BG97519A - METHOD FOR PREPARING A HUMAN IMMUNE FAILURE VACCINE FOR A VACCINE - Google Patents

Abstract

The acquired immunodeficiency syndrome (AIDS) vaccine contains human immunodeficiency virus type-1 (niv-1) surface proteins produced by cloning niv-1 surface genes from a vector system of baculovirus infected insect cells. Recombinant niv-1 proteins are purified, pooled, and then adsorbed onto aluminum phosphate adjuvant. The resulting form of an adsorbed recombinant niv-1 viral surface protein (HIV vaccine) is highly immunogenic in animals and secretes antibodies that bind to the niv-1 viral envelope and neutralizes the infectivity of the virus in vitro tests. Said spinal vaccine induces a novel immune and cellular immune response in infected patients and is useful in vaccine therapy to delay or prevent immune system disruption. 1 claim

Description

METHOD FOR ADULT COMPOSITION HA HIV VACCINE

This application is, in part, a continuation of US patent series $ 151,976, registered February, 3,1988, IP series 920,197 register October 16, 1986 / now series »585,266 / .These registrations and the summaries cited herein are included · in the abstracts.

DETAILS OF THE INVENTION

Human immunodeficiency virus type-1 (HIV-1) is a retrovirus that causes infection of the whole body with major pathology in the immune system and is the etiologic agent responsible for Acquired Immune Deficiency Syndrome / ΑΙΏ57. OfaZZC-SiHOUSti et al., 3j / e / IC is 220: 868-871 (1983); ROCHC et al., SCiFMCC 224: 497-500 (1984). Clinically isolated HIV-1 refers to the lymphadenopathy-related virus / Τε, ΟΖΙήΟ et al., Is / € WC 225: 69-72 (1984) and AIDS? -related virus et al.,

J6 / £> W225: 840-842 / 1984 /.

AIDS 'has become a pandemic disease and the development of vaccines has become a top priority for the health of people around the world. The at-risk percentage of people infected with HR-1 indicates a progressive loss of immune function due to depletion of lymphocytes. These cells, like certain nerve cells, have a surface molecule called CP 4. H1V-1 recognizes the CP 4 molecule by a receptor rolling on the envelope of the cell.

the blotches that penetrate these cells / finally / possibly / recycle them > kills. It is expected the objective - '-'. Ι T. j. kJCxiiIjjlcl ί- le /, invokes formation fc “· Lt / antibodies, sieve to be connect is the shell ya d! · · 1 * ·. to go ^- they shudder ecting Ια Τ _Δ Lead * Ocytes or <ια other malleable cells. Usually All the same are given to, h, .good people before

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*. : .ν::. ·? $ '$' ·>

• ^; ·· · * ♦.! - '· ·' · '··· ·' · · «... · · · ·.: Ί '· ··''··'. · · · · · ·

administer to patients, such as immune prophylaxis. However, it is logical to consider the use of an effective AIDS vaccine after being immunized as immunotherapy against the disease. S / M; y (/ _akaszgl: 473-476 / 1987 /.

'Piroko is widely believed that the ΗΓΖ-1 shell is the most promising ingredient in the creation of the AIDS vaccine.

fac-d / Ucot. _f 1566-1559 (1985);

Vogt * yb'tsh .Rinws In Jnf, Msh ·. 991-1000 No. & oijauti _t : 9276-9283. The HIV-1 envelope protein was initially synthesized as a glycoprotein with a molecular weight of 160,000 (gp 160). The percussion rn 160 cleaves to an external glycoprotein

- with a molecular weight of 120,000 (gp 120) and transmembrane glycoprotein with a molecular weight of 41,000 (gp 41). These envelope proteins are the major antigens for antibodies in AIDS patients. et al., Sg.iCWCG ', 228: 1094-1096 (1985). Native HIV-1 rn 120 has been shown to be immunogenic and capable of causing antibody neutralization in rodents, goats, rhesus monkeys and chimpanzees. ν / 1986 /.

