external glycoprotein (gpl20) and a 41,000 molecular weight transmembrane glycoprotein (gp41) .
- envelope proteins are the major target antigens for antibodies in AIDS patients. Barin, et al., Science. 228: 1094-1096 (1985) .
- the native HIV-1 gpl20 has been shown to be immunogenic and capable of inducing neutralizing antibodies in rodents, goats, rhesus monkeys and chimpanzees. Robey, et al., Proc. Natl. Acad. Sci. USA £1:7023-7027 (1986).
- HIV-1 envelope protein Due to the very low levels of native HIV-1 envelope protein in infected cells and the risks associated with preparing an AIDS vaccine from HIV-1 infected cells, recombinant DNA methods have been employed to produce HIV-1 envelope antigens for use as AIDS vaccines. Recombinant DNA technology appears to present the best option for the production of an AIDS subunit vaccine because of the ability to produce large quantities of safe and economical immunogens.
- the HIV-1 envelope protein has been expressed in genetically altered vaccinia virus recombinants. Chakrabarti, et al., Nature. 320: 535-537 (1986); Hu, et al., Nature. 320: 537-540 (1986); Kieny, et al. , Biotechnology. 4_:790-795 (1986).
- the envelope protein has also been expressed in bacterial cells (Putney, et al., Science. 234: 1392-1395 (1986)), in mammalian cells (Lasky, et al., Science. 21:209-12 (1986)), and in insect cells. Synthetic peptides derived from amino acid sequences in an HIV-1 gp41 have also been considered as candidate AIDS vaccines. Kennedy, et al. (1986). However, a -successful AIDS vaccine has not been produced using these materials and methods.
- the use of a baculovirus-insect cell vector system to produce recombinant HIV-1 envelope proteins is " one aspect of the invention disclosed in copending and coassigned U.S. patent application Serial No. 920,197 filed October 16, 1986 (now Serial No. 585,266) . See . also. Serial No. 151,976.
- the baculovirus system has been demonstrated to be of general utility in producing HIV-1 proteins and other proteins.
- the baculovirus Auto ⁇ rapha californica nuclear polyhedrosis virus (AcNPV) has been used as a vector for the expression of the full length gpl60 and various portions of the HIV-1 envelope gene in infected Spodoptera frucriperda (fall armyworm) cells (Sf9 cells) .
- the truncated gpl60 gene (recombinant number Ac3046)
- the protein produced from recombinant Ac3046 and a purification technique for the Ac3046 gene product that includes lentil lectin affinity chromatography and gel filtration chromatography.
- the gpl60 protein purified in this manner and aggregated to form particles was found to be highly immunogenic in rodent and primate species.
- the ideal AIDS vaccine in addition to the requirements of being substantially biologically pure and non-pyrogenic, should provide life-long protection against infection with HIV-1 after a single or a few injections. This is usually the case with live attenuated vaccines.
- live attenuated vaccines When killed bacteria or viruses, or materials isolated from them, such as toxoids or proteins, are used to make a vaccine, there often results a poor antibody response and only short term immunity.
- an additional component called an adjuvant, may be added.
- Adjuvants are materials which help stimulate the immune response.
- Adjuvants in common use in human vaccines are gels of aluminum salts (aluminum phosphate or aluminum hydroxide) , usually referred to as alum adjuvants.
- the present invention provides a vaccine and treatment methods for human immunodeficiency virus (HIV) , comprising the administration of recombinant HIV envelope protein to an infected or susceptible individual.
- HIV human immunodeficiency virus
- the envelope protein may be purified, aggregated, and combined with an adjuvant (e.g. , alum) for vaccine use.
- Fig. 1 illustrates the cloning strategy used to isolate the HIV-1 envelope gene (env) from the E. coli plasmid pNA2.
- the hatched regions are HIV-1 DNA sequences and the open regions are from the cloning vectors.
- the black region in the plasmid pl774 is constructed from synthetic oligonucleotides and was introduced as an Smal-- Kpnl fragment into the Smal-Kpnl sites of plasmid pl614. The sequence of this synthetic oligonucleotide is shown.
- Fig. 2 illustrates the strategy used to construct the recombinant plasmid vector (p3046) , which in turn is used to construct the baculovirus expression vector Ac3046.
- the plasmid p GS3 contains sequences (cross-hatched areas) from the baculovirus AcNPV on either side of a cloning site at position 4.00. This site has the unique restriction endonuclease sites for Smal, Kpnl, and Bgl l.
- the AcNPV polyhedrin promoter is in the 5' direction from the 4.00 position.
- the sequence 5' -TAATTAATTAA-3' is in the 3' direction, and has a translational termination codon in all three reading frames.
- the plasmid pl774 and the sequence of the synthetic oligonucleotide region is as described in Fig. 1.
- the plasmid p3046 contains all of p GS3 except for the sequences between the Smal and Bglll sites, where the HIV-1 envelope gene of pl774 is inserted.
- Fig. 3 shows the nucleotide sequences of the DNA flanking the Ac3046 gpl60 coding sequences.
