SK18993A3 - Vaccine and treatment method of hiv infection - Google Patents

Abstract

An Acquired Immunodeficiency Syndrome (AIDS) vaccine containing the Human Immunodeficiency Virus, Type-1 (HIV-1) envelope proteins is produced from cloned HIV-1 envelope genes in a baculovirus-insect cell vector system. The recombinant HIV-1 proteins are purified, assembled into particles and then adsorbed on an aluminum phosphate adjuvant. The resulting adsorbed recombinant HIV-1 virus envelope protein formulation (AIDS vaccine) is highly immunogenic in animals and elicits antibodies which bind to the HIV-1 virus envelope and neutralize the infectivity of the virus in in vitro tests. The above AIDS vaccine induces new humoral and cellular immune responses in HIV-infected patients and is useful as a form of vaccine therapy to delay or prevent the destruction of the immune system.

Description

TechnikyTechnology

The invention relates to a vaccine and a method of treatment for human immunodeficiency virus.

j. BACKGROUND OF THE INVENTION j. The human immunodeficiency virus type 1 / HIV 1 virus is retrovirus, which causes systemic infection with a major pathology e in the immune system and is the etiological agent responsible for

j! Acquired Immunodeficiency Syndrome (AIDS) - Earre-Sinoussi et al.

and ^Λ Science, 220 = 868-871 (1983), and F'opovic. et al., Science, 224 =

497-500 (1984). Clinically isolated HIV 1 may also be referred to as Lymphadenopathy-Associated Virus (Feorino et al., Science,

225 ^: 69-72 (1984) and the AIDS-related Cirue ”(Levy et al., Science i 225: 840-842 (1984).

cl.

ς AIDS has become a pandemic and vaccine development has become a top priority for human health worldwide. High percentage of persons

HIV-1 infected shows a progressive decrease in immune function due to T4 depletion. These T4 cells, as well as certain nerve cells, have molecules called CD4 on their surface. HIV-1 recognizes the CD4 molecule by means of receptors located on the vortex particle envelope, enters these cells and eventually replicates and kills the cell. An effective AIDS vaccine is expected to induce the production of antibodies that will bind to the HIV-1 envelope and prevent infection of T4 lymphocytes or other susceptible cells.

• .'í. · <

 • Vaccines are usually administered to healthy individuals prior to exposure to disease organisms such as immunoprophylaxis. It is

AIDS vaccines against disease.

it is also wise to consider uses effective in post-exposure immunization as immunotherapy

Salk, J., Nature

127: 473-476 (1987).

li.

r

It is generally believed that the IIIV-1 envelope (env) is the most promising candidate in the development of the AIDS vaccine. Francis and Petricciani, New Eng. J. Med., 1586-1559 (1985); Vogt and Hirsh, Reviews of Infectious Disease, 8 = 991-1000 (1986); Fauci, Proc. Natl. Acad. Sci. USA, 83 ^: 9278-9283. The HIV-1 coat protein is first synthesized as a 16,000 molecular weight glycoprotein (gp

160), gp160 precursor is molecular weight then cleaved to external glycoprotein

120000 (gp 120) and transmembrane; Ί

: ’· Ίύ N’ · :. ?.? · -I r '·.

molecular weight glycoprotein 41000 (gp 41). These coat proteins are mostly targets of antibodies in et al., Science, 228 = 1094-1096 (1985).

AIDS patients. Barin

Native HIV-1 gp 120 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 83: 7023-7027 (1986).

Due to the very low levels of the native HIV-1 envelope protein in infected cells and the risks associated with the preparation of an AIDS vaccine from HIV-1 infected cells. Recombinant DNA methods have been developed to produce HIV-1 envelope antigens for use as AIDS vaccines. Recombinant DNA technologies seem to have better possibilities for the production of AIDS under unit vaccines for the safe and economical production of high amounts of immunogens. The HIV-1 coat protein is expressed in genetically altered vaccine viral recombinants. Chakrabarti et al., Nature, 320 = 535-537 (1986), Hu et al.

Nature, 320 = 537-540 (1986), Kieny et al., Bioteclinology, 4 =

790-795 (1986). The envelope protein was also expressed in bacterial cells (Putney et al., Science, 234 = 1392-1395 (1986), in mammalian cells (Lasky et al. Science, 23- 209-12 (1986) and in insect cells.) Synthetic peptides derived from the amino acid sequences in HIV-1 gp41 were also considered candidates for AIDS vaccine.Kenedy et al., (1986) However, a successful AIDS vaccine was not produced using these materials and methods.

The use of the baci lovir-insect cell vector system to produce recombinant HIV-1 coat proteins is one? .

an aspect of the invention described in patent application no. 92) 197 filed October 16, 1986 (now No. 585 266). See also no. 151 and 976.

The baculovirus system has been shown to be generally useful in j

the production of HIV-1 proteins and other proteins. For example.

₍ Autographa caliulica baculovirus nuclear polyhedric virus (AcNPV) was used as a vector to express x full length gp160 and various portions of the HIV-1 envelope gene in infected Spodoptera frugiperda cells (Sf9 cells). a prior art related gpl60 i

é g genius (recombinant Ac3046 number), the protein produced by á recombinant Ac3046 and the purification technique for the Ac3046 gene product such that it includes lens lectin affinity chromatography and gel filtration chromatography. The Gp160 protein is purified in this way and aggregated to form pacticles

as highly immunogenic for rodents and primates.

;! Ideal AIDS vaccine. in addition to being totally biologically pure and non-pathogenic, it should provide long-term protection; · · * j protection against IIIV-1 after one or several injections. This is common in the case of living attenuated vaccines. If they are smiling

IN- x bacteria or or beliefs, or materials from them isolated. than clay 3 à toxoids or proteins , used to creation vaccine. lead no often ί l k puro against substance reply but n briefly immun • you. ··] ’ • To overcome or minimize these vaccine deficiencies knives

an adjuvant, called an adjuvant, may be added. These ingredients are materials that help stimulate the immune response. Adjuvants commonly used in human vaccines are gel / aluminum salts (aluminum phosphate or aluminum hydroxide), commonly referred to as alum adjuvant. Bomford et al., Adjuvaiits, Animal Cell Biotech, Vol 2 = 235-2.50, Academic Press Inc. ..

$ (London: 1985) i

SUMMARY OF THE INVENTION The present invention provides a vaccine and treatment methods for $ 3

human immunodeficiency virus (HIV), including the administration of a recombinant HIV envelope protein to an infected or susceptible individual. For suitable use, the coat protein may be purified.

it is aggregated and combined with an adjuvant (e.g. alum) for use in a vaccine.

Description of the attached images

The details of the present invention are given below with reference to r, Ϊ

(i) the attached figures;

J '? FIG. 1: Illustrates the cloning strategy used to isolate the envelope gene (env) from E. coli plasmid pN A2. The hatched regions ä are the 11IV-1 DNA sequences and the empty regions are from the cloning / vector. The black region in plasmid p174 is constructed from j synthetic oligonucleotides and was inserted as a SmaI-KpnI fragment into the SmaI-KpnI sites of plasmid p1614. Sequence of this ^? The synthetic plasmid is shown.

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Fig. 2. illustrates the strategy used for construction

/1 / recombinant plasmid vector (p3046), which in turn is used to construct the baculovirus expression vector Ac3046. Plasmid pMGS3 contains the sequence (cross-hatched regions) of α ' -from. baculovirus AcNPV on each side of the cloning site _at position 400. This site is the only restriction site for SmaI, KpnI, and BglII restriction endonucleases. The AcNPV polyhedrin promoter is 5'-direction from 4.00 position. The sequence 5 '-TAATTAATTAA-3' is in the 3 * -smid and has a translation termination codon in all three r reading frames. Plasmid p1774 and the sequence of the synthetic oligonucleotide region 1 are as in FIG. Plasmid p3046; contains the entire pMGS3 except for the sequences between the SmaI and BglII sites.

even those where it is embedded HIV-1 envelope gene pl.774. Fig. 3 shows nucleotide a DNA sequence flanking \ c3046 GPLGLAL coding sequence u. 3046 env.DNA sequence between +1

and +2264 is shown in FIG. 4th

Figures 4a-4k represent the actual DNA sequences of the HJV-j env gene segment together with the synthetic oligonucleotide sequences at the 5 'end of the env gene in Ac3046 (between +1 and +2264). The location of the restriction endonuclease sites is recorded above the DNA sequence and the determined amino acid sequence is recorded below the DNA sequence. The bases are numbered right and left.