, tteact 06 /. Ute 63: 7 ^ 23-7927

Due lnogo low levels of native H; -1 envelope protein n infected cells and the risks associated with the preparation ia CI ⁷ '' vaccine cm PG'-1 infected cells are used reksmbinantni D OF: -zts _and .and manufacturing know 7ΙΥ- 1 coated antigens in the production of SEP1 vaccines. Pekombiyaantnata D technology zvodstvoti ria AIDS vaccine poua _l .and uh ~. z. ly * io st and c <pluchshshaie me. naked, are quantities safe

u.Nogesh. n1 ^t . ^: “1 Flushing Protein C :: :: 10 Genetically Changing Recombinant Cow Disease Virus? Chdtkzc ^^^ str. , / ¼ ZNO: 535 - 537 (1986);

·· ···· // U, h et al., FatuZO, 320: 533-530 (1966); , et al, her / 790-795 / 1966 /. The envelope protein is also present

drawn from an amino acid sequence in HIV * 1 gp41 is also considered γΒ | AIDS vaccine has not been successfully produced to date using these materials and methods. The use of baculovirus with an insect cell carrier ι ₄ κ 1.31c> recombinant HIV-1 envelope protein is the only aspect of the invention disclosed in U.S. Patent No. 920,197, filed October 16, 2008. 1966 / now series No. 585,266 /. See also

C. nt is, / .. baculovirus has been shown to be most useful in the production of HIV-1 protein and other proteins. For example,

used as a carrier for expressing the full length rn 160 and varying amounts of HIV-1 envelope gene in infected cells of

pkzQ. 9 cells). The annexes to the foregoing patent also disclose the stem gay gp 160 (recombinant $ 1.80 Ac 3046), a protein produced recombinant Ac 3046, and a purified technique for the Ac3046 gene product, which includes a lens-like drug,

tinafin chromatography and gel filtration chromatography. In this way, the rn160 protein is purified and aggregated in the form of particles that are more immunogenic in rodents and primates.

The ideal AIDS vaccine, in addition to the requirements, for substantially biologically pure and non-pyrogenic, provides continued A so-called protection against HIV-1 infection after one or more injections. This is common in cases with an attenuated vaccine. When

• · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

- 4 ;, p. Ixsriums or viruses, or materials isolated from them, such as proteins or proteins, are used to produce the vaccine, the f'ccuo result is a poor antibody response and only short-lived immunity. - avoid or reduce these disadvantages of waxy cabbage .. one additional ingredient called dcalate may be added. Adjuncts are materials that help stimulate the lightning response. Adjunctives commonly used in Cho; .e - g vaccines are gels of aluminum salts / aluminum phosphate or

-7 ', 1 < RTI ID = 0.0 > hie &lt; / RTI &gt; oxide /, commonly used as an adjuvant, alum, and str., ^ Al ^ 1 (/.

^'T astoyatssto invention provides a vaccine and treatment methods chogesheiya and nulodedaiten virus / NG7 comprising administering pa ^ okolbinaateya NEW envelope protein on infected or susceptible to being individuals. D preferred inclusions, the envelope protein .. can be then purified, aggregated and combined with adjuvant / e.g. alum / for vaccine use.

ΙΤΛΊΗ0 DRAWINGS OF THE DRAWINGS

Details of this invention are summarized in the drawings below.

tour. 1 illustrates the cloning strategy used for isoH.ya.ie of NG ^m -1 envelope gene / os / cm E. colt plasmid plMAg. For the IIrihos'sts regions are the HIV-1 D MA sequence and the open regions are clonal mRNA carriers, the first region in plasmid p1774 was created from synthetic oligonucleotides and introduced as $ Weiss & kpvl / ragmelt in $ w (XI - Kp Plasmid p1614 shows the sequence of this synthetic nucleotide.

pura 2 illustrates the strategy used to prepare a recombinant plasma carrier / p304o / that is sequentially used

• · · · <<: creating the recombinant baculovirus expressing the carrier ·

AoU46. Plasmid ρΜθΓ3 contains a sequence / fields with crossed ',. ^ Ιχπ / cm baculovirus AcMPV on both sides of the cloning of the meat-' <, · | then on. position of h.o. This site is the only restriction en- -1 -.- / - I dinuclear site 3aSwOl, ΚρπΙ and Belli. The AcMPV polyhedrin activator is in a 5 * direction of 4.00 position. The series GR and- ΑΑΊΤΑΑΊΊΆΑ-3 is in 3 strand and has a terminal retransmission '

... * <. V ..... · H and all three read structures. The gulasmide p1B4 and the synthetic oligonucleotide sequence are described in FIG. 1. The plasmid p3046 contains all of pM @ S3 except the sequence between / gH] v, Ffyl II, where the HIV-1 envelope gene of p1774.J is inserted;

