LV10492B - Vaccine and treatment method for human immunodeficiency virus - Google Patents

Abstract

The acquired immunodeficiency syndrome (AIDS) vaccine, containing type I human immunodeficiency virus (HIV-I) shell proteins, is obtained from the cloned HIV-I shell protein genes in the baculovirus-insect cell vector system. The recombined HIV-I proteins are purified, rolled into particles, and after that adsorbed upon the aluminum phosphate adjuvant. As a result the obtained adsorbed recombined HIV-I virus shell proteins (AIDS vaccine) are highly immunogenic and cause the creation of antibodies in animals, which are related to the HIV-I virus shell, and in vitro tests can make viruses harmless. The above-mentioned AIDS vaccine causes new humoral and shell immune response in HIV infected patients and is used in the form of vaccine therapy to delay or eliminate the destruction of the immune system.

Description

LV 10492

VACCINE AND TREATMENT METHOD FOR HUMAN IMMUNODEFICIENCY VĪRUS

This application is a Continuation-in-part of U.S. Patent Application Serial No. 151,976 filed February 3, 1988 which is a Continuation-in-part of U.S. Patent application Serial No. 920,197 filed October 16, 1986 (now Serial

No. 585,266) . These applications and the references cited herein are incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

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 aģent 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)).

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-l show a Progressive loss of immune function due to the depletion of T4 lymphocytes. These T4 celis, as veli as certain nerve celis, have a molecule on their surface called CD4. HIV-1 recognizes the CD4 molecule through a receptor located on the envelope of the virus pārticies, enters these celis, and eventually replicates and kills the celi. An effective AIDS 2 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 celis.

Vaccines are generally given to healthy individuāls 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 immunotherapy against the disease. Salk, J., Nature. 327: 473-476 (1987).

It is widely believed that the HIV-1, envelope ("env") is the most promising candidate in the development of an AIDS vaccine. Francis and Petricciani. New Eng. J. Med.. 1586-1559 (1985); Vogt and Hirsh, Reviews of

Infectioug Disease. £: 991-1000 (1986); Fauci, Proc. Natl_._ Acad. Sci. USA. 83: 9278-9283. The 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 (gp4l). These envelope proteīns are the major target antigens for antibodies in AIDS patients. Bariņ, et al., Science. 228: 1094-1096 (1985). The native HIV-1 gpl20 p has been shovm to be immunogenic and capable of inducing neutralizing antibodies in rodents, goats, rhesus monkeys and chimpanzees. Robey, et al.f Proc,_Natl. Acad. Sci. USA £1:7023-7027 (1986).

Due to the very low Ievels of native HIV-1 envelope protein in infected celis and the risks associated with preparing an AIDS vaccine from HIV-1 infected celis, recombinant DNA methods have been employed to producē 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 producē 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., 3 LV 10492

Biotechnolocry. 4:790-795 (1986). The envelope protein has also been expressed in bacterial celis (Putney, et al., Science. 234: 1392-1395 (1986)), in mammalian celis (Lasky, et al., Science. 23.:209-12 (1986)), and in insect celis. 5 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 materiāls and methods. 10 The use of a baculovirus-insect celi vector system to producē recombinant HIV-1 envelope proteīns 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 15 No. 151,976.

The baculovirus system has been demonstrated to be of general utility in producing HIV-1 proteins and other proteins. As examples, the baculovirus Autograoha californica nuclear polyhedrosis virus (AcNPV) has been used 20 as a vector for the expression of the full length gpl60 and various portions of the HIV-1 envelope gene in infected Spodootera fruoiperda (fall armyworm) celis (Sf9 celis). Also disclosed in the prior copending patent applications is the truncated gpl60 gene (recombinant number Ac3046), the 25 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 pārticies was found to be highly 30 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. 35 This is usually the case with live attenuated vaccines. When killed bacteria or viruses, or materiāls isolated from them, such as toxoids or proteins, are used to make a vaccine, there often results a poor antibody response and 4 only short term immunity. To overcome or minimizē these deficiencies in a vaccine, an additional component, called an adjuvant, may be added. Adjuvants are materiāls which help stimul^te the immune response. Adjuvants in comrnon use in human vaccines are gels of aluminum salts (aluminum phosphate or aluminum hydroxide), usually referred to as alum adjuvants. Bomford, et al., "Adjuvants," Animal Celi Biotech. Vol. 2: 235-250, Academic Press Inc. (London: 1985) .

The present invention provides a vaccine and treatment methods for human immunodeficiency vīrus (HIV), comprising the administration of recombinant HIV envelope protein to an infected or susceptible individual. In a preferred embodiment, the envelope protein may be purified, aggregated, and combined with an adjuvant (β.σ.. alum) for vaccine use. »

BRIEF DESCRIPTION OF THE DRAWINGS

Details of this invention are set forth belov with reference to the accompanying drawings:

Fig. l illustrates the cloning strategy used to isolate the HIV-1 envelope gene (esv) from the E. coli plasmid pNA2. The hatched reģions are HIV-1 DNA seguences and the open reģions 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-KpnI 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 pMGS3 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, KpnI, and BglII. The AcNPV polyhedrin promoter is in the 5' direction from the 4.00 position. The seguence 5 LV 10492 5' -TAATTAATTAA-3' is in the 3' direction, and has a translational termination codon in ali three reading frames. The plasmid pl774 and the sequence of the synthetic oligonucleotide region is as described in Fig. l. The 5 plasmid p3046 contains ali of pMGS3 except for the seguences between the Smal and BglII 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 10 sequence between +1 and +2267 is shown in Fig. 4.

Figs. 4a-4t show the actual DNA sequence of the HIV-1 env gene segment along with the synthetic oligonucleotide sequences at the 5' end of the env gene in Ac3046 (b'etween +1 and + 2267) The locations of restriction endo-15 nuclease sites are listed above the DNA seguence and the predicted amino acid seguence is listed below the DNA sequence. The bases are numbered on the right and on the left.

Figs. 5a-5h compare the DNA sequences of the env . * 20 gene from Ac3046 with a published env gene seguence from LAV-i. 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-25 Hobson, et al.f Celi. 1£: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 (IJ55, KL55) or gpl20 (AB55, CD55, GH55). The ELISA titers 30 were measured against highly purified gpl20 and gpl60 proteīns. The specifically bound antibody was measured with a goat anti-human IgG HRP conjugate. The highest dilution of serum that gives a positive response in the tēst is the titer. 35 Fig. 7 is a Table summarizing the gpl60 Vaccine- induced immune responses of vaccinated seropositive patients. 6 \ f

Fig. Β (Α and Β) 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 individuāls.

Fig. 10 (A-C) shows the lymphocyte proliferation responses associated with vaccination.

Fig.. ll is a graph showing the percent change in CD4 celis in fesponders and non-responders over time.