Figures 5a-5d compare the DNA sequences of the env gene from Ac3046 to the published env gene sequence from LAV-1. The LAV-1 sequence is at the top and Ac3046 at the bottom. Line (1) below the LAC-1 sequence indicates that the sequence in Ac3046 is the same at this position. The DNA sequence numbering used is described by Vain-Hobson et al. Cell, 40 = 9-17 (1985) for LAV-1.

Fig. 6 shows ELIS A and point dilution of human HIV-1 antibody titers of positive serum (top graphs) and rhesus monkey sera (bottom graphs) from animals injected with gp160 (IJ55, KL55) or gp120 (AB55, CD55, GH55). Specifically bound antibody was measured with a goat anti-human IgG HRP conjugate. The highest serum dilution giving a positive response is the titer. ELISA titers measured against highly purified fp120 and gp160 proteins.

Fig. 7 is a table summarizing gp160 vaccine-induced innate responses of vaccinated seropositive patients.

Fig. 8 shows a vaccine induced anti-drug response directed against specific epitope of HIV envelope.

Figure 9 shows vaccine-induced T cell proliferative responses to gp160 in vaccinated seropositive individuals.

b

Fig. 10 (A-C) shows the proliferative lymphocyte response associated with vaccination.

.Obr. 11 is a graph showing the percentage of CD4 change. bins in response or non-response depending on time.

Recombinant HIV-1 gp160 envelope protein (rgp160), especially when adsorbed to an adjuvant such as alum (i.e. alurainium phosphate), has been found to be significantly useful as an AIDS vaccine. One aspect of the present invention is an AcNIPV expression vector having a coding sequence for a portion of the HIV-1 envelope gene comprising amino acids 1-757 found in recombinant clone # 1. 3046. Another aspect of the invention is the production of recombinant HIV-1 coat protein (and the protein itself) in insect cells - particularly the rpg160 protein encoded by the amino acid sequence (i.e., 03046).

Other aspects of the invention include purifying and forming recombinant coat protein particles from a gene product.

I of a recombinant baculovirus that produces 3046 protein j and adsorption of 3046 particles into aluminum phosphate aggregates.

and

The invention also encompasses a prophylactic and / or therapeutic vaccine for AIDS or HIV infection, and methods of preventing or treating AIDS or HIV infection.

Examples of the invention

The following examples illustrate the invention without limiting its scope.

Recombinant baculovirus Äutographa californica, a nuclear polyhedrosis virus (AnNPV), which contains a modified HIV-1 gp160 gene encoding amino acids 1-757 of the HIV envelope protein (recombinant Ac3046) is described in copending US patent application no. 920197 (now number 585260). The cloning steps used to construct the recombinant baculovirus containing the genes or portions of the HIV-1 genes are also described herein and are incorporated herein by reference.

The following is a detailed description of the genetic engineering steps used to construct the Ac3046 expression vector. The materials used, including enzymes and immunological reagents, were obtained from 6 commercial sources. Examples. showing how to make and use the invention are also disclosed.

Other recombinant coat proteins, referred to collectively as rgp160, are also contemplated and include recombinant proteins gp110 and gp41. Ac3046 is just one example of an expression vector and a recombinant protein in accordance with the invention.

Example 1

Construction of Baculovirus Recombinant Ac3046 Carrying

1IIV -1. coding sequence for amino acids 1-7 ';

Cloning and expression of foreign protein coding sequences in baculovirus vectors requires that the coding sequence be aligned with the polyhedrin promoter and the upstream sequences on one side and with the baculovirus coding | sequences on the other hand. The ranking is such that homologous recombination with the baculovirus genome results in the transfection of the foreign coding sequence aligned with the polyhedrin promoter j and the inactive polyhedrin gene.

Ί a Correspondingly, a variety of insertion vectors for use in HIV envelope gene constructs. Advertisement vector j MGS3. described below. was designed to replace the ATG translation initiation codon. The insertion of foreign sequences into this vector must be translated such that a translation frame, formed by the initiation codon, is maintained correct through foreign sequences.

í; The MGS3 insertion vector was constructed from an EcoRI-I plasmid-isolated DNA clone fragment, purified by the AcMNPV isolate? (VT-1). MGS3 was found to have the following structural features: a) 4000 bp sequence upstream of the ATG initiation <codon of the polyhedrin gene, b) a polylinker introduced by site directed mutagenesis, which contains the ATG initiation codon at position. corresponding to the polyhedrin codon and SmaI restriction sites. KPnI, BglII and the universal stop-codon segment, c) a 1700 bp sequence between the KpnI restriction site (which is within the polyhedrin gene) to the terminal LcoRI restriction site of the EcoRI clone. See fig. Second

Example 2

Construction of baculos of Irish recombinants carrying LAV env coding sequences

The recombinant plasmid designated NA2 (FIG. 1) consists of a 21.8 kb segment of the whole HIV provirus inserted into pIC11J. This clone was reported to be infectious because it could produce a virus for transfection of certain human cells. Adachi et al., J. Virol. 59 ^; 284-281 (1986). The complete envelope gene sequences contained in NA2 were derived from the HIV LAV chain. Barre-Sinoussi (1983).

The HIV-1 envelope gene was processed as described below and shown in FIG. 1. The envelope gene was first isolated from ΝΛ2 as a 3846 bp EcoRI / SacI restriction fragment cloned into the EcoRI / SacI restriction sites of pUC19. The resulting plasmid was designated p708.

The envelope gene was subsequently re-isolated as a 2300 bp KpnI restriction fragment and cloned into the KpnI restriction sites of pUC18. The resulting clone was designated p1614.

The Kpn restriction fragment in P1614 contains a slightly modified portion of the HIV envelope gene such that the 12.1 bp N-terminal coding sequence was deleted. The missing portion of the gene that contained the V signal peptide sequences. was replaced by insertion of a double-stranded synthetic oligomer. The inverted oligomer hol determined from the LAV amino acid sequence using a preferred polyhedrin gene codon procedure. To facilitate further manipulation, the Smal Nva was restrictive! a sequence co-inserted into the AT6 initiation codon site. The AT6 initiation codon l> ol replaced by a baculovirus vector. The resulting plasmid was designated as P1774.

As described in FIG. 2, restriction fragments of p17774 containing the coding sequences of the various domains of the HIV-1 envelope. were cloned into MGS insertion vectors (i.e., MGS3) if and when. The AT6 initiation column of the insertion vector was in frame with the codons of the envelope gene. The p3046 construct consisted of a SmaI / BamIII restriction fragment isolated from p1874, inserted into the SmaI / BglOO site of the plasmid vector pMGS3. This clone contains sequences coding for amino acids 1 to 757 gp.160 and utilizes the tin codon replaced by the MGS3 vector.

Example 3

Preparation and selection of recombinant baculovirus

The HIV env gene recombinant plasmid p3046 was precipitated with calcium phosphate with AcMNPV DNA (VT-1) and added to uninfected Spodoptera frugiperda cells. The chimeric gene was then inserted into the AcMNPV genome by homologous recombination. Recombinant viruses were identified by occlusion of negative plaque morphology. Such a cytopathic effect, but not subsequent plaque purifications, plaques exhibit an identifiable nuclear occlusion. Two additional were made to obtain pure recombinant viruses. Recombinant viral DNA was analyzed by site-specific insertion of HIV env sequences by comparing their restriction and hybridization characteristics to the wild-type viral DNA.

Example 4

Expression of IIIV env from recombinant baculoviruses in infected insect cells

Expression of the env sequence from recombinant viruses in insect cells should result in the synthesis of the primary translation product. This primary product will be composed of amino acids translated from codons replaced by a recombinant vector.

The result is a protein containing all the amino acids / encoded by the ATG initiation codon of the expression vector downstream of the polyhedrin promoter, to translate to a terminating signal on the expression vector (i.e., rpg10). The primary translation product Ac3046 can be read Met-Pro-Gly-Arg-Val at the end where Arg (position 4) is Arg at position 2 in the original LAV kJone. Met-Pro-Gly-codons are replaced as a result of the cloning strategy.