'. g. ya 3 shows the nucleotide sequence of t) MA,;

(bordering Ac3046 rp160 in code sequence. In Fig. 4 with a shown 3046 collapsed DMA sequence between +1 and +2264., * games 4a-4k show the current I PA sequence -. persecuted segment further cover plate of synthetic oli- ¹ 'gonukleotidna series of B'nai embedding gene in As3046 / between 2' - I + 1 and + 2264 /. Localization restrictive endonuklearni <

.-sols are registered above the I) MA sequence and suggest-;

; i sleep a .inacid sequence .is registered above the DMA sequence. Poses are numbered from left and right. · -J | jp3

Figures 5a-6oL compares the DMA sequence of the 1 '' j αο3Ύ6 envelope gene with the published IAV-1 envelope gene sequence.;.

The · -'- Cr I LA-1 sequence is from above and Ac3046 is from below. The line / 1 / -. · ..., .3 under the 1AV-1 sequence shows that the sequence in Ac3O46 is the same in this position. The numbering of the IAA IIIA sequence is the same, .lX. | as described by Wcdti L &lt; 3 &gt; et al., 40: 9-17 for LAV-1.

Figure 6 shows the end point of the solubility titres; method of human HIV-1 antibody positive serum / IF ^ GYa gr & fic · · · · · · · · · · · · · · · · · · · · · · · · ·· ·· ·· ·· k ccp.v ,; of rhesus monkeys / lower graph / from animals immunized with rp 160 HE / 55, KL 55 / or rp120 / AB 55,00 55, bH55 /. There are W $ L titles. measured against highly purified rp120 and rp160 The specific bound antibody was measured with goat anti-human ma, which gave a positive response in the test.

Figure 7 is a table summarizing the vaccine GP160-elicited immune response in vaccinated seropositive patients.

Figure P / A and B / shows a vaccine-evoked antibody response directed against specific HIV envelope epitopes.

Figure 9 shows a vaccine eliciting a T-cell proliferative response to rp160 in vaccinated seropositive patients. - y

Figure 10 (A-C) shows lymphocyte proliferative response - * '1 •. . . ·, * Associated with vaccination.

Figure 11 is a graph showing the percentage change in CD 4 cells reacting or reacting excessively. 'F' Ί

Recombinant HIV-1 rp160 envelope protein ("rrp 160") has been found to be particularly adsorbed on an adjuvant such as alum. aluminum phosphate / is especially useful as a spin vaccine. 3in aspect of this invention is an Ac & PV carrier having the code sequence for a portion of the HIV-1 envelope gene, which

it surrounds amino acids 1-757 found in recombinant <clone 3046. Another aspect of the invention is the production of

this recombinant HIV-1 enveloping irotex (and the protein itself) in insect cells - especially prp 160 encoded by amino acids®. <* · And sequence 1-757 / 03046 /. :

Other aspects of the present invention include purification and recombinant envelope protein particles / g. the product ia the recombinant baculonirus that produces • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

445 protein and adsorption of 3046 particles to aggregate on alumina ZISh 1 I, with _X <DG.

The invention also includes prophylactic and / or therapeutic vaccines for υΓίΙΙ and whether HIV infections and methods for preventing or treating LAG1 or HIV infection.

πς'Τ'ΈΗΟ DESCRIPTION AT I30PREGSHIE0

The following examples illustrate the invention without limiting its scope.

Recombinant polycha baculovirus, a cutaneous virus (Asyapv), which contains the HIV-1 stem gp 160 gene κο ,, ρραίΐ for amino acids 1-757 of the HIV envelope protein / pe1, with binary Ac304 &lt; / RTI &gt; No. 920,197 (now Serial No. 585,260). The cloned steps used to construct the recombinant baculovirus-containing genius or parts of HIV-1® genes are also shown here and included in the summary,

The following is a full description of the genius engineering stages they use

Hz. To create ya Ac3046 and a carrier. Materials used incl / v _V C

commercially available immunological reagents and immunological reagents. There are also examples showing how to make and use the invention.

Other recombinant envelope proteins, referring to but as prp 160, are also contemplated to include recombinant <RTI ID = 0.0> rp 120 </RTI> rp 41. Ac 46 is a single, zinc primer and recombinant coat protein according to the invention.