SUMMARY OF THE INVENTION

It has been discovered that recombinant HIV-1 gplBO envelope protein ("rgpl60"), especially when adsorbed onto an adjuvant such as alum (e.g.. aluminum phosphate) is particularly useful as an AIDS vaccine. 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 eguivalent to 1-757 of the sequence in Wain-Hobson (ibid ), which are amino acids 1-752 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 celis -- especially the rgpl60 protein coded for by the amino acid seguences 1-757 of Wain-Hobson (i.e./ 03046 having 752 amino acid residues).

Other aspects of this invention comprise purification and formation of recombinant envelope protein pārticies from the gene product of the recombinant baculovirus that. producēs the 3046 protein and adsorption of the 3046 pārticies 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.

DETAILEP DSSCRIPTION OF .THE.INVENTION

The following examples illustrate the invention vdthout limiting its scope.

The recombinant baculovirus Autoaraoha califomica nuclear polyhedrosis virus (AcNPV) which contains a truncated HIV-1 gpl60 gene coding for amino acids 1-757 of 7 LV 10492 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.

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

Other recombinant envelope proteīns, referred to collectively as rgplSO, are also contemplated, and include recombinant gpl20 and gp41 proteīns. Ac3046 is just one example of an expression vector and recombinant envelope protein according to the invention. EXAMPLE 1

Construction of the baculovirus recombinant Ac3046 bearina the HIV-1 coding seguences for amino acids 1-212

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 seguences 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 seguence aligned with the polyhedrin promoter and an inactive polyhedrin gene.

Accordingly, a variety of insertion vectors were designed for use in HIV envelope gene constructions. The insertion vector MGS3, described below, was designed to supply the ATG translational initiating codon. Insertion of foreign sequences into this vector must be engineered such that the translational frame established by the initiating codon is maintained correctly through the foreign sequences.

The insertion vector MGS3 was constructed from an EcoRI-I restriction fragment clone of DNA isolated from a δ plaque purified AcMNPV isolate (VJT-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-5 directed mutagenesis, which consists of an ATG initiating codon at a position of the corresponding polyhedrin codon, and restriction sites Sma.I, KpnI, BglII and a universal stop codon segment; (c) 1700 bp of sequence extending from the KpnI restriction site (which is internai to the polyhedrin 10 gene) through to the termiņai EcoRI restriction site of the EcoRI-I clone. See. β.σ.. Fig. 2. EXAMPLE 2

Construction of baculovirus recombinants 15 bearincr LAV env codina seguences_ 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 producē virus following transfection of 20 certain human celis. Adachi, et al., J. Virol. 59:284-291' (1986). The complete envelope gene sequences contained in NA2 were derived from the LAV strain of HIV. Barre-Sinoussi (1983).

The HIV -1 envelope gene was isolated and 25 engineered as described belov, and as shown in Fig. 1. The envelope gene was initially isolated from NA2 as a 3846 bp EcoRI/SacI restriction fragment and cloned into the EcoRI/SacI restriction site pUC19. The resultant plasmid was designated p708. 30 The envelope gene was subsequently reisolated as a 2800 bp KpnI restriction fragment and cloned into the KpnI restriction site of pUC18. The resulting clone was designated pl6l4.

The KpnI restriction fragment in pl614 contained 35 a slightly truncated piece of the HIV envelope gene such that 121 bp of the N-termiņai corresponding sequence was missing. This missing part in the gene, which included the signal peptide seguences, was replaced by insertion of a 9 LV 10492 double-stranded synthetic oligomer. The inserted oligomer was designed from the LAV amino acid seguence using preferred polyhedrin gene codon usage. To facilitate further manipulation, a new Smal restriction sequence was 5 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.

Referring to Pig. 2, restriction fragments from 10 pl774 containing coding sequences of various domains of the HIV-1 envelope were cloned into the MGS insertion vectors (e.cr.. MGS3) such that the ATG initiating codon of the insertion vector was in-frame with the codons of the envelope gene. Constmct p3046 consisted of the Smal/BamHI 15 restriction fragment isolated from pl774 inserted into the Smal/BglII 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. 20 EXAMPLE 3

Preparation and Selection of Recombinant Baculovirus The HIV env gene recombination plasmid p3046 was calcium phosphate precipitated with AcMNPV DNA (WT-1) and 25 added to uninfected Spodoptera frugiperda celis. 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 30 nuclear occlusions. Two additional successive plague purifications were carried out to obtain pure recombinant virus. Recombinant virai DNA was analyzed for site-specific insertion of the HIV env sequences by comparing their restrictions and hybridization characteristics to wild-type 35 virai DNA. EŽSMPLF..-.1

Expression of HIV env from recombinant 10 baculoviruseg in infected insect celis

Expression of HIV env sequences from the recombinant viruses in insect celis should result in the synthesis of primary translational product. This primary 5 product will consist of amino acids translated from the codons supplied by the recombination vector. The result is a protein containing aļl the amino acids coded for from the ATG initiating codon of the expression vector downstream from the polyhedrin promoter to the translational 10 termination signal -on the expression vector (e.a.. rgpl60). The primary translation product of Ac3046 should read Met-Pro-Gly-Arg-Val at the terminus where Arg (position 4} iš the Arg at position 2 in the original LAV clone. The Met-Pro-Gly codons are supplied as a result of the cloning 15 strategy. EXAMPLS 5

Nucleotide secruence of the ctp160 insert and flanking ΏΜ^ 20 The nucleotide seguence of the gplSO insert and flanking DNA was determined from restriction fragments isolated from virai exprešsion vector Ac3046 DNA. The sequencing strategy involved the following steps. The 3.9 kb EcoRV-BamHI fragment was purified by restriction 25 digestion of Ac3046 virai DNA. The Ac3046 virai DNA had been prepared from extracellular virus present in the media of celis being used for a production lot of vaccine.

As shown in Fig. 2, the 3.9 kb EcoRV-BamHI fragment consists of the entire gpl60 gene and 100 bp of 30 upstream and about 1000 bp of downstream flanking DNA. Of this, the nucleotide sequence of the entire gpl60 gene was determined, including 100 bp of upstream and 100 bp of dovmstream flanking DNA.

Briefly, the results of the sequencing revealed a 35 chimeric construct as predicted from the cloning strategy. The sequence of the gpl60 was essentially as reported by Wain-Hobson, et al. (1985). The seguence of 2256 bases between the presumed translation initiation and termination LV 10492 codons predicts 752 amino acid codons and 28 potential N-linked glycosylation sites. The estimated molecular weight of this rgpl60, including the sugar residues, is approximately 145,000.