Example 5 v

Nucleotide sequence of the gp110 insert and flanking DNA

F

The nucleotide sequence of the gp160 insert and flanking DNA is determined from restriction fragments isolated from the DNA in the Ac3046 T expression vector. The sequencing procedure includes the following steps. The 3.0 kb EcoRV-BamHI fragment was purified by restriction digestion of .3,346 viral DNA. Ac3046 viral DNA was prepared from the extracellular virus present in the cell medium used to produce the vaccine fraction. The 2.3 kb EcoRV-BamHI fragment is folded; of the entire gp160 gene and 100 bp above and about 1000 bp below flanking

DNA.

Briefly, the sequencing results revealed the chimeric j construct envisaged by the cloning strategy. Sequence! gp160 exactly corresponds to that described by Valn-Hobson et al (1985). The 2253 base sequence between the putative initiation sequence and the termination codon was determined by both 751 amino acids and the novel codons j and 28 of the potential N-linked glycosylated sites. The molecular weight of this rgp10, together with the sugar residues, is approximately 145,000.

Sequence analyzes of 200 bases of flanking DNA indicate corresponding insertion as shown in FIG. 3, 4 and 5. ¹ : Example 6 j.

I> 1.0 * t

i ä '

Gp160 amino acid sequence

Using the standard automatic Cdman degradation and HI1LC procedure, the N-terminal sequence of the first 15 residues was determined to be identical to the same sequence determined from the J »NA sequence. The N-terminal methionine is not present in the gp.1.60 protein. This is consistent with the observation that the AcNPV polyhedrin protein is also produced without the N-terminal methionine. A summary of the actual gp160 DNA and N-terminal protein sequences as determined by analysis of AcNPV 3046 DNA and purified gp160 are provided below (Table 1).

Table 1

LAV env gene in AcNPV 3046 expression vector

rest

2 3 4 5 6 7 8 9 10 11 12 Pro Gly Arg Val Lys Glu Lys Ty r G Ln His Leu 13 14 Trp Arg Trp Gly ATG CCC GGG CGT GTG AAG GAG AAG TAC CAA CAC CTG TGG CGT

TGG GGC

These results are compared to the original LAV-1 joint as follows (Table 2).

Table 2LAV env gene in the original LAV-1 clone

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Met Arg wall Lys Glu Lys Tyr gin II í. with Leu Trp Arg Trp Gly ATG AGA GTG AAG GAG AAG ΤΛΤ CAG CAC TTG TGG AGA TGG GGG

Example 7 f

Purification of recombinant gp160

One aspect of the present invention is a method used to extract and purify the recombinant HIV envelope protein encoded in an Ac3046 expression vector. Recombinant H1V-1 coat protein gp110 is produced in S. frugiperda cells within 4-5 days after Ac3046 infection. Purification of this rgplSO protein comprises the steps of:

1. Cell washing

2. Cell lysis

3. Goal filtration chromatography

4. Lentil lectin affinity chromatography

5. Dialysis

This example describes the purification of recombinant gp.LGO in 2 x 10 ⁹ Ac3046 infected cells.

1. Cell washing. The infected cells are washed in a buffer containing 50 mM Tris buffer (pH 7.5), 1 mM EDTA and 1% Triton X-100. Cells are resuspended in a buffer, homogenized using standard methods, and centrifuged at 5000 rpm ^-1. This procedure is repeated 3 times.

2. Cell lysis. The washed cells are lysed by ultrasound in 50 mM Tris buffer (pH 8.0-8.5), 4% deoxycholate and 1% beta-mercaptoethanol. Ultrasound is used by standard methods. After sonication, only remnants of the nuclear membrane intact and these are removed by centrifugation at 5000 rpm-iuin ^first The supernatant containing the extracted gp160 does not have intact cells, as evidenced by optical microscopic observation.

3. Goal filtration. Gel filtration is performed in a Pharmacla 5.0 x 50 cm glass column packed with Sephacryl (Ph. Irmacia). The total volume is about 1750 ml. At least 6 liters of 0.1N NaOH are allowed to pass through the column for 24 hours to depyrogenate and clean up the column and pipe connections. The eluent outlet from the column is connected to a UV flow cell and monitor and recorder (PharmaciaJ, and then the column is equilibrated with 4 liters of goal filtration buffer. The crude gp160 is loaded onto the column and developed with the goal filtration buffer.

The column separates the crude mixture into three main UV absorbing fractions. The first peak is between about 500 and 700 ml. a second between 700 and 1400 ml and a third between 1400 and 1900 ml buffer. The same profile is observed on small analytical columns where the first peak was determined to be a material having a molecular weight of H 200O000.

This peak is translucent due to the high molecular weight lipid concentration and lipid complexes. This peak also contains 10% to 20% gp1GO extracted from the infected cells.

Probably this fraction of gp1GO is complexed with it or with other cellular compartments therein to form high molecular weight aggregates.

The second broad peak contains a major amount of gp1GO and proteins having a molecular weight between about 18,000 and 200,000.

!

The third peak contains low protein and UV absorption mainly due to beta-mercaptoethanol in the sample.

If the second peak is first delegated from UV absorbance. The eluerite from the column is applied directly to the lens lectin column. If the second peak exits the column, the eluent is detached from the lentil lectin column and is discarded.

i i 4. Lentil lectin. Affinity Goal Chromatography Medium in; Lentin lectin (Lenti 1 Lectin-Sepharose 4B) was obtained crude from Pharinacia. Lentin lectin was isolated by Sephadex affinity chromatography to greater than 98% purity and then mobilized by coupling with Sepharose 4B using cyanogen bromide.

The matrix contains about 2 mg of ligaride per ml of gel. Column with? The lentil lecturer is a 5.0 x 30 cm glass column (Pharraacia).

j containing 125 ml of Lentil-Lectin-Sepharose gel. The affinity j of the matrix is reused after careful washing and regeneration

13 r according to the manufacturer's recommended procedure. If not in use. the gel in the column is stored in a solution of 0.9% NaCl, 1 mM MnCl 3, 1 mM CaCl 2, and 0.01% thimerosal. The column is washed and equilibrated with 250 ml of the lentil lectin buffer described above before each use.

The crude gp160 is applied to the column directly as it exits the gel filtration column (described above). When the crude gp160 is bound to the column, it is washed with 800 ml of Lentil Lectin Buffer. containing 0.1% deoxycholate. Under these conditions, all gp160 binds to the column. Lentil lectin buffer plus 0.3M alpha-methylmanoside is used to elute the vera. glycoproteins, which is monitored by UV monitors at the wavelength

280 nm.

5. Dialysis. Sugars and deoxycholate are removed by conventional dialysis.

Purification of gp160 from 1 liter of infected cells can be summarized in Table 3 below.

In another embodiment, conventional ion exchange chromatography (anionic or cationic) may be used instead of gel filtration.

Similarly, the order of steps is not essential - for example, gel filtration or ion exchange chromatography may be carried out after the step of purification with lentil lectin. Other agents may also be used according to the invention. For example, other ⁴ detergents may be used to purify the recombinant protein instead of deoxycholate. These include rionogenic detergents such as Tveen (polysorbate 20), Tveen 80, Lubrol and Triton X-100.

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Table 3

Summary of purification Stage v total protein (mg) ¹ gp160 protein (mg) % gpl60 total removal by accounts, nanty purification i Bun. pellet 1 .. 2., 3. 1-2000 20 1-2 cult, medium • washing gel 250 15 6th serum albumin, most nucl. acids and soluble cell. proteins f i 1 loss i a 120 12 12 lipids, nuclides and aggregates mol. weight. Sos. lectin T 14 10 70 non-glycosylated proteins dialysis 13 9 70 sugar, deoxycholate

excess Tri with buffer 1

j ¹ Total protein was evaluated from the absorbance at 280 nm.

_f Λ Example 8 .1

A. Assembly of gp160 particles

As one aspect of the present invention, it has been discovered that the gp160 antigen can be assembled into clay particles of 2000000 molecular weight during purification. Gpl60 protein is extracted from the cell as a mixture of 80-90% monomeric (160,000 molecular weight) and 10-20% polymeric (particle form) Water Separators Step ^f1 gel filtration removes the aggregated forms GPLGLAL. 1'urification of gp160 from this fraction (first peak from the gel filtration column) confirms that it is complexed with other cellular proteins, possibly 15 ^; · Ϊ́;

with membrane fragments. However, the gp160 antigen in the second peak of the goal filter column has a molecular weight of about 160000-300000 and is therefore primarily in monomeric or dimeric form.