Baccalaureate Recombinant Ac 3046 bearing the NG-1 encoded sequence for amino acids 1-757

4ic pound five and the expression "a foreign protein-encoded take •, the thoroughness of the Sakulvirus carrier requires the alignment of co-I • ripr-tiai and the sequence is the aolihydrin activator and up in the row cm is _A on the side and with baculovirus encoded-sequentially .. ovra stands.

7 The alignment is such that homologous recombination with the baculovirus genome results in the transfer of a dummy-encoded sequence aligned with the polyhydric activator and the inactive polyhydrin gene.

Numerous insertable carriers for the use of bHIV gene constructs have been created. The embedded MS $ 3 media described below is designed to support ATG retransmission and initial information. The insertion of a foreign sequence into this medium must be three-folded such that the retransmitted information is a structure established by the original.

The elongated fragment

CLO.H3 supports properly carrier MG $ 3 is constructed from Eco ^ I-DNA DNA-limiting isolated from purified ι-solvated Ac '/. NPV ixv.a following structural features: a / 4000βρ from through the strand.

at the top of the row rin? b / Polylinker inserted

ATG initiation .. a polyhedd gene extension by side of a conductive mutagenesis, which consists of an ATG initiation codon at the corresponding position, and restriction sites Ι, ΚρπΙ, stopxdon segment; c / 1700 bp from the restriction site sequence / which is internally located on the EcoRI limited occupancy site / a.ScoRIST POLYCHES ^ AND! codon Fvt II and universal lining from ΚρηΙ lhhedrn

Ixxxkh

And a clone.

the gene /

t.

* ίΣ * · · through

Creating Bachelor Recruits iOCCU-'x Uw \ ^t Wrap Encoders.txt Series

Recombinant plasmid designated MA2 segment of whole '-1IV-1 provirus inserted into

Fr.

become contagious when it produces the virus following !, and in certain human cells.

59: 254-291 (1986). '' yalata Gaia ebvivaed series NA2 propohzzhda of LA ^'r vyD of h;

contains'

Ί7. baiU-SiUMMi / 1 / C /.

q . -. · -. ' - ·, ··· <. ' .. .. · .. ·· - ·· · · · · · · · · · · · - · • · «· ♦ ·. . • · · · · · · · ♦ -4 · ·· ···? :. Ο * ··. ' οΐ »· \ 1 1 '- - » ...... · · · · · · · ·· · ·

of lyran kkv NA2 as 3846 bp EcoPI / N acI restriction fragment and cloned into the 03oRI / N acI restriction side | The resulting plasmid is designated p7O8.

The envelope gene was subsequently isolated again as the 2800 6p рρπΙ restriction fragment and cloned into the ΚρπΙ restriction side of pUCIS. The resulting clone is designated ρ1614.

ΚρπΙ the restriction fragment in p1614 contains a light stem particle of IG ^; gay envelope such that 121bp of the M-terminal corresponding sequence is missing. This missing particle in the gene that includes the signaling peptide sequence is replaced by inserting a double stranded synthetic oligomer. The inserted oligomer was created from the IAV amino acid sequence using a preferred Gulichin gene codon practice. To facilitate further manipulation, a new SmA1 restriction sequence was inserted in place of the ATG original xdoi. The ATG initial 'g' is depleted of the baculovirus carrier. The resulting plasmid was designated as p774.

According to Figure 2, there are also restricted p1774 bands containing a x-ray sequence from different regions

are; spirants of / ss & zhs / zhs / so that ATG nCp-jC.i K. & lnzak 1se'GIA LOSmPbL Cl'pj ivl ^ pciTc ·. With ccd ^ c ict ln obBiBl /.^ ia i'C ι. yΊ'ρυβ.ij / ijiT ό 'jbC'luH li SH L-T'jci 1 * -L - :. <iVshC · Ί € -' iT isolated from ^ 1774 in ^ TKx.ai in Srnal / kKxxKi ΕψΊΙ country of h .. <. i '.' the next carrier p / GSd, Tzze maple contains ι.ordica ks .._, prana for ί * '. sour 1-70 / la gp13C · and uses a terminal cedar το ί from 'G53 loo / ι fused.

r ''

HIV enveloping gel river yuyanten 'ı and with a patch of 3/4 6 with alo, and b splashed / tyrant with

.added to nso. wishes! hb ι

χ ·, ν '“ _t >' * 'Π ee inserted into the AcMMRT genome through a homologue © combination. Recombinant viruses were identified by negative exclusion of the mma ''''' ¹ * <' · '''* * ¹ * foil plaque. Such plaques exhibit an identifying cytopathic effect but no nuclear occlusion. Two additional sequences of plaque purification were performed to produce pure recombinant virus. Recombinant viral. DNA was analyzed for side-specific insertion of the HIV envelope sequence by comparing their restriction and hybridization characteristics / with the broad type of viral HA.