5 Sequence analysis of 200 bases of flanking DNA indicated correct insercion as shown in Figs. 3, 4 and 5. EXAMPLE 6

Amino Acid Searuence of arplSO 10 Using Standard automated Edman degradation and HPLC procedures, the N-termiņai sequence of the first 15 residues of gpl60 was determined to be identical to that predicted from the DNA seguence. The N-terminal methionine is not present on the gpl60 protein. This is consistent 15 with the observation that AcNPV polyhedrin protein is also produced without an N-terminal methionine. A summary of the actual gpl60 DNA and N-terminal protein sequences, as has been determined by analysis of the AcNPV 3046 DNA and purified gpl60, is as follows (Table 1). 20 TABLE 1 LAV env gene in the AcNPV 3046 expression vector Residue 2 3 4 5 6 7 8 9 10 11 12 13 14 25 Pro Gly Arg Vai Lys Glu Lys Tyr Gln His Leu Trp Arg Trp

Gly

ATG CCC GGG CGT GTG AAG GAG AAG TAC CAA CAC CTG TGG CGT TGG GGC 30 These results compare to the original LAV-l clone as follows (Table 2). TABLE 2 35 LAV env gene in the original LAV-l clone . Residue 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Met Arg Vai Lys Glu Lys Tyr Gln His Leu Trp Arg Trp Gly

ATG AGA GTG AAG GAG AAG TAT CAG CAC TTG TGG AGA TGG GGG 40 5 12 \ EXĀMPLE 7

Purif ication· of Recombinant gpl60

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 £. frugiperda celis during 4-5 days after infection with Ac3046. Purification of this rgpl60 protein involves the steps: 10 15 1. Washing the Celis 2. Celi Lysis 3. Gel Filtration Chromatography 4. Lentil Lectin Affinity Chromatography 5. Dialysis

This example describes the purification of the recombinant gpl60 from about 2 x 10’ Ac3046 infected celis. 1. Washing the celis. Infected celis are washed in a buffer containing 50 mM Tris buffer (pH 7.5), 1 mM EDTA and 20 1% Triton Χ-100. The celis are resuspended in this buffer, homogenized usiņg Standard methods, and centrifuged at 5000 rpm for 20 minūtes. This process is repeated 3 times. 2. Celi Lvsis. The washed celis are lysed by 25 sonication in 50 mM Tris buffer (pH 8.0-8.5), 4% deoxy- cholate and 1% beta mercaptoethanol. Sonication is done using Standard methods. After sonication, only remnants of the nuclear membrane are intact and these are removed by centrifugation at 5000 rpm for 30 minūtes. The supernatant 30 containing the extracted gpl60 has no intact celis, as determined by light microscopy observations. 3. Gel filtration. Gel filtration is done in a Pharmacia 5.0 x 50 cm glass column packed with a Sephacryl 35 resin (Pharmacia). The total bed volume is about 1750 ml. To depyrogenate and sanitize the column and tubing connections, at least 6 liters of 0.1 N NaOH is run through the column over a period of 24 hours. The effluent from the 13 LV 10492 column is connected to a UV flow celi and monitor and a chart recorder (Pharmacia) and then is eguilifarated with 4 liters of Gel Filtration Buffer. The crude gpl60 is loaded onto the column and is developed with Gel Filtration Buffer.

The column separates the crude mixture into three major UV absorbing fractions. The first peak comes off between about 500 and 700 ml, the second between 700 and 1400 ml and the third betveen 1400 and 1900 ml buffer. This same profilē is observed on small analytical co'lumns 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 celis. ;Apparently this fraction of gplSO is complexed to itself or other celi components to form high molecular weight aggregates.

The second broad peak contains the majority of the gpl60 and proteīns 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.

When the second peak is first detected from the tracing of the UV absorbance, 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. 4. Lentil Lectin. 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 14 4Β gel. The affinity matrix is reused after being thoroughly washed and regenerated by a procedure . recommended by the supplier. When not in use, the gel is stored in the column in a solution of 0.9% NaCl, 1 mM MnCl2, 1 mM CaCl2, and 0.01% thimerosal. * 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 gplSO is bound to the column, it is washed with 800 ml lentil lectin buffer containing 0.1% deoxycholate. Under these conditions ali of the gplSO 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. 5. Dialysis. Sugars and deoxycholates are removed by conventional dialysis.

The purification of gpl60 from 1 liter of infected celis can be summarized in the following table (Table 3).

In another embodiment, conventional ion exchange chromatography (anionic or cationic) may be used in place of gel filtration. Similarly, the order of steps is not critical: For example, gel filtration or ion exchange chromatography may follow the lentil lectin purification step. Other reaģents may also be used according to the invention. For example, 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 Χ-100. 15 LV 10492 TABLE 3 - Purification Summarv 5 10

Purification Step Total Protein (mg)1 gpl60 Protein (mg) %gpl60 Total Contaminants Removed Celi Peliet . 1-2000 20 1-2 Culture Medium l,2,3rd Wash 250 15 6 Serum Alburain, most Nucleic Acids, and Soluble Celi Proteins Gel Filtration 120 12 12 Lipids, Nucleic Acids, and high mol wt aggregates Lentil Lectin 14 10 70 Nonglycosylated proteins Oialyeis 13 9 70 Sugar, deoxycholate, excess Tris buffer

EXĀMFLE B 15 A. Agsemblv of crol60 Pārticies.

As one aspect of the present invention, it has been discovered that the gpl60 antigen can be assembled into pārticies of > 2,000,000 molecular weight during purifi-cation. The gpl60 protein is extracted from the celi as a 20 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 25 celi proteīns, possibly even with membrane fragments. However, the gpl60 antigen in the second peak off the gel filtration column has a molecular veight of about 160,000-300,000 and is, therefore, in predominantly monomeric or dimeric form. 30 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 v/hether it is eluted from the lectin column in 0.5% deoxycholate, which is about the 0.2% critical micelle con- 35 ‘ Total protein was estimated from absorbance at 280nm. 16 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. A cross-linking study by Schwaller, āl. (1989), demonstrated that gpl60 produced in insect celis is a tetramer of identical submits. The study also shows that gpl60 in HlV-infected celis and virus pārticies is tetrameric. Thus, the recombinant gpl60 pārticies 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. It is likely that, as non-glycosylated proteīns are removed from association with the gpl60 antigen during the binding and vrashing to the lentil lectin cblumn, the hydrophobic portions of gpl60 begin to form intermolecular associations. The deoxycholate is probably not bound to the gpl60 as the concentration can be ķept above the CMC and the antigen will stili form complexes. The assembly of this antigen into aggregates appears to be an intrinsic property of this protein once it is purified according to the invention. It is possible that the very hydrophobic N-terminal sequence that is present on the gpl60 protein contributes to the natūrai ability of this protein to form pārticies. After purification, the gpl60 complexes can be sterile filtered through a 0.2 micron cellulose acetate filter vithout significant loss of protein. B. Analysis of Particle Formation.

An analysis of purified gpl60 pārticies by electron microscopy demonstrates that they are protein-like, spherical pārticies of 30-100 nM.