The formation of aggregates or polymers of gp160 occurs during the development of the lens lectin column. The antigen was determined to form aggregates. when eluted from the lectin column in 0.1% deoxycholate, which is about 0.2% Critical Mouse Concentration (CMC) for deoxycholate, or when gp160 is eluted from the column in 0.1% deoxycholate.

* Aggregate size is measured on a FPLC Superose 12 column (Pharmacia) with high resolution. Samples from representative batches of purified gp160 had a size predominantly equal to or greater than 2000000 molecular weight of the blue-dextran size standard.

A cross-link study by Schvaler et al. (1989) demonstrates that gp160 produced in insect cells is a tetramer of identical subunits. The study also shows that gp160 in HIV-infected ₍ / cells and virus particles) is tetrameric. Recombinant gp160 particles can have tertiary and quaternary structures that are

They are similar to those found in native HIV gp160.

♦

An appropriate 3-dimensional structure could be important for the generation of ‘epitopes that require the exact composition of gp160; It is likely that as non-glycosylated proteins are removed from the association of j with the gp1GO antigen during binding and washing on the lentil lectin column, the hydrophobic portions of gp160 begin to form intemolecular associations. Deoxycholate is unlikely to bind to gp160 when the concentration is maintained above CMC and the antigen will still form complexes. The assembly of this antigen into aggregates is an intrinsic property of the protein in the purification according to the invention. It is possible that a very hydrophobic N-terminal frequency is present in the gp160 protein, consistent with the natural ability of the protein to form particles. Po ΐ

For purification, gp160 complexes can be filtered through a 0.2 micron cellulose acetate filter to yield significant protein loss.

B- Particle formation analysis

Electron microscopy analysis of purified gp160 particles demonstrates that they are protein-like, spherical particles of 30-100 nM.

As another test for the presence of particles, gp160 was analyzed by gel filtration. About 100 micrograms of gp160 was applied to a Superose 12, FPLC gel filtration HR 10/30 * column (Pharmacia Inc-). This column was first calibrated with molecular weight standards. The protein profile of this column is highly reproducible. The elution volume is reversed relative to the molecular weight of the protein standards. The column separates monomeric gp160 from polymeric forms and secretes globular proteins of 2 x 10 ⁶ molecular weight. In developing this column, substantially all of the purified gp1610 elutes in an empty volume and has a size (12 x 10 ⁶ ¢ 2000000) molecular weight.

Example 9

A. Adsorption of gp160 to alum

The efficacy of insoluble aluminum compounds as immunological adjuvants depends on the complete adsorption of antigens to the solid phase. It has been discovered in the present invention that alum compositions could be more effective at adsorption of gp160 but at a pH that does not reduce the efficacy of the gp160-alum complex as an immunogen. The factors controlled during the formation of this alum aluminum gel (phosphate) composition are ^: 1. The optimum pH for adsorption of antigens to alum is about 5.0. However, it has been discovered that gp160 loses immunogenicity at pH 6.5 as compared to pH 7.5 such that alutoi is produced at pH 7.1 10 0.1. It was discovered that essentially

100% gp1GO will still adsorb to alum at this pH.

2. The ionic strength of the NaCl present is relatively low and is less than 0.15 M.

3. There is a molar excess of aluminum chloride to sodium phosphate to ensure the absence of free phosphate ions in the supernatant.

4. The gp160 antigen is added to the freshly prepared alumn to stop crystal growth and minimize particle size.

The procedure is performed with 200 ml of alum and the adsorption of purified gp160 to alum is such that the final antigen concentration is 40 µg / ml as shown below.

B. Reagent Preparation (200 ml of total prepared lot)

V ^sa Prepare the following solution in 100 ml sterile, pyrogen-free bottles or beakers. The salts for solution 1 and solution 2 and sodium hydroxide were mixed and filtered through 0.2 microin cellulose acetate filters. into 100 ml sterile, pyrogen-free bottles

solution 1 A1C1 _3-6 H ₂ 0 0,895 9 NaHAc - 3H ₂ 0 0,136 g dissolve in 40 ml of water for injection (VFI) 0.2 m i krometer f i 1ter solution 2 Na3P0 * i. 12 H ₂ 0 1.234 g dissolve in 40 ml VFI, 0.2 m i crometer f i 1ter

Dissolve 3 NaOH in 100 ml of VFI,

0.2 micrometer f. J. ter

Solution 4 Tris

2,0 g

Dissolve 1.25 g in 100 ml of VFI, add 1 ml to 90 ml of VFI, adjust to pH 7,5 with 0,5 N HCl and adjust to 100 ml;

using VFI

The solutions were autoclaved for 30 min, sucked slowly. Cool to room temperature.

and

C. Formation of alum

1. Add Solution 1 (aluminum chloride-sodium acetate) to the preparation vial using a 25 ml sterile disposable pipette. Record the volume of solution 1 and start stirring the solution.

ί

I. 2. Add solution 2 (sodium phosphate) to the vial using ml sterile, disposable pipettes and continue stirring] to form a precipitate and record the volume of solution 2.

j i

3. Add solution 3 (sodium hydroxide) and continue for 5 minutes; in mixing. A 0.5 ml sample is taken and the pH is measured. If the pH is less than 7.0, an additional 0.5 ml of sodium hydroxide is added, stirred for an additional 5 minutes and the pH is again measured. Continue until the pH is between 7.0 and 7.2.

'I' / I '· ^first · 4. Determine the total volume added to the container (Solution 1 + Solution 2 + Solution EXAMPLE 3), then a sterile V L ^R I to adjust the volume of the

J i; . 100 ml.

Immediately add 8.0 micrograms of purified gp1GO in 100 mL of 1 mM Tris pH, directly to the preparation vial.

6. Stir for at least 20 minutes, then disperse the prepared vaccine into sterile vials.

J

Example 10 ΐ Immunogenicity of gp160 absorbed on alum (specific Ab response)? • J

A suitable method for detecting immunogenicity of r-j antigen.

i ·!

The preparation (vaccine) is a measurement of the drug-specific response in a group of mice receiving a single dose of antigen. - At the end of 4 weeks, mice were bled or measured for serum levels of antibodies to specific antigens (usually antigens used to immunize animals). a test, e.g. ELISA (enzyme binding immunoadsorbent assays).

Immunogenicity in mice with purified gp160 and no adjuvant at pH 6.0 and pH 7.5 adsorbed to alum (as described in Example 9) or mixed with Freud's complete adjuvant is summarized in Table 4 below.

Table 4

Group gpl60 seroconversion a by ALISA gpl60 adjuvant CAR. no. OD ² % (M / N) -

1 µg no. pil 7.5 8702 0,140 57% 4/6 no. pH 6.0 8702 0,110 26% 2/7 alum 8702 1,000 90% 9/10 alum 8705 2.285 100% 6/6 Freund. 8604 1,108 83% 5/6 Freund. 8702 1.396 100% 7/7 0.1 µg Freund. 8604 0,434 67% 4/6 alum 8705 1.003 (.7% 4/6 ² Mouse i are killed 28 days after im'unizáci i and serum are tested at dilution : 1: 10 i v ELISA test to purif ikovariemu gpl60. Similarly

results are obtained using a commercial ELISA (Genetics Systems Inc. = ELIA TM ELISA) against native HIV-1 proteins at a 1 ^: 400 serum dilution.

³ Number of seroconverted mice (P) to total number tested (M).

Mice immunized with a single 1.0 microgram dose of gp1GO antigen without any adjuvant elicited an anti-gp160 drug response (see table above). However, it is more potent against the drug response observed in a group of mice immunized with 1.0 microgram of gp1GO adsorbed on alum as an adjuvant. A single dose of less than 0.1 microgram of gp160 mixed with complete Freud's adjuvant or formulated with aluminum will be seroconverted to H 50% of immunized mice. Although the gp160 antigen was immunogenic in mice as an unformulated antigen at pH 7.5 and pH 6.0 in such a small amount, immunogenicity was lost at a lower pH.

Example 11

Immunogenicity of gplGO adsorbed on alum (ELISA serum study)

The ability of said vaccine to elicit an important biological property, the formulated gp160 vaccine, was to confirm that the following adjuvant was performed.