- the 3046 env DNA sequence between +1 and +2264 is shown in Fig. 4.
- Figs. 4a-4k show the actual DNA sequence of the HIV-1 env gene segment along with the synthetic oligonucleo ⁇ tide sequences at the 5' end of the env gene in Ac3046 (between +1 and +2264) .
- the locations of restriction endo- nuclease sites are listed above the DNA sequence and the predicted amino acid sequence is listed below the DNA sequence.
- the bases are numbered on the right and on the left.
- Figs. 5a-5d compare the DNA sequences of the env gene from Ac3046 with a published env gene sequence from LAV-l.
- the LAV-1 sequence is on the top and Ac3046 is on the bottom.
- a line (1) below the LAV-1 sequence indicates that the sequence in Ac3046 is the same in this position.
- the DNA sequence numbering used is that described by Wain- Hobson, et al., Cell. 40:9-17 (1985) for LAV-1.
- Fig. 6 shows the ELISA end point dilution titers of human HIV-1 antibody positive sera (top graph) and rhesus monkey sera (bottom graph) from animals immunized with gpl60
- F g- 7 is a Table summarizing the gpl60 Vaccine- induced immune responses of vaccinated seropositive patients.
- Fig. 8 shows vaccine-induced antibody responses directed against specific HIV envelope epitopes.
- Fig. 9 shows the vaccine-induced T-cell proliferative responses to gpl60 in vaccinated seropositive individuals.
- Fig. 10 shows the lymphocyte proliferation responses associated with vaccination.
- Fig. 11 is a graph showing the percent change in CD4 cells in responders and non-responders over time.
- recombinant HIV-1 gpl60 envelope protein (“rgpl60”) , especially when adsorbed onto an adjuvant such as alum (e.g. , aluminum phosphate) is particularly useful as an AIDS vaccine.
- alum e.g. , aluminum phosphate
- One aspect of this invention is an AcNPV expression vector having the coding sequence for a portion of the HIV-1 envelope gene which encompasses the amino acids 1-757 found in the recombinant clone No. 3046.
- Another aspect of the invention is the production of that recombinant HIV-1 envelope protein (and the protein itself) in insect cells -- especially the rgpl60 protein coded for by the amino acid sequences 1-757 (i.e., 03046) .
- aspects of this invention comprise purification and formation of recombinant envelope protein particles from the gene product of the recombinant baculovirus that produces the 3046 protein and adsorption of the 3046 particles to aggregates of aluminum phosphate.
- the invention also comprises prophylactic and/or therapeutic vaccines for AIDS or HIV infection and methods of preventing or treating AIDS or HIV infection.
- the recombinant baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) which contains a truncated HIV-1 gpl60 gene coding for amino acids 1-757 of the HIV envelope protein (recombinant Ac3046) is described in copending, coassigned U.S. application Serial No. 920,197 (now Serial No. 585,260).
- the cloning steps employed to construct the recombinant baculovirus-containing genes or portions of genes from HIV-1 are also disclosed there and are incorporated by reference.
- recombinant envelope proteins referred to collectively as rgpl60
- rgpl60 recombinant envelope proteins
- gpl20 and gp41 proteins recombinant gpl20 and gp41 proteins.
- Ac3046 is just one example of an expression vector and recombinant envelope protein according to the invention.
- Cloning and expression of foreign protein coding sequences in a baculovirus vector requires that the coding sequence be aligned with the polyhedrin promoter and upstream sequences on one side and with baculovirus coding sequences on the other side.
- the alignment is such that homologous recombination with the baculovirus genome results in transfer of the foreign coding sequence aligned with the polyhedrin promoter and an inactive polyhedrin gene.
- insertion vectors were designed for use in HIV envelope gene constructions.
- the insertion vector MGS3 was constructed from an
- MGS3 EcoRI-I restriction fragment clone of DNA isolated from a plaque purified AcMNPV isolate (WT-1) .
- MGS3 was designed to consist of the following structural features: (a) 4000 bp of sequence upstream from the ATG initiating codon of the polyhedrin gene; (b) a polylinker introduced by site- directed mutagenesis, which consists of an ATG initiating codon at a position of the corresponding polyhedrin codon, and restriction sites Smal, Kpnl, Bglll and a universal stop codon segment; (c) 1700 bp of sequence extending from the
- a recombinant plasmid designated NA2 (Fig. 1) consists of a 21.8 kb segment of an entire HIV-1 provirus inserted into pUC18. This clone was reportedly infectious since it could produce virus following transfection of certain human cells. Adachi, et al., J. Virol. 59:284-291
- NA2 were derived from the LAV strain of HIV. Barre-Sinoussi
- the HIV-1 envelope gene was isolated and engineered as described below, and as shown in Fig. 1.
- the envelope gene was initially isolated from NA2 as a 3846 bp
- the resultant plasmid was designated p708.
- the envelope gene was subsequently reisolated as a 2800 bp Kpnl restriction fragment and cloned into the Kpnl restriction site of pUC18. The resulting clone was designated pl614.