As an additional tēst for the presence of pārticies, purified gpl60 was analyzed by gel filtration. 17 LV 10492

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 profilē from this column'is highly 5 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* molecular weight. When developed on this column, essentially ali of 10 the purified gpl60 elutes in the void volume and is, there-fore, > 2 x 10* (2,000,000) molecular veight in size. EXĀMPLE 9 A. Adsorotion of gpl60 to Alum. 15 The effectiveness of insoluble aluminum compounds as immunologic adjuvants depends on the completeness of adsorption of the antigens on the solid phase. As part of the present invention it was discovered that alum compositions could be made that would efficiently adsorb the 20 gpl60 but at a pH that would not reduce the potency of the gplSO-alum complex as an immunogen. The factors controlled during the formation of this alum (aluminum phosphate gel) composition are: 25 30 1. The optimal pH for adsorption of antigens to alum is about 5.0. However, it was discovered that the gplSO 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 gplSO will stili adsorb to the alum at this pH. 2. The ionic strength from the NaCl present is rela-tively low and is less than 0.15 M. 3. Tnere is a molar excess of aluminum chloride rela-tive to sodium phosphate to assure that there is 35 18 \ an absence of free phosphate ions in the supernatant. 4. The gpl60 antigen is added to freshly formed alum to stop crystal growth and minimizē the size of the pārticies.

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 /ig/ml, as outlined below. B. Preoaration of Reaģents (200 ml total formulated

Prepare the following Solutions in 100 ml sterile, pyrogen-free bottles or beakers. Mix the salts for Solution 1 and Solution 2 and the sodium hydroxide and filter through 0.2 micron cellulose acetate filters into 100 ml sterile, pyrogen-free bottles.

Solution 1 AlClj. 6H20 0.895 grams NaHAc. 3HļO 0.136 grams Dissolve in 40 ml water for injection (WFI), 0.2 micron filter Solution 2 Na3P0.12H20 1.234 grams Dissolve in 40 ml WFI, 0.2 micron filter Solution 3 NaOH 2.0 grams Dissolve in 100 ml WFI, 0.2 micron filter Solution 4 Tris 1.25 grams Dissolve in 100 ml WFI, add 1 ml to 90 ml WFI, adjust pH to 7.5 with 0.5N HC1, and bring to 100 ml with WFI

Autoclave the Solutions for 30 min; slow exhaust. Cool to room temperature. C. Formation of Alum 1. Add Solution l (aluminum chloride-sodium acetate to the i'ormulation vessel using 25 ml sterile, 19 LV 10492 disposable pipets. Note the volume of Solution 1 and begin stirring the solution. 2. Add Solution 2 (sodium phosphate) to the vessel using 25 ml sterile, disposable pipets and continue stirring as the precipitate forms and note the volume of Solution 2. 3,. Add 3 ml Solution 3 (sodium hydroxide) and continue stirring for 5 min. Take a 0.5 ml sample and measure the pH. If the pH is less than 7.0, add an additional 0.5 ml sodium hydroxide, stir for another 5 minūtes and measure the pH again. Continue until the pH is between 7.0 and 7.2. 4. Determine the total volume added to the formulation vessel (Solution 1 + Solution 2 + Solution 3), then add sterile WFI to bring the volume to 100 ml. 5. Immediately add 8,000 micrograms of purified gplSO in 100 ml of 1 mM Tris pH 7.5 directly into the formulation vessel. 6. Continue stirring for a minimum of 20 minūtes, then dispense the formulated vaccine into sterile vials. BXAMPLE 10

Immunogenicitv of Alum Absorbed erplSO (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 vhich have been given a single dose of antigen. At the end of 4 weeks the mice are bled and the serum antibody Ievels to a specified antigen (usually the antigen used to immunize the animal) are measured by a Standard antibody tēst, e.g. an ELISA (enzyme linked immunosorbent assay). 20

The immunogenicity in mice of purified gpl60 vrith no adjuvant at pH 6.0 and pH 7.5 adsorbed vrith alum (as described in Example 9) or mixed vrith Freund's Complete Adjuvant ι are summarized • below (Table 4). 5 TABLE 4 Group gpl60 Mean ELISA Seroconversion qd160 Adiuvant Lot# OD1 % (P/N)2 10 1 μ% None, pH 7,5 8702 0.140 57% 4/6 None, pH 6.0 8702 0.110 26% 2/7 Alum 8702 1.000 90% 9/10 Alum 8705 2.285 100% 6/6 15 Freund's 8604 1.108 83% 5/6 Freund's 8702 1.396 100% 7/7 0.1 Mg Freund's 8604 0.434 67% 4/6 Alum 8705 1.003 67% 4/6 20 Mice immunized vrith a single 1.0 microgram dose of gpl60 antigen vrithout any added adjuvant vri.ll elicit an antibody response against gpl60 (see table above). However, a much stronger antibody response is seen in groups of mice immunized with 1.0 microgram of gpl60 adsorbed to the alum 25 adjuvant. A single dose of less than 0.1 microgram of gpl60 mixed vrith complete Freund's or formulated with alum will seroconvert > 50% of the immunized mice. Although less so, 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 30 immunogenicity at the lower pH. 40 1

The mice were bled 28 days post immunization and the sera tested at 1:10 dilution in an ELISA assay against gel-35 purified gpl60. Similar results were obtained using a commercial ELISA (Genetic Systems Inc.? EIA® ELISA) assay against the native HIV-1 proteīns at a serum dilution of 1:400. 2

The number of seroconverted mice (P) to the total number tested (N). 21 LV 10492 EXAMPLE 11

Immunogenicitv of Alum Ab9orbed ctp160 {ELISA Serum Study)

The ability of a candidate vaccine to elicit an » 5 irnmune response is a very important biological property. To confirm that the alum formulated gplSO vaccine was immunogenic in animals and to confirm that the alum adjuvant increased this immunogenicity, the following experiment was performed. 10 On day 0, 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'a adjuvant (CFA) . On day 28 the mice were bled and the sera examined by ELISA (1:10 dilution) for 15 the presence of antibodies to gpl60.

Results from the sera drawn on day 28 are summarized in the table below (Table 5) . In ali groups, greater than 50% of the mice showed seroconversion. At ali doses the number of sero-conversions and the average serum 20 absorbance (0D4J0 nm at a 1:10 dilution in the ELISA assay) were higher with gpl60 adsorbed to alum than those obtained in mice immunized with gpl60 alone.