The aC immune response is great

To confirm that; s and 1 µm immunogenic in animals increases this immunogenicity,

On day 0, mice (group 10) were injected with a single dose (0.5 micrograms, 1.0 micrograms, or 5 micrograms) of gp160 alone, gp160 adsorbed on alum or gp110 in Compi f Freud's Adjuvant (CFA). On day 28, the mice were bled and the serum was subjected to an ELISA (10 dilution) for the presence against gp160.

The serum results obtained on day 28 are summarized in the table below (Table 5). Seroconversion was demonstrated in more than 50% mice in all groups. In all doses, the number of seroconversion. and the mean serum absorbance (OD 50 nm at a 1: 10 dilution in ELISA) were higher for gp160 adsorbed on alum than the same as obtained from mice immunized with gp160 alone.

These results demonstrate that the alurn adjuvant clearly enhances the immunogenicity of the gp160 antigen.

Table 5 days after injection

0.5 ud dose average 1.0 µg dose average 5.0 µg dose average 9 » s; P / N ⁴ OD ³ P / N FROM P / N FROM gi Ί60 9/10 .407 7/10. 699 7/10 .430 gplí> 0 • 9/10 .547 8/10. 797 10/10 1,347 GPLGLAL (Alun) * 10/10 1,130 10/10 1 , 967 10/10 1,317 GPLGLAL (CFA)

⁴ Number of seroconverted mice (P) compared to total (N) tested 28 days after immunization with 0.5 micrograms, 1 microgram or 5 micrograms of VaxSyn TM HIV-1.

⁵ The mean absorbance value (OD ^ go) in seroconverted mice as measured by gp160 ELISA at a 1: 10 dilution of serum.

Example 12

Neutralization data

HIV-1 neutralization assays are an accepted method for determining whether an anti-drug preparation will inhibit HIV-1 virus upon infection of cultured susceptible human lymphocyte cells. Antisera from animals immunized with gp1GO were tested in the HIV-1 neutralization assay and the results are summarized in Table 6 below.

à 'j' *

ä í

! table The animal 6 identification immunogen / adjuvant micrograms ⁶ neutralization titer ⁷ rhesus 0.55 gp! 20 / alum 16/8/8 1 = 80-1: 160 í ' and rhesus H55 gp! 20 / alum 16/8/8 1/80 - 1 = 160 rhesus L55 gpl60 / aluin .16 / 8/8 and i: 80 and i · mouse pool 3 gp120 / Γreund .25 / .25 / .25 1:40 - 1:80 1 ! • mouse pool 8 GPLGLAL / Freund . 1 /. 1 /. 1 1:40 - 1 = 80 s • j & guinea pig IgG purification gp! 60 / Freund 10/10/10 1: 320

⁶ gpl60 or gpl20 micrograms administered during the first j / second j / third immunization ⁷ Highest dilution of antiserum that inhibits infection by 50% t relative to HIV-1 infected cells exposed to serum

; non-immunized animals.

Guinea pigs, rabbits, rhesus monkeys were also immunized with gp160 Cza -Cαa. use of alum or Freud's adjuvant). In general, immunization of these animals produced a good anti-drug response against the &lt; 9 &gt; IV-1 envelope proteins.

• | · 'Example 13' I • I <Immunogenicity in chimpanzees · ('Genetically are chimpanzees! Closest human relatives and in the present

They are the only animal model for HIV-1 infection.

In safe / immunogenicity assays in three chimpanzees, two chimpanzees were immunized with 40 µmgrams and gp160 in the vaccine. Each received a second immunization 4 weeks with micrograms or 80 micrograms gp160. The control animal was immunized simultaneously with 1 ml of saline. Weekly serum samples were analyzed from each of the three chimpanzees for anti-gp1GO antibodies and for IIIV-1 viral antigens using three immunoassays, ELISA against pure gp.160 1 '>

developed by Micro GeneSyy Inc., Vestern-BIott analysis and commercial HIV-1 ELISA. The results of these analyzes are described below.

A. ELISA (MGSearch HIV 160)

ELISA test, MGSearch HIV-1, MGSearch is a trademark of MicroGeneSys, Inc. from Meriden, Connecticut, USA, is an immunosorbent assay against gp.160 and is described in related U.S. patent application Ser. 920197 (now No. 585266).

Serum samples taken before immunization and 11 weeks following the primary iinunizácii were diluted ^1: 10-1 = 100,000 and then incubated with nitrocellulose strips, containing 100 lig purified gpl60 per well. The final dilution titer is the highest dilution in which the assay was positive for anti gp160 antibodies as detected by a goat anti-human IgG-alkaline phosphatase conjugate.

• Serum samples from control animals and pre-immune serum from immunized animals were negative. Chimpanzee 80 which received ¹ microgram dose of ^4, was positive at a dilution of 1: 100 two-week chimp which received the 40 microgram dose was positive at a dilution of 1 '10 4th week. Anti-gp.160 antibody titers were β

; they increased up to week 5, in which the final dilution titers were approximately 1 - 10000Ô and 1 2000000. The antibody titers of both i animals decreased only slightly over 6-11 weeks.

i. This type of response is the same as quantitatively and qualitatively as the anti-substance response commonly observed in chimpanzees vaccinated against the human hepatitis virus vaccine.

B.

B. Commercial ELISA

Obviously, MGSearch HIV 160 ELISA and Vesternblot analysis in serum from chimpanzees immunized with VaxSyn (registered trademark of MicroGeneSys, Inc. for the AIDS vaccine described herein) that they seroconverted and have antibodies against recombinant gp1GO. To determine whether they also generate anti-HIV antibodies that recognize native viral envelope proteins, pre-immune serum and serum from weeks 1 to 11 were tested on a licensed, commercial ELISA test kit, the LAV EIA TM test kit from Genetic System Corporation, Seattle, Vashington. Animals immunized with 80 micrograms of gp1GO were positive at a t 100 dilution at week 2 and sustained an increase against the drug level up to 6 weeks. Animals immunized with 40 micrograms were positive at a 1: 100 dilution at 6 weeks.

Example 14

Antibody distribution between gp120 and gp41

It is important to find out whether the anti-gp1GO drug response in the vaccinated animal is directed against gp4L, gp120 or against fibrobes. Various immunological methods, including radioimmunoprecipitation (RIP), immunofluorescence (Ι1Γ), Western blot (VB) analysis, and quantitative ELISA against three different recombinant envelope antigens were used to detect and measure antibody distribution against different regions of H1V-1 envelope proteins.

Fig. 5 includes the immunoceactivity of three different recombinant antigens: (ART / TAB) (1) gp120-delta (modified recombinant HIV-1 gp120 with about 40 missing amino acids at the C-terminus of the molecule); (ART / TAB) (2) gp1.20 (full-length recombinant HIV-1 gp120) and (ART) TAB / (3) gp1GO.

Human sera from 50 IIIV-1 antibody positive individuals>

and 3‘ pooled human sera were highly reactive with gp.160, moderately reactive with gp120 and little or no anti-substance non-reactive with gp120 modified. Modified gpL20, which represents more than 90% of the HIV-1 external glycoproteins, is likely to contain protective determinants. The observation that human AIDS positive sera have several antibodies to this envelope region is consistent with the fact that the immune response to viral infection is not completely protective and that human positive series usually exhibit low levels of neutralizing activity in vitro.

□ in contrast, rhesus monkeys, immunized with either gp.1.60 inunugene. or modified gp120, have antibodies that strongly react with the modified gp120 portion of the HIV-1 envelope. This difference in the distribution of vortex envelope antibody recognition sites and the high titers observed in monkeys can be attributed to the fact that monkey serum has high neutralization titers.

A quantitative determination of the immunoreactivity of these three recoinbinant envelope antigens with human and immune rhesus monkey sera is shown in FIG. All monkey sera that were tested had high titers of antibodies against the modified gp120 antigen (gp120-delta), including those from animals immunized with gp160.

These results demonstrate that recombinant gp160 increases

a substance response in rhesus monkeys that is different from that usually found during natural infection. In the gp120-delta region of gp160 there are epitopes that are effectively recognized in immunized monkeys and which are not recognized by the human immune system during infection. These new epitopes may be important for protection against HIV-1 and may be an important property of recombinant gplGO for the prevention and treatment of HIV infection.