- the Kpnl restriction fragment in pl614 contained a slightly truncated piece of the HIV envelope gene such that 121 bp of the N-terminal corresponding sequence was missing. This missing part in the gene, which included the signal peptide sequences, was replaced by insertion of a double-stranded synthetic oligomer.
- the inserted oligomer was designed from the LAV amino acid sequence using preferred polyhedrin gene codon usage. To facilitate further manipulation, a new Smal restriction sequence was concomitantly introduced in place of the ATG initiating codon. The ATG initiation codon will be supplied by the baculovirus insertion vector.
- the resultant plasmid was designated pl774.
- restriction fragments from pl774 containing coding sequences of various domains of the HIV-1 envelope were cloned into the MGS insertion vectors (e.g.. MGS3) such that the ATG initiating codon of the insertion vector was in-frame with the codons of the envelope gene.
- Construct p3046 consisted of the Smal/BamHI restriction fragment isolated from pl774 inserted into the Smal/Bglll site of the plasmid vector pMGS3. This clone contains sequences coding for amino acids 1 through 757 of gpl60 and uses a termination codon supplied by the MGS3 vector.
- the HIV env gene recombination plasmid p3046 was calcium phosphate precipitated with AcMNPV DNA (WT-1) and added to uninfected Spodoptera frugiperda cells. The chimeric gene was then inserted into the AcMNPV genome by homologous recombination. Recombinant viruses were iden ⁇ tified by an occlusion negative plaque morphology. Such plaques exhibit an identifiable cytopathic effect but no nuclear occlusions. Two additional successive plaque purifications were carried out to obtain pure recombinant virus. Recombinant viral DNA was analyzed for site-specific insertion of the HIV env sequences by comparing their restrictions and hybridization characteristics to wild-type viral DNA.
- Expression of HIV env sequences from the recombinant viruses in insect cells should result in the synthesis of primary translational product.
- This primary product will consist of amino acids translated from the codons supplied by the recombination vector.
- the result is a protein containing all the amino acids coded for from the ATG initiating codon of the expression vector downstream from the polyhedrin promoter to the translational termination signal on the expression vector (e.g.. rgpl60) .
- the primary translation product of Ac3046 should read Met- Pro-Gly-Arg-Val at the terminus where Arg (position 4) is the Arg at position 2 in the original LAV clone.
- the Met- Pro-Gly codons are supplied as a result of the cloning strategy.
- the nucleotide sequence of the gpl60 insert and flanking DNA was determined from restriction fragments isolated from viral expression vector Ac3046 DNA.
- the sequencing strategy involved the following steps.
- the 3.9 kb EcoRV-BamHI fragment was purified by restriction digestion of Ac3046 viral DNA.
- the Ac3046 viral DNA had been prepared from extracellular virus present in the media of cells being used for a production lot of vaccine.
- the 3.9 kb EcoRV-BamHI fragment consists of the entire gpl60 gene and 100 bp of upstream and about 1000 bp of downstream flanking DNA.
- the nucleotide sequence of the entire gpl60 gene was determined, including 100 bp of upstream and 100 bp of downstream flanking DNA.
- the sequence of the gpl60 was essentially as reported by
- One aspect of the present invention is the procedure used to extract and purify the recombinant HIV-1 envelope protein coded for in the Ac3046 expression vector.
- the recombinant HIV-1 envelope protein gpl60 is produced in S.. frugiperda cells during 4-5 days after infection with Ac3046. Purification of this rgpl60 protein involves the steps: 1. Washing the Cells
- Dialysis This example describes the purification of the recombinant gpl60 from about 2 x 10 9 Ac3046 infected cells.
- the first peak comes off between about 500 and 700 ml, the second between 700 and 1400 ml and the third between 1400 and 1900 ml buffer. This same profile is observed on small analytical columns from which it has been determined that the first peak is material that has a molecular weight of > 2,000,000.
- This peak is translucent due to a concentration of high molecular weight lipids and lipid complexes. This peak also contains from 10% to 20% of the gpl60.extracted from the infected cells. Apparently this fraction of gpl60 is complexed to itself or • other cell components to form high molecular weight aggregates.
- the second broad peak contains the majority of the gpl60 and proteins with molecular weights of between about 18,000 and 200,000.
- the third peak contains little protein and the majority of the UV absorption is due to the beta mercapto- ethanol in the sample.
- the effluent from the column is applied directly onto the lentil lectin column. Once the second peak has come off the column, the effluent is disconnected from the lentil lectin column and directed to waste.
- the lentil lectin affinity gel media (Lentil Lectin-Sepharose 4B) was purchased in bulk from Pharmacia. The lentil lectin was isolated by affinity chromatography on Sephadex to greater than 98% purity and then was immobilized by coupling to Sepharose 4B using cyanogen bromide. The matrix contains about 2 mg ligand per ml of gel.
- the lentil lectin column is a 5.0 x 30 cm glass column (Pharmacia) containing 125 ml lentil lectin-Sepharose 4B gel.
- the affinity matrix is reused after being thoroughly washed and regenerated by a procedure recommended by the supplier.