• ADF 4

Expression of HIV envelope by recombinant baculoviruses in infected cells is not insects.

Expression of the HIV envelope sequence of recombinant viruses in insect cells would result in synthesis of-. that of the primary trach-slice product. This initial product consists of codon-translated amino acids substituted by the recombinant carrier. The result is a protein containing all amino acids encoded by the Α expression codon | the gray vector at the bottom of the determining signal on the expression vector / carrier / -Hirg! prn 160. The primary ¹ trachslatsionen product As3046 must decipher Mei- Pt, o- $ iy- - ^bw and £ of the terminal where Ax ^. / position4 / s Atg, position 2 is the original IAV clone. Met- Ρνο-β / y codons are replaced by the cloning strategy.

EXAMPLE 5

Nucleotide sequence of glycoprotein / gp / 160 µm chat and flanking DMA.

The nucleotide sequence of rp160 inserted and flakable HA was determined by restriction fragments isolated from the viral expression carrier Ac3046 US. A coherent strategy involves the following steps. The 3.9 ks EccKV-ButpHI fragment was purified

by limiting the dissolution of Ac3O46 viral DNA. The AC304b viral DNA is prepared from an extracellular virus present in the medium of cells used to produce a group of vaccines.

As shown in Figure 2, 3.9, the EcoPV-Ba HI fragment consists of the entire rp160 gene and 100 µr from the top and _; about 1000 pips from the lower flank. CD: 'Thus, the nucleotide sequence of the entire rn gene was determined including ΙΟΟορ from the upper and 10Q δρ from the lower flanking HA.

Wed In short, the results of the sequence show the desired construction, as predicted by the caching strategy. The sequence of gp160 is important, as reported by fyhov ^et al., (1985). The sequence of 2253 bases between the putative initial translation and the transcriptional codons provides 751 amino acid codons and 28 potential N-linked carbohydrate sites. The calculated molecular weight of this prnlSO, including the sugar residues, is approximately 145,000.

Sequential analyzes of 200 bases of flanking ON A showed a transverse insertion, as shown in Figures 3,4,5. PRINTER 6

Amino acid sequence of rn! 60

Using a mill; automatic EcUan degradation r .'- 'PIC

procedures the N-terminal sequence is defined

the first 15 leftovers and it is the same as predicted by JNA the sequence. The N-terminal junction is not present in the gp 160. Observed is that the AeNPV polyhedron protein is a synthetic protein

derived without N-terminal hardware. Summary of actual rp150 DNA and N-terminal protein sequences have been determined

divided by analysis !: of AcNPV 3046 J) NA and purified rn! 60

/Table 1/

TABLE 1

TM ' ^Mr Messrs. RGSh ggy p ίπΛΤΡΓ twn ->pr' aviip raotpg: /-tpp.^frp/

- 12th c

? ¹ Table 1

V

ACYUT3046 expression vector / carrier /

7 8 9 10 11 12 13 <J

LAV envelope gene

The rest

3 4 „He _t , _p / ¾ t &'bb ogtste AAGefiG fifiG w sal- && ste Tc & bbb These results are compared with the original LAU41 clan cact® follows (Table 2).

IAV envelope gene

The rest

Table 2 of the original I / AV-l clone

2 3 4 No. VaC & S & / and Xj Ίνίζί / Η His Τζϋ аa j> p £ &

ffn $ fiSA- SVG ftfiC QAC M & iXTCfiQ C-ASTG6 T&S fi $ -T <S6 && G

7 8 9 10 11 12: 18

EXAMPLE 7

Purification of recombinant rn1bO

One aspect of the present invention is the procedure for extracting and purifying a recombinant HIV-1 envelope protein encoded for an Ac3C46 expression vector (carrier). The recombinant HIV-1 envelope protein rp150 is produced in Cells for 4-5 days after Ac304b infection. Purification of this ρππΙβΟ involves

1 · Washing

The cellular stages are as follows:

of the cells

9_ * 7 ·

LYSIS

Gel filtration chromatography

Pentyl lactin chromatography

Fr. Analysis a

1ozi example describes the purification of the recombinant gp16S cm about 2 x 1C ~ As304b; n _g .sltirani cells.