Example 15

Therapeutic administration of the vaccine

Clinical trials with 30-HIV seropositive patients were performed to determine the effect of vaccination with HIV gp160 cloned (produced in a baculovirus system as described above) on HIV-infected individuals.

Vaccination with recombinant gp160 resulted in an increase in the gp160 HIV-specific humoral and cellular immune response by 19 out of 30 (63%) HIV seropositive volunteers. 14 of 15 (93%) volunteers who received 6 doses of vaccine showed an increase in total gp1GO antibodies. For this, ceconibiriant HIV proteins (i.e., rgp41, rgp120, rgp110 and mixtures thereof) can be advantageously administered in a method of treating human HIV-infected patients.

The effective amounts of the HIV protein used in this embodiment of the invention can be determined in accordance with methods well known in the art, such as those set forth below. Usually, such an effective amount should be in the range of about 1 microgram to about / 100 micrograms per kilogram of body weight of the patient.

The frequency of administration can also be determined by known methods. The preferred route of administration is parental, ie. intravenous, intramuscular, intradermal, etc. , as is well known to those skilled in the art.

•

Ä. Selection of volunteers

30 volunteers with HIV infection were obtained. Only seropositive patients with early-stage HIV infection as defined by Valter Read scale 1 or 2 (CD4 cells not less than 400 for 3 months with or without lymphadenopathy) were eligible (Kedfield et al. New Engl. JMed. 314: 131-132 (1986)). In addition, the entry criteria limited volunteers to adults between 18 and 50 years of age, with normal complete blood counts, an unidentified organ disease, without. o alcohol or drug addiction for more than 12 months and who have not received anti-retroviral or even a standard drug. All patients underwent a 2-year baseline assessment before randomly assigned to treatment groups. No volunteer received anticetroviral or immunomodulatory drugs during the trial.

of the 30 volunteers were men. 4 women. 14 were white, black and 3 Hispanic. The average age was 29 years (range

- 49). There were 8 Valter Reed volunteers in the group and 22 volunteers were Valter Reed Grade 2. The baseline 'average CD4 count was 668 (range 388-1639). The mean time between the initial diagnosis and the onset of the study was 24 months * (range 3-4 months).

* B. Vaccine production and immunization chart

As described herein, the test vaccine contained a non-infectious subunit of gp160 derived glycoprotein as a baculovirus-expressed recombinant protein.

The immunogenic protein was produced in Lepidoptera insect cells, was biochemically purified and was adsorbed onto aluminum phosphate for the final vaccine formulation.

The three formulated doses of gp160 were used ^: 40 micrograms per milliliter, 160 micrograms per milliliter and 320 micrograms per milliliter. The injection volume for the 40 µg and 160 µg doses was 1 ml; 2 ml of 320 µg per milliliter were used to deliver a 640 µg injection dose.

Thirty volunteers were divided into 6 groups of 5 volunteers. Two immunization procedures were evaluated: procedure

A - with vaccination on days 0, 30 and 120 and procedure B - with vaccination on days 0, 30, 60, 120, 150 and 180. For both immunization procedures (A and B), there were three groups receiving different doses of vaccine ( table 7). All vaccines were injected intramuscularly into the deltoid muscle. The duration of the test was 10 months: 2 months of baseline follow-up and 8 months of follow-up

-ú

M • i'í

Ί ŕ!

follow-up, following initial vaccination.

Table 7

Immunization procedure ·

day 0 30 60 120 150 If it 0 procedure group 1 40 40 40 group 3 160 160 160 group 5 640 640 640

procedure B

group 2 40 40 40 160 160 160 group 4 160 160 160 640 640 640 A group 6 640 640 640 640 640 640

C. Determination of safety and toxicity

Each volunteer was interviewed and tested on days 0, 1, 2, 3, 15 and 30 after each injection. Volunteers were proven with regard to temperature, chills, vomiting, nausea, arthralgia (joint pain), myalgia (muscle pain), restlessness.

itching (tingling), head. The assay included ertem.

difficulty breathing, dizziness and pain with local injection site reactions.

swelling, itching, pain and touch sensitivity.

discoloration of the skin, peeling of the skin, changes in regional lymphadenopathy, changes in function in the injected limb, and subcutaneous nodular formation at the injection site. Complete blood counts, serum composition, coagulation profile and urine analysis were also determined monthly.

In vitro, cellular immune functions were determined by T-cell phenotyplysis (total lymphocytes, CD4 and CD8 cell phenotypes) as described by Rickmen et al. Clinical Immuno. 52 ^: 85-95 (1989); Birx et al., J. Acquir. Immune Defic. Syndrome. 4 ^: 188-186 (1991). T-cell proliferative responses to pokeveed and Con A mitogens and control antigens (Candida albicans and tetanus) were also determined. Birx et al., Supra. In vivo, cellular immune functions were determined by a late-skin sensitivity test for control antigens (ie, mumps, aethetic toxoid, Candida albicans, and tricliophytone).

Quantitative viral cultures of peripheral blood cells and plasma were determined as described by Búrke et al., J. Acquir. Immune

Deficit. Syndrome. 3 = 1159-1167, 1991. DNA polymerase chain reaction reactions of CWages et al., J. Med. Virol. 33 = 58-63, 1991) and serum levels of p24 antibody were determined to monitor in vivo HIV virus activity.

No systemic toxicity was noted, but local reactogenicity was recorded in 87 percent of subjects (<13 in each vaccine group).

Local reactions included leakage, sensitivity to touch and transient subcutaneous nodules of 11-formation

Rarely regional adenopathy. None at the injection site.

an increase in the subject was not refused another injection.

No frequency difference observed after primary immunization, next injection or dose.

There was no adverse effect or by measurement in 'vitro mitogen and aiitigénprol iferatívne j are also tested response _t the late response of the skin to read to ivelosti, or by acceleration of quantitative CD4 cell depletion. Initial Average Number of Cl) 4 i

ä · cells were 716 and 605 responding and non-responding to the vaccine, j. During the 140-day assay, the mean number of CD4 j cells in the vaccine responders was minus 0.2%; whereas, among non-responders, the average CD4 f cell count decreased by 7.3 percent (Fig. 11). HIV-induced vaccine

L * immunogenicity was not associated with recording acceleration of CD4 decrease in any individual throughout the course of the assay.

? To assess the possibility of increasing HIV replication and viral levels in subjects as a result of vaccination, they were measured in vivo

J 'vortex activity by quantitative plasma and vortex cultures,

PBMC by polymerase DNA strand construction and serum levels of p24 antigen. Quantitative cultures and DNA polymerase chain reaction reactions did not demonstrate alteration during this assay. Serum p24 antigen was not detectable in subjects.

D. Determination of immunogenicity

Antibodies directed against whole HIV proteins were measured using both the recombinantly produced gp160, p66, p24 viral gene products and whole viral lysates of the prototype HIV strain MN. Dot-blot and Western-blot techniques as described by Tpubin et al., Proc. Natl. Acad. Sci. USA 76 = 1350-4354 (1979). Antibody responses to specific epitopes of the envelope were also measured (see Figure 7).

In FIG. ‘7, epitopes 88 (amino acids 88-98 in gp120) and 448C (amino acids 448-514 in gp120) were selected because antibodies directed against these regions of gp120 were described as corresponding to the early phase of HIV infection.

The epitopes 106 (amino acids 106-121 in gp120), 241 (amino acids 241-272), 254 (amino acids 308-322), 422 (amino acids 422-454) and 735 (amino acids 735-752) were selected for their predicted functional importance. Epitopes 106-422 are comprised in CD4 binding, epitopes 241, 254 and 735 are comprised in a specific neutralization group, epitopes 300-308 are comprised in type-specific neutralization.

Epitope 582 (amino acids 582-602) was selected as a control because they represent the immunodominant envelope domain in natural HIV infection. Other epitopes under investigation are 49 (amino acids 49-128) and 342 (amino acids 342-405).

In FIG. 7, shaded fields indicate a documented change in immune response directed against the HIV envelope. Shaded boxes with <=) indicate primary humoral response, shaded boxes with (+) mean secondary to drug response. (-) means negative antibody - negative for specific eptitope for a post immunization but without quantitative change. Shaded cells with (.) Indicate a new T cell proliferative response to the subsequent immunization with gp160. (.) alone means no cellular response to gp160, while hb means high background (non-interpretable) and nd indicates not performed.