- the gel When not in use, the gel is stored in the column in a solution of 0.9% NaCl, 1 mM MnCl 2 , 1 mM CaCl 2 , and 0.01% thi erosal.
- the column is washed and equilibrated with 250 ml lentil lectin buffer described above before each use.
- the crude gpl60 is applied to the column directly as it is eluting from the gel filtration column as described above. Once the crude gpl60 is bound to the column, it is washed with 800 ml lentil lectin buffer containing 0.1% deoxycholate. Under these conditions all of the gpl60 binds to the column. Lentil lectin buffer plus 0.3M alpha-methyl mannoside is used to elute the bound glycoproteins which is monitored through a UV monitor at a wavelength of 280 nm.
- gpl60 The purification of gpl60 from 1 liter of infected cells can be summarized in the following table (Table 3) .
- conventional ion exchange chromatography anionic or cationic
- gel filtration or ion exchange chromatography may follow the lentil lectin purification step.
- Other reagents may also be used according to the invention.
- other detergents may be used to purify the recombinant protein in place of deoxycholate. These include nonionic detergents such as Tween 20 (polysorbate 20), Tween 80, Lubrol, and Triton X-100.
- EXAMPLE 8 A. Assembly of gp!60 Particles.
- the gpl60 antigen can be assembled into particles of > . 2,000,000 molecular weight during purifi ⁇ cation.
- the gpl60 protein is extracted from the cell as a mixture of 80-90% monomeric (160,000 molecular weight) and 10-20% polymeric (particle form) .
- the gel filtration step removes the aggregated forms of gpl60. Attempts to purify the gpl60 from this fraction (first peak off the gel filtration column) suggest that it is complexed with other cell proteins, possibly even with membrane fragments.
- the gpl60 antigen in the second peak off the gel filtration column has a molecular weight of about 160,000- 300,000 and is, therefore, in predominantly monomeric or dimeric form.
- the formation of aggregates or polymers of gpl60 occurs during the development of the lentil lectin column. It has been determined that the antigen forms aggregates whether it is eluted from the lectin column in 0.5% deoxycholate, which is about the 0.2% critical micelle con-
- Total protein was estimated from absorbance at 280nm. centration (CMC) for deoxycholate, or whether the gpl60 is eluted from the column in 0.1% deoxycholate.
- the size of the aggregates are measured on a high resolution FPLC Superose 12 column (Pharmacia) . Samples from representative lots of purified gpl60 have a size that is predominantly equal to or greater than the 2,000,000 molecular weight of a blue dextran size standard.
- gpl60 produced in insect cells is a tetramer of identical submits.
- the study also shows that gpl60 in HIV-infected cells and virus particles is tetrameric.
- the recombinant gpl60 particles may have tertiary and quaternary structures that are similar to those found in the native HIV gpl60. Proper 3-dimensional structure could be important for the formation of epitopes that require correct folding of gpl60.
- purified gpl60 was analyzed by gel filtration. About 100 micrograms of gpl60 was applied to a Superose 12, FPLC gel filtration HR 10/30 column (Pharmacia, Inc.) . This column was first calibrated with protein molecular weight standards. The protein profile from this column is highly reproducible; the elution volume is inversely proportional to the molecular weight of the protein standards. The column separates the monomeric gpl60 from the polymeric forms and excludes globular proteins of > 2 x 10 6 molecular weight. When developed on this column, essentially all of the purified gpl60 elutes in the void volume and is, there ⁇ fore, > 2 x 10 6 (2,000,000) molecular weight in size.
- the effectiveness of insoluble aluminum compounds as immunologic adjuvants depends on the completeness of adsorption of the antigens on the solid phase.
- alum compositions could be made that would efficiently adsorb the gpl60 but at a pH that would not reduce the potency of the gpl60-alum complex as an immunogen.
- the factors controlled during the formation of this alum (aluminum phosphate gel) composition are:
- the optimal pH for adsorption of antigens to alum is about 5.0.
- the gpl60 lost immunogenicity at a pH of 6.5 in comparison to a pH of 7.5 so the alum is made at a pH of 7.1 ⁇ 0.1. It was discovered that essentially 100% of the gpl60 will still adsorb to the alum at this pH.
- the ionic strength from the NaCl present is rela ⁇ tively low and is less than 0.15 M.
- the gpl60 antigen is added to freshly formed alum to stop crystal growth and minimize the size of the particles.
- the procedure to make 200 ml alum and adsorb puri ⁇ fied gpl60 to the alum is such that the final concentration of antigen is 40 ⁇ g/ml, as outlined below.
- Solution 1 aluminum chloride-sodium acetate to the formulation vessel using 25 ml sterile, disposable pipets. Note the volume of Solution 1 and begin stirring the solution.
- Solution 2 sodium phosphate
- Immunogenicity of Alum Absorbed gpl60 (Specific Ab Response) An accepted method to determine the immunogenicity of an antigen preparation (vaccine) is to measure the specific antibody response in groups of mice which have been given a single dose of antigen. At the end of 4 weeks the mice are bled and the serum antibody levels to a specified antigen (usually the antigen used to immunize the animal) are measured by a standard antibody test, e.g. an ELISA (enzyme linked immunosorbent assay) .