These results demonstrate that the alum adjuvant significantly increased the immunogenicity of the gplSO 25 antigen. 30 35 5 22 TABĪR-ž - -2B-_Days Post-Iniection

0.5 μς Dose 1.0 μg Dose 5.0 μ<3 Dose Mean Mean Mean P/N4 QD1 2 3 £ZH ΩΒ ĒM QO gpl60 9/10 .407 7/10 .699 7/10 .430 gpl60 (alum) 9/10 .547 8/10 .797 10/10 1.347 gpieo (CFA) 10/10 1.130 10/10 1.967 10/10 1.317 EXAMPLE 12 10

Neutralization Data HIV-1 neutralization assays are an accepted method to determine whether an antibody preparation will inhibit 15 the HIV-1 virus from infecting susceptible human cultured lymphocyte celis. 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). 20 25 30 1 * The number of mice that seroconverted (P) compared to 2 total number tested (N) at 28 days after being immunized 35 with 0.5 micrograms, l micrograms or 5 micrograms of VaxSynea HIV-1. 3

The mean absorbance (OD430) of the mice that seroconverted as measured by the sponsor's ELISA assay against 40 gpl60 at a 1:10 dilution of serum. 23 LV 10492 TABLE 6

Animal Identification Immunogen/ Adjuvant Micrograms1 2 Meutraliz-ing Titer3 Rhesus G55 · gpl20/Alum 16/8/8 1:80-1:160 Rhesus ΗΞ5 gpl20/Alum 16/8/8 1:80-1:160 Rhesus L55 gpl60/Alum 16/8/8 > 1:80 Mice Pool 3 gpl20/Freund's .25/.25/.25 1:40-1:80 Mice Pool 8 gpl60/Freund's .l/.l/.l 1:40-1:80 G. Pig Purified IgG gpl60/Freund's 10/10/10 1:320 10 Guinea pigs, rabbits and rhesus monkeys have also been irtununized 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-1 envelope proteīns. 15 EXAMPLE 13

Immunogenicity in Chlmpanzees

Genetically, the chimpanzee is man's closest rela-tive and is currently the only animal modei for infection of 20 HIV-1. In a safety/immunogenicity trial in three chimpanzees, two chimpanzees were immunized with 40 micrograms or 80 micrograms of gpl€0 in an alum formulated vaccine. Each received a booster immunization at 4 weeks with 40 micrograms and 80 micrograms of gpl60, respectively. 25 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 virai antigens using three immunological assaysi an ELISA assay against purified gpl60 developed by 1 30 4 Micrograms of gpl60 or gpl20 administered during the 2 first/second/third immunization. 3

The highest ciilution of antisera that will inhibit the 35 Infection by 50% relative to HIV-1 infected celis that were exposed to serum from non-immunized animals. 24 \

MicroGeneSys, Inc., Western Blot analysis, and a, commercial HIV-1 ELISA assay. The results of these analyses are described below. A. ELISA (MGSearch HIV 160)

The ELISA assay, MGSearch HIV 160, MGSearch being a trademark of MicroGeneSys, Inc. of Meriden, Connecticut, U.S.A., is an immunosorbent assay against gpl60 and is described in copending coassigned U.S. patent Application Serial No. 920,197 (now No. 585,266).

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 μg purified gpl60 in a spot. The end point dilution titer is the highest dilution in which the tēst was positive for anti-gpl60 antibody as detected with a goat anti-human IgG-alkaline phosphatase conjugate.

The serum 9amples 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. The antibody titer in both aniraals dropped just slightly during weeks 6-11.

This type of response is similar both quantitatively and qualitatively to antibody responses commonly observed in chimps that have been vaccinated with a human Hepatitis B Virus vaccine. B. Commercial ELISA Tēst

It was clear from the MGSearch HIV 160 ELISA and Western blot analyses of sera from the VaxSyn1 immunized 1

VaxSyn is a trademark of MicroGeneSys, Inc. for the AIDS vaccine described herein. 25 LV 10492 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 1 virai envelope proteīns, the pre-immune sera and sera from 5 weeks 1 through 11 were tested in a licensed, commercial ELISA tēst kit, the LAV EIA1* tēst kit of Genetic System Corporation, Seattle, Washington. The animal immunized with 80 micrograms of gpl60 was positive at a 1:100 dilution by week 2 and continued to show an increase in antibody Ievel 10 through week 6. The animal immunized with 40 micrograms was positive at a 1:100 dilution by week 6. EXĀMPLE 14

Distribution of Antibodies Betveen crol20 and crp41 15 It is important to determine whether the antibody responses against gpl60 in a vaccinated animal is directed against gp41, gpl20 or both. A variety of immunological methods, including radioimmunoprecipitation (RIP), immuno-fluorescence (IF), Hestera blot analysis (WB), and quantita-20 tive ELISA against three different recombinant envelope antigens were employed to detect and measure for the distribution of antibodies against various reģions of the HIV-1 envelope proteins.

Fig. 6 summarizes the immunoreactivity of three 25 different recombinant antigens: (ART] [TAB] (1) gpl20-delta (truncated recombinant HIV-1 gpl20 with about 40 amino acids missing from the C-teminus of the molecule); [ART] [TAB] (2) gpl20 (full length recombinant HIV-1 gpl20; and [ART] [TAB] (3) gpl60. 30 Human sera from 50 HIV-1 antibody positive in dividuāls and 3 pooled human sera were highly reactive with gplSO, moderately reactive with gpl20 and little or no antibody reacted with truncated gpl20. It is likely that the truncated gpl20, which represents more than 90% of the 35 HIV-1 extemal glycoprotein, contains protective determi-nants. The observation that human AIDS positive sera have few antibodies to this region of the envelope is consistent with the fact that the immune response to virai infection is 26 not fully protective and that human positive sera usually exhibit a low-Ievel of neutralizing activity in vitro.

In contrast, rhesus monkeys immunized with either the gpl60 iīnmunogen or with. the truncated. gpl20 have 5 antibodies Chat react strongly with the truncated gpl20 portion of the HIV-1 envelope. This difference in distribu-tion of antibody recognition sites along the virai envelope and the higher titers observed in the monkeys may account for the fact that the monkey sera had high neutralizing 10 titers. A quantitative assessment of the immunoreactivity of these three recombinānt envelope antigens with human and immune rhesus sera is presented in Fig. 7. Ali the monkey sera tested had high titer antibody against the truncated 15 gpl20 antigen (gpl20-delta), including those from animals immunized with gpl60.

These results dēmonstrate that the recombinānt gpl60 elicits an antibody response in rhesus monkeys that is different than what often occurs during natūrai infection. 20 There are epitopes in the gpl20-delta region of gp-160 that are efficiently recognized in the immunized monkeys that are not seen by the human immune system during infection. These new epitopes may be important for protection against HIV-l, and could be an important property of the recombinānt gpl60 25 for prevention and treatment of HIV-infection. EXRMPLB 15

Therapeutic Vaccine Ādministration A clinical trial with 30 HlV-seropositive human 30 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 individuāls.

Vaccination with the recombinānt gpl60 led to an augmentation in the gpl60 HlV-specific humoral and cellular 35 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. Therefore, recombinānt HIV proteins 27 LV 10492 (i.e., rgp41, rgpl20, rgpl60 and admixtures thereof) can be advantageously administered in a method to treat a human patient infected by HIV.

The effective amounts of HIV protein used in this 5 embodiment bf the inventioņ can be determined according to techniques well known in tlļe 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 10 also be determined by known means. In a preferred embodiment, 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. 15 A. Volunteer Selection

Thirty volunteers with HIV infection were recruit-ed. Only seropositive volunteers with early stage HIV infection, defined as Walter Reed Stage 1 or 2 (CD4 celi 20 count not less than 400 for greater than 3 months, with or without lymphadenopathy) were eligible for enrollment. (Redfield, et al., New Εησί.. J. Med. 314: 131-132 (1986). Additional entry criteria limited .volunteers to adults between the ages of 18 and 50, with a normai complete blood 25 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. Ali patients underwent a 2 month baseline evaluation prior to randomization into treatment groups. No volunteers received 30 any antiretroviral or immunomodulatory drugs during the trial.