Neutralizing activity was measured against three prototype isolates (HIV-IIIB, KF and MN) in the syncitium inhibition assay as described by Nara, Nature, 333: 469-470 (1988). HIV specific cell responses were measured by known proliferative lymphocytes

3 i -f technique using gp160, p24 and baculovirus and control protein expression systems. (Birx, supra.).

E- Vaccine responsive and non-responsive

Subjects were classified as vaccine responsive only when a reproducible selective increase in both cellular and humoral immune responses against specific epitopes of the HIV envelope was associated with the vaccine series (Fig. 7). A vaccine-induced humoral response has been defined! as seroconversion to specific epitopes of the HIV envelope and / or a secondary immune response to a second injection to specific epitopes of the envelope. Vaccine-induced cellular immunity has been defined! as the development of a new, reproducible vaccine-associated proliferative response to gp1GO (the definition of vaccine responses is highly limiting in terms of scientific judgment of this experiment: e.g., to estimate the feasibility of post-infectious immunization). Subjects in which Sei did not develop either a humoral or cellular proliferative response, or who developed only a humoral or only cellular proliferative response to the gp160 epitope or HIV envelope were classified as non-responding.

F. Vaccine-induced humoral response

As shown in FIG. 7, 19 of 30 subjects (63%) showed a vaccine-induced increase as gp160 HIV specific humoral and cellular immune responses. These 19 were classified as responding to the vaccine. 4 of IJ 11 'are moved as neodpovedúcich huinorálna or cellular immune response ^j. All 6 subjects who failed to demonstrate any vaccine-induced response received only 3 doses (procedure 3). No changes in binding of antibodies to 1IIIV polymerase (p66) _? or structural (p24) gene products or non-HJV tetanus control antigen have not been demonstrated. No anti-baculoviral-Depidoptera cell control protein antibody was developed in any subject.

_t The increase in envelope antibody (gpl60) was delegated by the 13 subjects by Western Blot using the whole virus lysate HIV-MN. Changes were related to the immunization program. For three of the 15 subjects (20%) of Procedure A and 10 of the 15 subjects of Procedure i; 'B (67%) developed an increase in antibodies against envelope proteins' (p = 0.025 in Fisher's exact assay, performed twice in each

; each). All 13 subjects also seroconverted to specific β envelope epitopes.

Ί

I i

In contrast, 10 subjects who did not seroconvert> to any specific envelope epitope were not shown

Ϊ: increase in Wester-blot envelope antibodies. The remaining

The 7 subjects that seroconverted to specific envelope proteins did not demonstrate any change in antibodies against the ₄ whole Western blot envelope. No changes in antibodies directed against non-enveloped HIV were observed i

<· No subject.

teenagers

Fourteen of the 15 subjects (93%) in procedure B (6 doses) showed an increase in total gp160 antibodies compared to 7 of the 15 subjects -i ¢ 47 in procedure A (3 doses) (P == 0.01 Fisher test, performed (Fig. 7). .

"with ·.

As shown in FIG. 8, it is pre-immunizing to a post?

3] the prevalence of individual gp160 specific epitopes not as follows:

epitope 49 (27 to 70%), epitope 88 (28 to 52%), epitope 106 (50 to 87%), epitope 214 (0 to 14 percent), epitope 25 i (0 to 13 percent), epitope 300 <47 to percent), epitope 308 (42 to 69 percent), epitope

342 (0 to percent), epitope 422 (3 to 10 percent), epitope

448C (73 percent) and epitope

735 (17 j to 33 percent). Vaccine-induced seroconversion was recorded against all specific epitopes except 582 (FIG.

j 7). Antibodies (seroconversion) directed against epitopes 241, 254; or 342 were delegated only after vaccination.

Í

J '

Ϊ l

i i

I í

s

I ä <.4 í

Á: ľ

y 'l'

Ϊ

Secondary immune responses were detected to the following epitopes 8888, 106, 300, 448C and 582. The prevalence of antibodies directed against epitope 582 was 100% prior to vaccination and only one subject (3 percent) showed a secondary immune response.

The type of vaccine induced against the envelope epitope agent was varied (Fig. 7). A primary anti-drug response (seroconversion) to at least one etiotope occurred in 20 subjects, 14 out of 15 received procedure B and 6 out of 15 randomly selected procedures (P = 0.005 Fisher test, twice performed). Entities of the procedure

And seroconverted to only 15 of 110 (14%) potential etiopies.

against which they had no pre-immunization antibodies. Subjects of Procedure B seroconverted to 60 out of 129 (47%) (P <0.0001

Fisher test, twice performed). Seroconversion to three or more envelope epitopes occurred in 9 subjects (60%) randomized to Procedure B, but only in 2 subjects (13%)

Ô randomly selected for procedure A (P = 0.02 Fisher test, twice, transferred).

Serum neutralizing activity against three different chains (HIV-IIIB, MN and RF) was determined on day 0, 90 and 195 in 7 subjects. 4 of 5 vaccine responders also showed an increase in neutralizing activity to three or more isolators. The vaccine responders also demonstrated the ability to inhibit the formation of syncytia compared to non-responders.

G. Vaccine-induced cellular responses

The changes in cellular immune response were related to

Average diameter comparison of pre-vaccination and post-vaccination index of lymphocyte stimulation by the Vilcoxon series summary test.

(Basic) (LSI) use

21 of 30 subjects (70 µl) developed a new proliferative response to gp1SO after immunization (Fig. 7).

Fig. 9 illustrates proliferative responses to gp160, p24, and baculovirus control

Protein in four typical time-responsive vaccines.

For all subjects, gp160 induced proliferation increased from baseline LSI 3 to LSI 10 (calculated from the average of 4 values after the last immunization). In contrast, no protein change was noted for proliferative responses directed against HIV p24 protein or control baculovirus.

Vaccine-indicated changes in the mean LSI values for all subjects, subjects of the vaccine responding subgroup, and subjects in the groups according to the immunization procedure are shown in FIG. 10th

The change in difference in the proliferative response to the gp160 responsive vaccine was significant and unresponsive (<

0.001, Wilcoxon, once transferred).

gp160 for IL-induced responses according to the procedure

B (6 doses) were greater than those induced by Procedure A (3 doses (P

0,10, Wilcoxon, cancel

Nineteen of the 21 subjects who developed proliferative responses to gp160 also developed an anti-substance response (responding to the vaccine).

Max i has an average mean lymphocyte stimulating index (LSI) per gp160.

observed in all vaccine responders was 50.1.

for everyone

However ^? responses to the vaccine were different peak values within the vaccination range

LSI 4 to 170 (FIG. 7), according to the time relationship and course of cellular responses to gp1b0 (FIG. 9).

H. Discussion of results

Despite the limited sample size in this assay, several factors have been shown to be associated with immunogenicity of the vaccine. Six out of 15 (40 percent) subjects in procedure A versus 13 of 15 (87 percent) subjects in procedure B were responding to the vaccine (P = 0.02, Fisher test, twice). Of the 16 subjects with a baseline mean CD4 count greater than 600 per milliliter, 13 (8.1 percent) responded to the vaccine, compared to 6 out of 14 (43 percent) subjects whose mean baseline CD4 count was less than 600 cells per milliliter. For example, 5 of the 6 subjects of Procedure B (6 injections) were responding to the vaccine compared to only 1 of 8 who received 3 injections (Procedure A) (P = 0.03 Fisher test, twice) (Table 8).

Table 8 gp160 immune to vaccine responding by baseline CD4 count and immunization procedure

IN

f l. CD4 count N matching (%) mismatched (%) J procedure .4 > 600 7 5 (71) 2 (29) 500 - 600 5 1 (20) 4 (80) j. • j · <500 3 0 (0) 3 (100) 4 group total 15 6 (40) 9 (60) in procedure B s' .4 > 600 9 8 (89) 1 (1 L) 500 - 600 2 2 (100) 0 (0) clay <n I ' <500 4 3 (75) 1 (25) group total 15 13 (87) 2 (13) D • J. 1 y pretty 30 19 (63) 11 (37) ií y * therapeutic the use of vaccines was introduced by Pastenn • m century for 1iečbu acute rabies. Usefulness of this

however, it has not been studied much in the treatment of other infections

Examples of post-infectious modification of viral specific immunity (such as post-hepatitis A or

B exposures) are not well documenting the transmissibility of infections.

studies of people that would demonstrate this procedure in either advanced or chronic

The invention provides a virus-specific immune modification by active immunization after infection.