- the immunogenicity in mice of purified gpl60 with no adjuvant at pH 6.0 and pH 7.5 adsorbed with alum (as described in Example 9) or mixed with Freund's Complete Adjuvant are summarized below (Table 4) .
- mice immunized with a single 1.0 microgram dose of gpl60 antigen without any added adjuvant will elicit an antibody response against gpl60 (see table above) .
- a much stronger antibody response is seen in groups of mice immunized with 1.0 microgram of gpl60 adsorbed to the alum adjuvant.
- a single dose of less than 0.1 microgram of gpl60 mixed with complete Freund's or formulated with alum will seroconvert > 50% of the immunized mice.
- the gpl60 antigen was immunogenic in mice as an unformulated antigen at pH 7.5 and at pH 6.0, but there was a loss of immunogenicity at the lower pH.
- mice were bled 28 days post immunization and the sera tested at 1:10 dilution in an ELISA assay against gel- purified gpl60. Similar results were obtained using a commercial ELISA (Genetic Systems Inc.; EIA 611 ELISA) assay against the native HIV-1 proteins at a serum dilution of 1:400.
- mice (groups of 10) were injected with a single dose (0.5 micrograms, 1.0 micrograms, or 5.0 micrograms) of gpl60 alone, gpl60 adsorbed to alum or gpl60 in complete Freund's adjuvant (CFA) .
- CFA complete Freund's adjuvant
- mice Results from the sera drawn on day 28 are summarized in the table below (Table 5) .
- Table 5 Results from the sera drawn on day 28 are summarized in the table below (Table 5) .
- the number of sero-conversions and the average serum absorbance were higher with gpl60 adsorbed to alum than those obtained in mice immunized with gpl60 alone.
- HIV-1 neutralization assays are an accepted method to determine whether an antibody preparation will inhibit the HIV-1 virus from infecting susceptible human cultured lymphocyte cells. Antisera from animals immunized with gpl60 were tested in an HIV-1 neutralization assay and the results are summarized in the table below (Table 6) .
- Guinea pigs, rabbits and rhesus monkeys have also been immunized with gpl60 (using alum or Freund's as an adjuvant) . In general, the immunization of these animals has produced a good antibody response against the HIV-i envelope proteins.
- the chimpanzee is man's closest rela ⁇ tive and is currently the only animal model for infection of HIV-1.
- two chimpanzees were immunized with 40 micrograms or 80 micrograms of gpl60 in an alum formulated vaccine. Each received a booster immunization at 4 weeks with 40 micrograms and 80 micrograms of gpl60, respectively.
- a control animal was vaccinated at the same time with a l ml saline solution.
- Weekly serum samples were analyzed from each of the three chimpanzees for antibodies to gpl60 and to HIV-1 viral antigens using three immunological assays, an ELISA assay against purified gpl60 developed by
- Serum samples taken before immunization and for the 11 weeks following the primary immunization were diluted from 1:10 to 1:100,000 and then incubated with nitrocellulose strips containing a 100 ⁇ g purified gpl60 in a spot.
- the end point dilution titer is the highest dilution in which the test was positive for anti-gpl60 antibody as detected with a goat anti-human IgG-alkaline phosphatase conjugate.
- the serum samples from the control animal and from the pre-immune sera of the immunized animal were negative.
- the chimp which received the 80 microgram dose was positive at a 1:100 dilution by week 2 and the chimp which received a 40 microgram dose was positive at a 1:10 dilution by week 4.
- the antibody titers to gpl60 continued to increase until week 5, at which time the end point dilution titers were approximately 1:100,000 and 1:2,000,000 respectively.
- VaxSyn is a trademark of MicroGeneSys, Inc. for the AIDS vaccine described herein. chimpanzees, that they had seroconverted and have antibodies against the recombinant gpl60. To determine if they were also making anti-HIV antibody which recognized the native viral envelope proteins, the pre-immune sera and sera from weeks 1 through 11 were tested in a licensed, commercial ELISA test kit, the LAV EIATM test kit of Genetic System Corporation, Seattle, Washington. The animal immunized with 80 micrograms of gpl ⁇ O was positive at a 1:100 dilution by week 2 and continued to show an increase in antibody level through week 6. The animal immunized with 40 micrograms was positive at a 1:100 dilution by week 6.
- Fig. 6 summarizes the immunoreactivity of three different recombinant antigens: [ART] [TAB] (1) gpl20-delta (truncated recombinant HIV-1 gpl20 with about 40 amino acids missing from the C-terminus of the molecule) ; [ART] [TAB] (2) gpl20 (full length recombinant HIV-1 gpl20; and [ART] [TAB] (3) gpl60.
- Human sera from 50 HIV-1 antibody positive in ⁇ dividuals and 3 pooled human sera were highly reactive with gpl60, moderately reactive with gpl20 and little or no antibody reacted with truncated gpl20.
- gpl20 which represents more than 90% of the HIV-1 external glycoprotein, contains protective determi ⁇ nants.