Twenty-six of the 30 volunteers were men; 4 were women. Fourteen were Caucasian, 13 Black, and 3 Hispanic. The mean age was 29 (range 18-49). At enrollment 8 volun-35 teers were Walter Reed Stage 1 and 22 volunteers were Walter Reed Stage 2. The baseline mean CD4 count was 668 (range 388-1639). The mean time betveen initial diagnosis and study entry was 24 months (range 3 months to 49 months). 28 B. Vaccine Product and Immunization Schedule

As described herein, the tēst vaccine comprises a non-infectious subunit glycoprotein derived from gpl60 as a baculovirus expressed recombinant protein. The immunogenic protein was produced in Lepidopteran insect celis, was biochemically purified, and was adsorbed to aluminum phosphate for final vaccine formulation.

Three dose formulations of gplSO were used: 40 micrograms per milliliter, 160 micrograms per milliliter and 320 micrograms per milliliter. The injection volume for both the 40 μg and 160 μ<3 dosages was 1 ml; 2 ml of 320 μ$ per milliliter was used to deliver the 640 μ% 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). Ali 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. TABLE 7 - Immunization Schedule Amount of gpl60 Administered (μ$)

Day 0 30 60 120 150 190 Schsduls-A Group l 40 40 40 Group 3 160 160 160 Group 5 640 640 640 Group 2 40 40 40 160 160 160 Group 4 160 160 160 640 640 640 Group 6 640 640 640 640 640 640 29 LV 10492 C. Assessment of Safetv and Toxicitv

Each volunteer was interviewed and examined on days 0, l, 2, 3, 15 and 30 after each injection. Volunteers were queried concerning fever, chills, nausea, vomiting, 5 arthralgia (painful joints), myalgia (muscular pain), malaise, urticaria (hives), vrheezing, dizziness, or head-ache. Examinations to asse9s local reactions at the site of injection included erythema, svelling, itching, pain and tenderness, skin discoloration, skin breakdown, change in 10 regional lymphadenopathy, change in function of the injected extremity, and subcutaneous nodule formation at the site of injection. Monthly complete blood counts, serum chemis-tries, coagulation profilē and urine analysis were also assessed. 15 la vitro cellular immune function was assessed by T-cell phenotyping (total lymphocyte, CD4 and CD8 celi phenotypes) as described in Rickman, et al., Clinical Immuno. 52; 85-95, 1989; Birx, et al., jL. Accruir. Immune Defic. Syndr. 4; 188-196, 1991). T-cell proliferative 20 response to mitogens (pokeweed and Con A) and control antigens {Candida albicans and tetanus) was also evaluated. Birx et al, supra. In vivo cellular immune function was assessed by delayed hypersensitivity skin testing to control antigens (i.e.. mumps, tetanus toxoid, Candida albicans and 25 trichophyton).

Ouantitative virai cultures of peripheral blood mononuclear celis (PBMC) and plasma were assessed as described in Burke, et al., it Accruir. Immune Defic. Syndr. 2: 1159-1167, 1991. DNA polymerase chain reaction (Hages, 30 et al., iL_ Med. Virol. 33: 58-63, 1991) and serum p24 antigen Ievels were assessed to monitor ifl vivo HIV virai load.

No evidence of systemic toxicity was observed, but local reactogenicity was noted in 87 percent of the subjects 35 (13 in each vaccination group). Local reactions included induration, tenderness, and transient subcutaneous -nodule formation at the injection site; an increase in regional adenopathy was rarely noted. No subject refused a booster 30 \ injection. No difference in the frequency of local reac-tions was observed for primary immunization, booster injection, or dosage.

No evidence of adverse -effects on the immune system was demonstrated as measured in vitro by mitogen and antigen specific proliferative responses, in vivo by delayed hypersensitivity skin testing responses, or by acceleration of guantitative CD4 celi depletion. Baseline mean CD4 celi counts were 716 and 605 for vaccine responders and non-responders, respectively. Mean CD4 celi counts from study days 180-240 were 714 and 561, for vaccine responders and non-responders, respectively. During the course of the 240-day trial, the net change in mean 034 celi counts for vaccine responders was a minus 0.2 percent, while among vaccine non-responders the mean 034 celi count declined by 7.3 percent (Figurē 11). Vaccine induced HIV immunogenicity was not associated with evidence of accelerated 034 decline in any individual subject throughout the entire course of the trial.

To assess the possibility of increased HIV replication and virai load in subjects as a consequence of vaccination, in vivo virai activity was measured by guantitative plasma and PBMC virai culturēs, PBMC DNA polymerase Chain reaction, and serum Ievels 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. D. Assessment of Immunooenicitv

Antibodies directed against whole HIV proteīns were measured using both recombinant produced virai gene products gpl60, p66, p24 and whole virai lysate of prototype HIV strain MN. Dot blot and Nestem Blot techniques were used, as described in Toubin, et al., Proc. Nati. Acad... Sci. USA 7£: 4350-4354 (1979) . Antibody responses to specific envelope epitopes were also measured (see Fig. 7).

In Fig. 7 epitopes 88 (amino acids 88-98 in gpl20) and 448C (amino acids 448-514 in gpl20) were selected 31 LV 10492 because antibody directed against these reģions of gpi20 are reported to correlate with early stage HIV infection.

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 natūrai HIV infection. Additional epitopes investigated included 49 (amino acids 49-128); and 342 (amino acids 342-405).

In Fig. 7, a shaded box signifies a documented change in the HIV envelope-directed immune response. Shaded boxes with (-) signify a primary humoral response; shaded boxes with (+) signify a secondary humoral response; (-) signifies antibody negative to specific epitope pre and post immunization; and a (+) signifies antibody positive to specific epitope pre and post immunization, but Vithout a quantitative change. Shaded boxes with (.) signify new T-cell proliferative response to gpl60 following immunization. A (.) alone signifies no cellular response to gpl60; while hb signifies "high background" (not interpretable); and nd signifies "not done.”

Neutralization activity was measured against three prototype isolates (HIV-IIIB, RF and MN) in a syncytium inhibition assay as described in Nara, Nature. 333 :469-470 (1988). HIV specific cellular responses were measured by known lymphocyte proliferation assay technigues using gpi60, p24 and baculoviral expression system control protein (Birx, supra) . 32 E. Vaccine Responder9 and Non-Responders

Subjects were classified as vaccine responders onlv if a reproducible selective increase of both a cellular and humoral immune response against HIV envelope specific epitopes were associated with the vaccination series (Fig. 7). 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.’ Subjects who developed neither a humoral nor a cellular proliferative response or who developed only a humoral or only a cellular proliferative response to gpl60 epitopes or HIV envelope were classified as non-responders. F. Vaccine Induced Humoral Responses

Referring to Fig. 7, 19 of the 30 subjects (63 percent) demonstrated a vaccine induced augmentation of both. gpl60 HIV specific humoral and a cellular immune responses. Th'ese 19 were classified as "vaccine responders". Four of the 11 "non-responders" developed only a humoral or a cellular immune response. Ali 7 subjects who failed to demonstrate any detectable vaccine induced response received only 3 doses (Schedule A). No changes in antibody binding to HIV polymerase (p66), or structural (p24) gene products or the non-HIV control antigen tetanus were detected. No anti-baculoviral Lepidopteran celi control protein antibody developed in any subject.