1. Cell washing. The infected pellets were washed in buffer containing 5C < 3 &gt; Trier boil / pK 7.5 /, 1 mm MH. 15 · 15 1rvn X-100. The cells are suspended in this buffer, hemogenesis?

at sa

wounds using standard methods and centrifugation at. 5QQO for 20 min. This process is repeated three times.

2. Cell lysis. The washed cells were lysed by sonication in .50 mM Tris buffer / pH 8.0-8.5 / 4% deoxycholate and beta mercaptoethanol. The communication is performed using standard methods. After sonication, only the remnants of the nuclear memorial are removed intact and removed by centrifugation at

minutes. The supernatant containing the rn160 extracts lacks whole cells, which was determined by light microscopy observations.

3. Gel filtration. Gel filtration was performed on a 5.0 x 50 cm glass column coated with ^ bhGC'tyi J fyez / htCiCftl /

The total bulk volume is about 1750 ml. In order to depyrogenate and self-colonize the column and the connecting tubes, 0.1 M KeOON is passed through the column for 24 hours. The column flow was coupled to a UV cell stream and a monitor and recorder / PhQZfytGCd & J and then equilibrated with a 4 liter gel filter buffer. The crude GP160 was added to the column and reversed with gel filtration buffer.

The column separates the crude mixture into three major 1 (7 absorbent fractions. The first peak peaks between about 500 and 700 ml. The second - between 700 and 1400 ml. And the third - between 1400 and 1900 ml. Buffer. of which the first peak is determined to be a material with a molecular weight greater than or equal to 2,000,000.

This peak is translucent due to the high molecular weight concentration of the lipid and lipid complexes. This peak also contains 10 dS 206 gp160 extracted from infected cells. Undoubtedly, this fraction of rn 160 is complex to itself or other cellular components to form a high molecular weight of aggregates.

The second large peak consists of a preponderance of gp1 theories with molecular weights between © kol © 18, (XX) and 200,000.

The third peak contains little protein and the predominant N / V absorption is due to beta mercaptin ethanol in the sample. *

When the second peak is first observed by tracing the V absorption, the column flow is applied directly to the lentil lectim column. Once the second peak has exited the column, the edema is destroyed by the lentile lectin column and disposed of as waste. '

4. Lentil lectin. Lentil is the volatile affinity gel medium

4B / was obtained in a large volume of P (alpha) CfZ Lentil lectin was isolated by affinity chromatography over more than 98% purity and then immobilized by coupling with 3e // <RTI ID = 0.0> cyan bromide. The atrix contains about 2mg of ligand per 1ml of gel. The lentil lectin column is a 5o0 x 30 cm glass column! Pg / dBG (b) containing 125 ml lentyl lectin -? 1? 4B gel. The affinity matrix is reused after being thoroughly washed

and recovered through a procedure recommended by the substitute.

When the goal is not used, it is stored in a solution of 0.9% HCl, 1mM MnCl2 _and K CaCl2, and 0.01% thymersal. The column was washed and equilibrated with 250 ml of lentyl lectin buffer, described above, before each use.

The crude rp160 was applied to the left-hand column as purified by the gel filtration column described above. Once the crude rm 160 was bound to the kg of the cloth, washed with 800 ml of lentyl lectin booster containing C.1% deoxycholate. In these long wavelengths 280> / m, the whole * rn16C binds to the column. Lentil lectin buffer plus 0.3 And alpha-methyl mannoside is used to purify the bound glycoproteins that are monitored by a monitor

5. Tlizide, Welds, and deoxycholates are removed by conventional dialysis.

The purification of mp1b0 from 1 liter of infected cells can be explained in the following table (Table 3).

In another application, conventional ion exchange chromatography (anionic or cationic) may be used in place of gel filtration. The order of the steps is not strictly limiting: for example, gel filtration or ion exchange chromatography may be after lentil lectin purification step. Other reagents may be used according to the invention. For example, other detergents may be used to purify the recombinant protein instead of deoxycholate. They include non-ionic detergents such as / and 50? 20 (Polysorbate 20), 75 ^^ 77 80, ^ / z ^^ ^and 7x / 't £> 77 X-100.