- human AIDS positive sera have few antibodies to this region of the envelope is consistent with the fact that the immune response to viral infection is not fully protective and that human positive sera usually exhibit a low-level of neutralizing activity in vitro.
- rhesus monkeys immunized with either the gpl60 immunogen or with the truncated gpl20 have antibodies that react strongly with the truncated gpl20 portion of the HIV-1 envelope. This difference in distribu ⁇ tion of antibody recognition sites along the viral envelope and the higher titers observed in the monkeys may account for the fact that the monkey sera had high neutralizing titers.
- a clinical trial with 30 HIV-seropositive human patients was conducted to determine the effects of vaccina ⁇ tion with cloned HIV gpl60 (produced in the baculovirus system as described above) on HIV infected individuals.
- Vaccination with the recombinant gpl60 led to an augmentation in the gpl60 HIV-specific humoral and cellular immune responses of 19 out of 30 (63%) HIV seropositive volunteers. Fourteen out of 15 (93%) volunteers receiving 6 doses of the vaccine demonstrated an increase in their total gpl60 antibody.
- recombinant HIV proteins i.e., rgp41, rgpl20, rgpl60 and admixtures thereof
- the effective amounts of HIV protein used in this embodiment of the invention can be determined according to techniques well known in the art, such as those presented below. In general such effective amounts may range between about 1 microgram and about 100 micrograms per kilogram body weight of the patient.
- the frequency of administration can also be determined by known means. In a preferred embodi ⁇ ment, administration is via the parenteral route, i.e., intravenously, intraperitoneally, intramuscularly, intradermally, etc., as is well known by those of ordinary skill in the art.
- Additional entry criteria limited volunteers to adults between the ages of 18 and 50, with a normal complete blood count, no evidence of end organ disease, no alcohol or drug abuse over the preceding 12 months, and who were not receiving anti-retroviral or immunomodulatory drugs. All patients underwent a 2 month baseline evaluation prior to randomization into treatment groups. No volunteers received any antiretroviral or immunomodulatory drugs during the trial.
- the test vaccine comprises a non-infectious subunit glycoprotein derived from gpl60 as a baculovirus expressed recombinant protein.
- the immunogenic protein was produced in Lepidopteran insect cells, was biochemically purified, and was adsorbed to aluminum phosphate for final vaccine formulation.
- Three dose formulations of gpl60 were used: 40 micrograms per milliliter, 160 micrograms per milliliter and 320 micrograms per milliliter.
- the injection volume for both the 40 ⁇ g and 160 ⁇ g dosages was 1 ml; 2 ml of 320 ⁇ g per milliliter was used to deliver the 640 ⁇ g dose injec ⁇ tions.
- the thirty volunteers were distributed into six groups of five volunteers each. Two immunization schedules were investigated: Schedule A, with vaccination on days 0, 30, and 120; and Schedule B, with vaccination on days 0, 30, 60, 120, 150 and 180. Within each immunization Schedule (A or B) there were three groups which received different dosages of vaccine (Table 7 below) . All vaccinations were administered by intramuscular injection into the deltoid muscle. The duration of the trial was 10 months: a 2 month baseline evaluation, and an 8 month follow-up evaluation after the initial vaccination.
- T-cell phenotyping total lymphocyte, CD4 and CD8 cell phenotypes as described in Rickman, et al., Clinical Immuno. 52: 85-95, 1989; Birx, et al. , iL. Acquir. Immune Defic. Syndr. 4: 188-196, 1991
- T-cell proliferative response to mitogens pokeweed and Con A
- control antigens Candida albicans and tetanus
- In vivo cellular immune function was assessed by delayed hypersensitivity skin testing to control antigens (i.e.. mumps, tetanus toxoid, Candida albicans and trichophyton) .
- PBMC peripheral blood mononuclear cells
- Vaccine induced HIV immunogenicity was not associated with evidence "of accelerated CD4 decline in any individual subject throughout the entire course of the trial.
- in vivo viral activity was measured by quanti ⁇ tative plasma and PBMC viral cultures, PBMC DNA polymerase chain reaction, and serum levels of p24 antigen. Quantita- tive cultures and DNA polymerase chain reaction assays demonstrated no alteration during this trial. Serum p24 antigen was undetectable in the subjects.
- epitopes 88 amino acids 88-98 in gpl20
- 448C amino acids 448-514 in gpl20
- Epitopes 106 (amino acids 106-121 in gpl20) , 241 (amino acids 241-272) , 254 (amino acids 254-272) , 300 (amino acids 300-340) , 308 (amino acids 308-322) , 422 (amino acids 422-454) and 735 (amino acids 735-752) were selected because of their putative functional importance.
- Epitopes 106 and 422 have been implicated in CD4 binding; epitopes 241, 254 and 735 have been implicated in group specific neutraliza- tion; and epitopes 300 and 308 have been implicated in type- specific neutralization) .