Increases in envelope antibody (gpl60) were detected in 13 subjects by Western Slot using the whole virus lysate HIV-MN. The changes were related to the immunization schedule. Three of 15 subjects (20 percent) on Schedule A, and 10 of 15 subjects (67 percent). Schedule B 9 This definition of a vaccine responder is highly restrictive in light of the scientific objectives of this trial: e.g.. to assess the feasibility of post-infection immunization. 33 LV 10492 developed an antibody increase to envelope proteins (P=0.025 by Fisher's exact tēst, two-tailed). Ali 13 subjects also seroconverted to specific envelope epitopes.

Coņversely, of the 10 subjects who failed to 5 seroconvert to any envelope specific epitope, none exhibited an increase in envelope antibody by Western Blot. The remaining 7 subjects who seroconverted to specific envelope epitopes demonstrated no change in whole virus envelope antibody by Western Blot. No changes in antibody directed 10 against non envelope HIV proteins were observed in any subject.

Fourteen of 15 subjects (93 percent) on Schedule B (6 doses) demonstrated an increase in total gpl60 anti-body, as opposed to only 7 of 15 subjects (47 percent) on 15 Schedule A (3 doses) (P-0.01 Fisher's, two-tailed). (Fig. 7) .

As shown in Fig. 8, the pre-immnnization to post-vaccination prevalence of each gpl60 specific epitope respectively was as follows: Epitope 49 (27 to 70 percent), 20 Epitope 88 (28 to 52 percent) , Epitope 106 (50 to 87 percent) , Epitope 214 (0 to 14 percent), Epitope 254 (0 to 13 percent), Epitope 300 (47 to 77 percent), Epitope 308 (42 to 69 percent), Epitope 342 (0 to 27 percent), Epitope 422 (3 to 10 percent), Epitope 448C (73 to 87 percent), and 25 Epitope 735 (17 to 33 percent). Vaccine induced seroconversion was noted against ali of the specific epitopes except 582 (Fig. 7). Antibodies (seroconversion) directed against Epitopes 241, 254 or 342 were only detected folloving vaccination. 30 Secondary immune responses were detected to the following epitopes: 88, 106, 300, 448C, and 582. The prevalence of antibody directed against epitope 582 was 100 percent pre-vaccination and only one subject (3 percent) demonstrated a secondary immune response.

The pattern of vaccine induced HIV antibody to envelope epitopes was variable (Fig. 7). Primary antibody responses (seroconversion) to at least one epitope occurred in~ 20 subjects; 14 of 15 receiving Schedule B, and 6 of 15 35 34 randomized to Schedule A (P*0.005 Fisher's, two-tailed) . Schedule A subjects seroconverted to only 15 of 110 (14 percent) of the potential epitopes to which they had no preimmunizat’ion antibodies. Schedule B subjects seroconverted to 60 of 129 (47 percent) (P<0.0001 Fisher's, two-tailed). Seroconversion to three or more envelope epitopes occurred in 9 subjects (60 percent) randomized to

Schedule B but only 2 subjects (13 percent) randomized to

Schedule A (P=0.02 Fisher's, two-tailed).

Serum neutralization activity against three distinct strains (HIV-IIIB, MN, and RF) 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 demonstrated an increased ability to inhibit syncytium formation compared to non-responders. G. Vaccine Induced. Cellular Responses

Changes in cellular immune response were based on a comparison of mean pre-vaccination (baseline) and post-vaccination lymphocyte stimulation indices (LSI) using a Wilcoxon rank sum tēst.

Twenty-one of 30 subjects (70 percent) developed a new T celi proliferative response to gpl60 post-immuniza-tion (Fig. 7) .

Figurē 9 illustrates proliferative responses to gpi60, p24 and a baculovirus control protein in four typical vaccine responders over time. For ali subjects 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) . In contrast, 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 ali subjects, for subjects subgrouped by vaccine responsiveness,

I and for subjects grouped by immunization schedule are illus-trated in Figurē 10. 35 LV 10492

The change in proliferative response to gpl60 was significantly different between vaccine responders and non-responders (<0.001, Wilcoxon, one tailed). The gplSO proliferation responses induced by Schedule B (6 doses) were · greater than those induced by Schedule A (3 doses) (P<0.10, Wilcoxon, one tailed).

Nineteen of the 21 subjects who developed proliferative responses to gpl60 also developed a humoral response (vaccine responders). The maximum mean lymphocyte stimula-tion index (LSI) to gpi60 observed for ali vaccine responders was 50.1. However, each vaccine responder's response was variable (peak values ranging from a LSI of 3 to 171) (Fig. 7), as was the temporal relationship to vaccinatioh of the magnitude and duration of the cellular responses to gpl60 (Figurē 9). H. Discussion of Results

Despite the limited sample size of this trial, several factors were demonstrated to be associated with vaccine immunogenicity. Six of 15 (40 percent) of the subjects on Schedule A versus 13 of 15 (87 percent) of the subjects on Schedule B were vaccine responders (P-0.02 Fisher's, two-tailed) (Fig. 7). Of the 16 subjects with a mean baseline 004 count greater than 600 per milliliter, 13 (81 percent) were vaccine responders, as opposed to 6 of 14 (43 percent) subjects whose mean entry CD4 count was less than 600 celis per milliliter (P-0.07 Fisher's, two-tailed).

As summarized in Table 8, multiple immunizations improved immunogenicity, even among patients with baseline 004 counts less than 600 celis per milliliter. For example, 5 of 6 subjects on Schedule B (6 injections) were vaccine responders as compared to only 1 of 8 who received the 3 injection regimen (Schedule A) P-0.03 Fisher's, two-tailed) (Table 8). 36 \ ΤΑΒΙιΕ 8 GP 160 Vaccine Immune Responeiveneas by Baseline CD4 Count and Immuni2ation Schedule C04 Count Ņ # Responders (%) # Non Responders (%)

SCHEDULE *A >600 7 5 (71%) 2 (29%) 500-600 5 1 (20%) 4 (80%) <500 3 0 (0%) 3 (100%) Subtotal 15 6 (40%) 9 (60%) SCHEDULE B >600 9 8 (89%) 1 (11%) 500-600 2 2 (100%) 0 (0%) <500 4 3 (75%) 1 (25%) Subtotal 15 13 (87%) 2 (13%) TOTAL 30 19 (63%) 11 (37%)

The therapeutic use of vaccines was introduced by Pasteur in the I9th century for the treatment of acute 30 raJbies infection. But the utility of this approach in the treatment of other infections has not been extensively explored. Although there are other examples of post infection modification of virai-specific immunity (such as after hepatitis A or B exposure), there are no well'docu-35 mented studies in man which demonstrate the feasibility of this approach for an established or chronic virai infection.