TABLE 3- SUMMARY OF Purification

Purifier floor General protein gp160 / mg / $ gp160 total Removed contacts- minants Round cell / DM /. 1-2000 / 20 1-2 Average culture 1 P Q-TO washing 250 15 6 Albumin serum, many nucleic acid soluble cellular proteins Gel filter- tion 120 12 12 lipids, nucleic acids and high molecular weight aggregates Lentil Lectin 14 1 1 1 1 1 ο 1 1 t-H 1 1 1 1 1 1 1 1 1 70 non-glycosylated proteins

: then

Dialysis sugar, deoxycholate, excess Tris buffer

EXAMPLE 8

A. Collection of particles from rn160

According to one aspect of the present invention, it is discovered that the GP160 antigen can be collected in particles with a molecular weight greater than or equal to 2,000,000 ps during purification. The glycoprotein-rn160 is extracted from the cell as a mixture of 80-90% monomer / 160,000 mol. weight / and 10-20% polymer / particles /. The gel filtration step makkk1G removes the coupled forms of rp160. Attempts to purify rp160 from this fraction (the first peak coming out of the gel filter column) suggest that it is complex with other cellular proteins, even possible with membrane fragments. However, the rp160 antigen in the second peak upon exit from the gel filter column has a molecular weight of about 160,000-300,000 and is therefore in a predominantly myiomeric or dimeric form.

the formation of aggregates or polymers of gp160 occurs during the formation of the lentyl lectin column. The a.sigenic forms were determined to aggregate either by separation from the lectin column being C, 5% deoxycholate, which is about 0.2% of my critical front concentration (QoL) for deoxycholate or rp160 dividing the knee at 0, 1F deoxycholate.

Aggregate size / age is measured at? << D12 elbows / (/ _t .p y y, _{z z} /. Samples from represented groups of purified gp16C shat size, which breaks the same or ps-large cm 2 , P? C, C0C 'molecular weight per std. Second size blue dextran.

The doctrine of cross ties is not '' / 'in. e? et al., (1969) showed that the g1O2 produced by the insect cells is a tetramer of identical composition. It also indicates that: yy: 16C in the infected CIS. ' cells Livirus particles ^are tetraperic.

Thus, recombinant dp60 particles may have tertiary and quaternary structures similar to those found in native HIV dp160.

A proper three-dimensional structure may be important for the formation of epitopes that require the correct folding of dp6O. It is likely that as non-glycosidation proteins are removed from binding to the rp160 antigen during binding and washing to the lentile lectin column, the hydrophobic portions of rp1b0 begin to form intermolecular compounds. The deoxychol is probably not bound to rn160 as the concentration can be maintained above the KMC and the antigen can still form complexes. The coupling of these antigens into aggregates is an intrinsic property of the protein once purified according to the invention. The very hydrophobic K-terminal sequence represented on rn160 may contribute to the natural possibility of. this protein to re-particle. After purification, the rp160 complexes can be sterile filtered through a 0.2 micron cellulose adetate filter without syphi-ficant protein loss.

* 5. Analysis of particle formation

Analysis of the purified dgDbO particles by electron microscopy shows that they are protein-like spherical particles of 3O-100.

^g ATs dS11T.l; L'1elen iccx presence of particle irrechis-june

P1160 was analyzed by gel filtration. About 10 micrograms of PLB ^

'.And '14. . l>

"S * /. · '4 (

PRSII. · IIJ

proteins with molecular

gsporpd · snale ia msklskulnu weight of protein standards. The forehead divides the Menomeric protein rn160 into the pellmer forms and

B <Ca I1C “GLACh ·. iiJly rs

X

gp160 strains are released in free volume and their molecular weight is greater than or equal to 2 x 10 ^ (2,000,000).

EXAMPLE 9

A. Adsorption of gn1bO to alum

The effectiveness of insoluble aluminum compounds as immunological adjuvants depends on the completeness of solid phase antigen assay. As part of the present invention it has been found that the compounds of alum can efficiently adsorb gp1bO but in pH, which will reduce the ability w ^';: w minutes of gp1b0-alum complex as an immunogen. The factors controlling the formation of this alum (aluminum phosphate gel) are:

1. The optimum pH for adsorption of the antigen from the alum is k about 5.0. However, rp 160 was found to lose its immunogenicity at pH 6.2 as compared to pH 7.5, so alum was made at pH 7.1 - 0.1. It was found that pH 100 / of rn16C was absorbed by alum! A.