- Epitope 582 (amino acids 582-602) was selected as a control because it represents the immunodominant envelope domain in natural HIV infection. Additional epitopes investigated included 49 (amino acids 49-128); and 342 (amino acids 342-405) . '
- a shaded box signifies a documented change in the HIV envelope-directed immune response.
- Shaded boxes with (.) signify new T- cell proliferative response to gpl60 following immunization.
- a (.) alone signifies no cellular response to gpl60; while b signifies "high background” (not interpretable) ; and nd signifies "not done.”
- Vaccine induced humoral immunity was defined as seroconversion to HIV envelope specific epitopes and/or a secondary booster immune response to envelope specific epitopes.
- Vaccine induced cellular immunity was defined as the development of a new, reproducible, vaccine associated, proliferative response to gpl60. 9 Subjects who developed neither a humoral nor a cellular proliferative response or who developed only a humoral or only a cellular prolifera ⁇ tive response to gpl60 epitopes or HIV envelope were classified as non-responders.
- each gpl60 specific epitope was as follows: Epitope 49 (27 to 70 percent), Epitope 88 (28 to 52 percent) , Epitope 106 (50 to 87 percent) , Epitope 214 (0 to 14 percent) , Epitope 254 (0 to
- Serum neutralization activity against three distinct strains was determined on days 0, 90 and 195 in 7 subjects.
- Four of 5 vaccine responders demonstrated increasing neutralizing activity to one or more isolate.
- the vaccine responders also demon- strated an increased ability to inhibit syncytium formation compared to non-responders.
- Figure 9 illustrates proliferative responses to gpl60, p24 and a baculovirus control protein in four typical vaccine responders over time.
- the gpl60 induced proliferation increased from a baseline mean LSI of 3 to an LSI of 10 (calculated utilizing the mean of 4 values following the last immunization) .
- no change was noted for proliferative responses directed against HIV p24 protein or the control baculovirus protein.
- Vaccine induced changes in mean LSI values for all subjects, for subjects subgrouped by vaccine responsiveness, and for subjects grouped by immunization schedule are illus ⁇ trated in Figure 10.
- the change in proliferative response to gpl60 was significantly different between vaccine responders and non- responders ( ⁇ 0.001, Wilcoxon, one tailed).
- the gpl60 proliferation responses induced by Schedule B (6 doses) were greater than those induced by Schedule A (3 doses) (P ⁇ 0.10, Wilcoxon, one tailed) .
- the therapeutic use of vaccines was introduced by Pasteur in the 19th century for the treatment of acute rabies infection. But the utility of this approach in the treatment of other infections has not been extensively explored.
- post infection modification of viral-specific immunity such as after hepatitis A or B exposure
- the invention provides virus-specific immune modification by active immunization after infection.
- an HIV envelope gene derived gpl60 vaccine augmented the human host directed viral-specific humoral and cellular responses in 19 of 30 early HIV infected persons.
- a proliferative' response to HIV envelope proteins rarely occurs in natural HIV infection. However, after immunization with gpl60, specific T-cell proliferative respon-ses were documented in 21 (70 percent) of the subjects. The reason for this difference is unclear.
- the new proliferative response may be directed against an envelope epitope(s) unique to the vaccine (as a result of vaccine production methodology or alternative in vivo antigen processing) .
- the protein used in the proliferation assay may not stimulate primary T-cell proliferative responses against homologous "wild type" envelopes of natural virus.
- additional evidence that vaccination boosts the host cellular immune response has been obtained: selected vaccine responders demonstrated HIV-IIIB type-specific cytotoxic T-cell responses following booster immunization.
- the vaccination results in this study were also compared with a database of ten infected and untreated individuals matched for age, ethnic group, and baseline CD4 cell count.
- the mean CD4 count decreased by 8.7 percent in this reference group, decreased by 7.2 percent in the subjects assigned to Schedule A, and increased by 0.6 percent in subjects assigned to Schedule B.