Here, the invention provides virus-specific immune modification by active immunization after infection. Specifically, an HIV envelope gene derived gpl60 vaccine 40 augmented the human host directed virai-specific humoral and cellular responses in 19 of 30 early HIV infected persons.

This study qualitatively and quantitatively measured distinct antibody responses to specific HIV epitopes in natūrai infection versus post infection immuni-45 zation. In this way, an accurate detemination of vaccine induced humoral immunogenicity in already infected persons was documented in 70 percent of the subjects. For example, twenty subjects (19 vaccine responders and 1 vaccine non-responder) seroconverted to specific envelope epitopes. 37 LV 10492

Seroconversion associated only with vaccination (epitopes 241, 254, and 342) occurred in 10 subjects.

Additionally, variations in humoral responses to this vaccine,. as characterized by epitope mapping, will permit prospective cause and effect analysis of specific aritibody responses, and allow unique opportunities to characterize potential immunoregulatory mechanisms not elicited during a natūrai infection.

Although the in vivo relevance of serum neutraliz-ing activity is presently unknovn, the observation of increased neutralizing activity against disparate HIV strains (IIIB, RF, MN) in 4 of 5 vaccine responders suggests that post-infection immunization induced changes in func-tional antibodies. The tēst vaccine induced increases in serum neutralization capacity against distinct HIV strains and will potentially aid in the definition of group specific neutralization epitopes. A proliferative response to HIV envelope proteins rarely occurs in natūrai 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. One possibility is that the new proliferative response may be directed against an envelope epitope(s) unique to the vaccine (as a result of vaccine production methodology or altemative in vivo antigen Processing) . Altematively, the protein used in the proliferation assay may not stimulate primary T-cell proliferative responses against homologous "wild type" envelopes of natūrai vīrus. However, additional evidence that vaccination boosts the host cellular irranune response has been obtained: selected vaccine responders demonstrated HIV-IIIB type-specific cytotoxic T-cell responses following booster immunization.

The factors responsible for vaccine immunoresponsiveness in HIV infected persons remain to be clarified. Even in early HIV infection, individuāls respond suboptimally to a variety of vaccines as compared to matched Controls. This hyporesponsiveness has been related to early 38 B celi dysregulation and T-cell dysfunction. Here, vaccine immunoresponsiveness was associated with baseline 034 celi count, which ia consistent with the hypothesis that the immunological status of the host is an important determinant 5 of vaccine responsiveness. However, the immunization schedule within specific T-cell count intervāls aleo influenced vaccine responsiveness: Schedule B (6 injec- tions) was superior. Indeed, the decreased vaccine response observed in the subjects with lower CD4 celi counts could be 10 improved by an increased number of vaccinations which suggests that further modifications in dosage, regimen, adjuvants or formulation, could be anticipated to further improve host immunoresponsiveness.

Although concerns have been raised about the 15 safety of active immunization of HIV infected persons with KIV specific vaccine products, there was no evidence of immune-specific toxicity. Quantitative cultures, DNA polymerase chain reaction assays and serum antigen assays show an increased ifl vivo HIV load An excellent ifl vivo 20 marker of HIV replication, the rāte of CD4 celi decline, was favorably influenced arnong the subjects, especially those classified as vaccine responders. The change in mean 0)4 counts for responders was -0.2 percent and was -7.3 percent for non-responders. The data demonstrates that post-25 infection immune responsiveness was not associated with an increase in 0)4 destruction and suggests an association with decreased HIV replication in vivo.

The vaccination results in this study were also compared with a database of ten infected and untreated 30 individuāls matched for age, ethnic group, and baseline 0)4 celi count. The mean 0)4 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. These results 35 indicate that post-infection vaccination with recombinant HIV envelope protein is feasible, and furthermore the jresult are encouraging with respect to the prophylactic uses of such vaccines.

Claims (23)

Use of a recombinant HIV envelope protein for the manufacture of a medicament for the treatment of HIV-induced illnesses. 2. The use of claim 1, wherein said medicament comprises administering from 1 to 100 mcg of said protein per 1 kg of body weight. 3. The use of claim 1, wherein said medicament comprises administering from 10 to 4000 mcg of said protein per 1 kg of body weight. 4. The method of claim 1, wherein said medicament comprises administering 40 to 1280 mcg of said protein per kg body weight. 5. The use according to claim 3 for administering at least three doses of said medicament. 6. The use according to claim 3 for administering at least six doses of said medicament. 1 Use according to claim 5, wherein said medicament is administered at a interval of 30 to 60 days. 8. The method of claim 6, wherein said medicament is administered at a interval of 30 to 60 days. Use of recombinant HIV envelope protein for the manufacture of a medicament for treating HIV infected people by administering said protein in an amount that promotes an increase in HIV-specific cells and humoral immune responses. The use of claims 1, 3 or 5 for the use of said protein having a mass of about 145,000. Use according to claims 1, 3 or 5 for use as said protein in at least one of the proteins gp160, gp120 or gp41. Use according to claims 1, 3 or 5, for use as a protein of the baculovirus vector Ac3046 expressed in said protein insect cells. 13. The use of claims 1, 3 or 5, wherein said protein is in association with at least 2,000,000 molecular weights. The use of claims 1, 3 or 5, wherein said protein is included in the composition with excipients. 15. Use of Recombinant HIV Coating Protein for the manufacture of a medicament for treating HIV-infected people by administering said protein bound to the alum in associated bodies with a molecular weight of at least about 2,000,000. Use according to claims 1, 3, 5 or 15, which provides the production of said protein in a baculovirus-insect cell expression system. Use according to claim 15, wherein said protein is secreted from a fraction consisting of recombinant proteins gp160, gp120 and gp41, said protein having a molecular weight of 145,000 and expressed in vector Ac3046. The use according to claim 15, wherein said protein having about 757 consecutive gp160 amino acids is used, completely disabling about 40 consecutive gp160 terminal amino acids. Use according to claim 15, wherein said protein is administered at a dose of 1 to 4000 mcg. 20. A therapeutic HIV vaccine comprising the recombinant protein of the HIV envelope associated with the alum, wherein the recombinant protein forms a body of at least 2,000,000 molecular weight. 21. A composition according to claim 20, wherein the recombinant protein of the HIV envelope is administered at a dose of 10 to 4000 mcg at one time. 22. A composition according to claim 21, wherein the recombinant protein is produced in a baculovirus-cell expression system. 23. The composition of claim 21, wherein the recombinant protein comprises about 757 consecutive amino acids of the gp160, gp160 peptide having truncated amino acids from about 757 residues, and having at least 40 terminal gp160 amino acids. 3