The gene derived gp160 vaccine enhances the viral humoral and cellular response by infection.

More precisely, from HIV envelope in a human host controlled in 1S) of 30 persons with early HIV

This study quantitatively and quantitatively measures individual antibody responses to specific HIV infections and, in post-infection immunization, documented accurate vaccine epitope determination in natural

In this case, 70% of subjects had induced humoral immunogenicity in already infected subjects.

For example, twenty subjects (19 vaccine responders and 1 not) seroconverted to specific epitopes of the envelope. Only vaccination-related seroconversion (epitopes 241, 254, 342) occurred in only 10 subjects.

Furthermore, variations in humoral responses to this vaccine.

as characterized by epitope mapping, it will allow for a possible rationale and provide the potential for efficient drug-specific response analysis unique in characterizing the immunoregulatory mechanisms of natural infection not induced during

Although the importance of in vivo serum neutralizing activity is currently present. unknown activity of the vaccine induces the observation of increased neutralization against individual HIV strains (IIIB, RF, MN> in 4 of 5 responders suggesting that post-infectious immunization changes in functional antibodies.

It will induce HIV strain a and will potentially assist in defining group specific neutralizing epitopes.

- 30 Proliferative response to HIV envelope proteins is exceptionally found in natural HIV infection. However, following gplGO immunization, the documented specific T cell proliferative response was 21 (70 percent) subjects. The reason for this difference is unclear. One possibility is that the new proliferative response may be directed against a single vaccine unique epitope (s) (as a result of the vaccine-produced methodology or alternative antigen formation in vivo). Alternatively, the protein used in the fermentation assay may not stimulate a primarily T-cell proliferative response against a homologous wild-type envelope of a wild-type virus. However, additional examples have been obtained that vaccination supports the host's immune response ^: selected for vaccination responding individuals demonstrated an HIV-IIIB type-specific cytotoxic cellular response following further immunization.

The factors responsible for vaccine sensitivity in HIV infected persons remain clear. In an early 11IV infection, individuals respond suboptimally to vaccine variants as compared to controls performed. This reduced sensitivity was related to early B cell dysregulation and T cell dysfunction. Here, the vaccine sensitivity was associated with a baseline CD4 cell count consistent with the hypothesis that the immunological state of the host is an important determinant of vaccine sensitivity. However. the immunization program, within a range of specific T cell counts, also affects the sensitivity to the vaccine: post B (6 injections) was superior. Of course, the reduced vaccine response observed in subjects with lower CD4 cell counts may be increased by increasing the number of vaccinations that imply further modification of the dose, regimen, adjuvant or formulation, and could be associated with a further improvement in host sensitivity.

Although the debate on the safety of active immunization has been intensified

HIV indicated persons is tη the proper case of culture, six construction

HIV-specific vaccine product, not immunospecific toxicity. Quantitative DNA strand polymerase and serum antigen assays show an increase in in vivo HIV burden. Excellent in vivo marker of HIV replication, degree of CD4 cell decline. has been successfully influenced by objects. especially those classified as vaccine sensitive The change in mean CD4 numbers for responders was -0.2 percent and was -7.3 percent for non-responders. The data show that post-infectious immune sensitivity was not associated with an increase in CD4 destruction and offer association with decreased HIV replication in vivo.

The vaccine results of this study were also compared to a database of ten infected and untreated individuals, broken down by age, ethnic group, and baseline CD cell count. The mean CD4 count decreased by 8.7 percent in this reference group, decreased by 7.2 percent in subjects undergoing procedure A, and increased by 0.6 percent in subjects undergoing procedure B. These results indicate that post-infectious vaccination with recombinant IIIV envelope protein it is transferable and further encouraging the prophylactic use of such a vaccine.

Claims (4)

PATENTED INFLUENCED IMMUNODEFFICIENCES (CIVIV), characterized in that the infected individual is recombinantly In IV both hunts protein. 2. The method of claim 1, wherein the recombinant protein is administered at a dose of from about 1 to 100 micrograms per kilogram of body weight. A method according to claim 1, characterized in that. wherein the recombinant protein is administered at a dose of from about 10 micrograms to about 4000 micrograms. The method of claim 1, wherein the recombinant protein is administered at a dose of from about 40 micrograms to about 1280 micrograms. The method of claim 3, s j ú c and s a by being administered at least three d avky. The method of claim 4 j ú c and s a by being e at least six dose. The method of claim 5; u j ú <. : i s a because of each dose is administered v in t ej? v a 1 e about 30 until 60 days. The method of claim 6 u j ú c : i s a because of each dose is administered in an interval of about 30 until 60 days. A method of treating individuals infected with a human immunodeficiency virus (ILIV) virus, characterized by. comprising administering a recombinant HIV envelope protein to an infected individual in an amount sufficient to elicit an increased ILIV-specific cellular or humoral immune Ή. <responses. '1 The method of claim 1. v y z n a n j ú c i sa because of recombinant protein is a produced expression system baculovirus-insect cell. The method of claim 3, v y z n a n j ú c i sa because of recombinant protein is a . produced expression system baculovirus-insect cell. The method of claim 5, v y z n a n j ú c i sa because of recombinant protein is a produced expression system baculovirus-insect cell. The method of claim 1, v y z n a n j ú c i sa that the recombinant protein has a molecular weight of about 145,000. 14. The method of claim 3, wherein the recombinant protein has a molecular weight of about 145,000. 15. The method of claim 5, wherein the recombinant protein has a molecular weight of about 145,000. 16. The method of claim 1, wherein the HIV envelope protein is at least one of gI160, gp120 gp41. 17. The method of claim 3, wherein the HIV envelope protein is at least one of gp160, gp120, gp41. The method of claim 5, wherein the HIV envelope protein is at least one of gp160, gp120, gp41. ? 19. The method according to of claim 1 , v y z c i sa T that recombinant protein is a expr i innovated insect cell with baked cheese vector Ac3046. 20. The method of of claim 3 . v y z c i sa T that recant even nantný protein is a expr iinovaný insect cell with baculovirus vector Ac3046. 21. The method of claim 5 wherein the recombinant protein is expressed by both insect cells and the baculovirus vector Ac3046. 22nd The method of claim 1, wherein the recombinant protein is agglomerated into particles of at least a molecular weight 2,000,000th The method of claim 3, wherein the recombinant protein is agglomerated to have a molecular weight of at least 2,000,000th The method of claim 5, wherein the recombinant protein is agglomerated to a molecular weight of at least 2,000,000th The method of claim 1, wherein the recombinant protein is combined 26. The method of claim 3, wherein the recombinant protein is combined 27. The method of claim 5, wherein the recombinant protein is a combination additive. 28. A method of treating individuals infected with immunodeficiency (CHIV). wherein the recombinant IIIV coat protein is recombinant protein formed into and as particles, an additive having a) um. wherein the molecular weight is at least about 2000000. The method of claim 28, wherein the recombinant protein is produced by an expression baculovirus-insect cell. 30. The method of claim 28, wherein the recombinant protein is selected from the group consisting of recombinant gp160, recombinant gp120, recombinant gp41, recombinant 11IV coat protein having a molecular weight of about 145,000, and recombinant protein expressed by the Ac3046 vector. 31. The method of claim 28, wherein the recombinant protein comprises about 757 consecutive amino acids of gp160 and substantially eliminates up to 40 consecutive amino acid terminus of gp160. 32. The method of claim 28, wherein the recombinant protein is administered at a dose of from about 10 µg to about 4000 ng- II3). An HIV vaccine therapeutic composition comprising a recombinant HIV envelope protein and an additive, wherein the recombinant protein is formed into particles having a molecular weight of at least about 20,000,000. 34. The composition of claim 33; m. wherein the recombinant HIV envelope protein is present in an amount of from about 10 µg to 4000 µg per dose. 35. The composition of claim 34, wherein the recombinant protein is produced in a baculovirus-insect cell expression system. 36. The composition of claim 34, wherein the recombinant protein comprises about 757 consecutive amino acids of gp160 and substantially secretes about 40 terminal amino acids of gp160.