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SK18993A SK18993A3 (en) | 1991-06-11 | 1992-06-10 | Vaccine and treatment method of hiv infection |
PL29784992A PL297849A1 (en) | 1991-06-11 | 1992-06-10 | Vaccine against and method of treating the patients infectedwith hiv virus |
BR9205290A BR9205290A (en) | 1991-06-11 | 1992-06-10 | VACINE AND METHOD FOR TREATING HUMAN IMMUNODEFICIENCY VIRUS INFECTION |
NO93930453A NO930453L (en) | 1991-06-11 | 1993-02-09 | HUMAN IMMUNITY DEVICES VACCINE AND TREATMENT METHOD |
FI930577A FI930577A (en) | 1991-06-11 | 1993-02-10 | VACCIN OCH BEHANDLINGSMETOD FOER HIV-INFEKTION |
BG97519A BG97519A (en) | 1991-06-11 | 1993-03-10 | Method for the preparation of a hiv vaccine composition |
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US5464933A (en) * | 1993-06-07 | 1995-11-07 | Duke University | Synthetic peptide inhibitors of HIV transmission |
WO1996040191A1 (en) | 1995-06-07 | 1996-12-19 | Trimeris, Inc. | The treatment of hiv and other viral infections using combinatory therapy |
US5759809A (en) * | 1993-12-23 | 1998-06-02 | University Technologies International, Inc. | Methods of expressing proteins in insect cells and methods of killing insects |
US6017536A (en) * | 1993-06-07 | 2000-01-25 | Trimeris, Inc. | Simian immunodeficiency virus peptides with antifusogenic and antiviral activities |
US6060065A (en) * | 1993-06-07 | 2000-05-09 | Trimeris, Inc. | Compositions for inhibition of membrane fusion-associated events, including influenza virus transmission |
US6077662A (en) * | 1996-11-27 | 2000-06-20 | Emory University | Virus-like particles, methods and immunogenic compositions |
US6214540B1 (en) | 1997-03-26 | 2001-04-10 | University Of Maryland Biotechnology Institute | Chemokines that inhibit immunodeficiency virus infection and methods based thereon |
US6319504B1 (en) | 1996-06-24 | 2001-11-20 | University Of Maryland Biotechnology Institute | Treatment and prevention of HIV infection by administration of derivatives of human chorionic gonadotropin |
US6395541B1 (en) | 1996-05-23 | 2002-05-28 | The Rockefeller University | Methods for the identification of compounds capable of inhibiting HIV-1 viral replication employing murine cell lines expressing human topoisomerase I |
US6548631B1 (en) | 1997-09-16 | 2003-04-15 | BIOMéRIEUX, INC. | Macrophage derived chemokine (MDC) as an anti-viral agent for the treatment and prevention of lentivirus infection |
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US6750008B1 (en) | 1999-07-09 | 2004-06-15 | Trimeris, Inc. | Methods and compositions for inhibition of membrane fusion-associated events, including HIV transmission |
USRE40786E1 (en) * | 1984-03-16 | 2009-06-23 | The United States Of America As Represented By The Secretary Of The Army | Vaccines against intracellular pathogens using antigens encapsulated within biodegradable-biocompatible microspheres |
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CN102961760B (en) * | 2011-09-01 | 2017-07-18 | 常州文松生物技术有限公司 | Overlapping peptide is used as reagent and its application for preparing Skin-test and detection specific cellular immunity |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0327180A2 (en) * | 1988-02-03 | 1989-08-09 | MicroGeneSys, Inc. | Vaccine containing polypeptides derived from the envelope gene of human immunodeficiency virus type 1 |
-
1992
- 1992-06-03 IL IL10209292A patent/IL102092A/en not_active IP Right Cessation
- 1992-06-08 ZA ZA924150A patent/ZA924150B/en unknown
- 1992-06-10 MX MX9202781A patent/MX9202781A/en unknown
- 1992-06-10 SK SK18993A patent/SK18993A3/en unknown
- 1992-06-10 HU HU9300686A patent/HUT68355A/en unknown
- 1992-06-10 AU AU21931/92A patent/AU2193192A/en not_active Abandoned
- 1992-06-10 JP JP5501040A patent/JPH06501851A/en active Pending
- 1992-06-10 PL PL29784992A patent/PL297849A1/en unknown
- 1992-06-10 CA CA002087732A patent/CA2087732A1/en not_active Abandoned
- 1992-06-10 EP EP92913889A patent/EP0542998A1/en not_active Withdrawn
- 1992-06-10 WO PCT/US1992/004980 patent/WO1992022654A1/en not_active Application Discontinuation
- 1992-06-11 PT PT100584A patent/PT100584A/en not_active Application Discontinuation
- 1992-06-11 CN CN92104517A patent/CN1068266A/en active Pending
- 1992-07-01 IE IE187592A patent/IE921875A1/en not_active Application Discontinuation
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1993
- 1993-02-10 FI FI930577A patent/FI930577A/en not_active Application Discontinuation
- 1993-02-11 LT LTIP335A patent/LT3365B/en not_active IP Right Cessation
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1994
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Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0327180A2 (en) * | 1988-02-03 | 1989-08-09 | MicroGeneSys, Inc. | Vaccine containing polypeptides derived from the envelope gene of human immunodeficiency virus type 1 |
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BG97519A (en) | 1994-03-24 |
MX9202781A (en) | 1993-04-01 |
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PL297849A1 (en) | 1994-01-24 |
IL102092A (en) | 1996-11-14 |
CN1068266A (en) | 1993-01-27 |
FI930577A (en) | 1993-03-24 |
AU2193192A (en) | 1993-01-12 |
FI930577A0 (en) | 1993-02-10 |
LT3365B (en) | 1995-07-25 |
HUT68355A (en) | 1995-06-28 |
PT100584A (en) | 1993-07-30 |
SK18993A3 (en) | 1993-10-06 |
HU9300686D0 (en) | 1993-06-28 |
JPH06501851A (en) | 1994-03-03 |
EE9400183A (en) | 1995-12-15 |
ZA924150B (en) | 1993-02-24 |
LTIP335A (en) | 1995-01-31 |
CA2087732A1 (en) | 1992-12-12 |
AU4028895A (en) | 1996-02-22 |
IL102092A0 (en) | 1993-01-14 |
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