2. The ionic strength of USC1 is represented relatively low and is less than C.15.

ύ. There is a saline excess ia luminium chloride o.sodium chloride to ensure the absence of free phosphate iodine.

Mi.px.Lci .. j a.1'11 Ge-tt t, .-., Οτγηοη to iipjiCiio formed alum to stop the growth of m crystals and to reduce to min ,, - u size ia particles.

/ the procedure for obtaining a lLP. art. and absorbed © pre—

Bcc. 131167 Oh ogiptsh.this is chilis, hh k ^ spnats. the concentration of the la antigen is 4 ^^ -. / 'hC, itc with the cnc from the .αθ female - ^ olu ·

G- /

F. We prepare reagents (200 nl general formulation group)

Preparation of the following solutions in 100 ml sterile, <W> yn V .MWTP ~

our pmpets or bottles. Mixing Salts for Dissolve

Solution 2 i. Sodium hydroxide and Titration over 0.2 .mu.l of acetadisphenic acid in 100 ml sterile, apyrogein vials. Solution! 'And &&. 6Η2θ 0.895 grams

..— 82 ^ 51322 -______________ — __— 0.136 grams. _

Dissolve in 40 ml water for injection (VZJ), 0.2 micron filter

Solution 2 J ^ a ^ POa ^ HgO 1.2M grams' filter __

Solution 3 Laon 2.0 grams.

Dissolve in 100 ml of ESI, 0.2 micron filter

Solution 4 Tris 1.25 grams

Dissolving in 100 ml VZI, adding 1 ml to 90 ml VZK, adjusting pH to 7.5 with 0.5 M HCl and taking 109 ml with EJ

Solution slurry for 30 minutes; slow release. Room temperature cooling.

B. The image in the ad ie on stp.a

1. Add Solution 1 (aluminum chloride-sodium acetate) to the designated vessel using 25 ml sterile pipettes. Notes

is the volume of. Solution 1 and stir. 2. Adding Solution 2 Lottery ^ cc ^ at / nm. containers, saiiki 25 ..Ήι ejernal sawdust; ndid-lie is confused as it is form a precipitate and slip away. Solution 2. ύ. addition of 3 cm Restraser (sodium hydroxide) and pro- due to stirring for 5 minutes. Ezema was 0.5; pH measure. Ax pI is ps-max · of 7.0, 0.5lb &lt; tb &gt; sodium

ί hydroxide, stirred for a further 5 minutes, and pH again. This is repeated until the pH is between 7 _m 0 and 7.2.

.

'4. Determination of total volume added to the designated vessel (Solution 1% Solution 2 -F Solution 3), then sterile BZL was added to give 100 ml volume.

5. Immediately add 8,000 micrograms of purified gp!

100 ml of 1mM Tris pH 7.5 in the designated vessel.

6. Continue stirring for. at least 20 minutes afterwards, the created vaccine is distributed into sterile ampoules.

. example ίο

Immunogenicity of alum 160 g / Specific Ab response /

An accepted method for determining the immunogenicity of an antigen preparation (vaccine) is to measure a specific antibody response in groups of mice given single doses of the antigen. At the end of 4 " ^ϊα week, the blood and serum antibody ϊζεο to a specific antigen are taken from mice (usually the antigen used to immunize the animal) is measured by a standard antibody test, e.g. SLLfA.

'The muyogenicity in mice of purified gp160 without adjuvant at pH 6.0 and pH 7.5 adsorbed® with alum (as described in Example 9) or mixed with complete adjuvant are noted below (Table 4/).

Table 4 Seroconversion The group gp160 Adjuvant gp160 E1ISA rpjne 8 / P / K / ³ IjU ^, nel, pi 7.5 £ 702 0.140 Ο 4/6 no, pHbC 8702 0.11C 26 < 2/7 Oyster 8702 1,000 qrw 9/10 Oyster £ 705 with per; 100 / 6/6 8604 1.108 (3 / 5/6

I

Mice immunized with a single dose of 1 microgram of rp1bO antigen without the addition of any adjuvant elicited an antibody response against rp160 (see table above). A differently stronger amtibody response was observed in groups of mice immunized with 1 microgram of rp160 adsorbed on alum. A single dose of less than 0.1 micrograms D1160 mixed with a bullet of 7 m </i> g or formed with alum is seroconverted greater than or equal to 50% in Ionized mice. GP160 immunogen is immunogenic in mice as an undetermined antigen at pH 7.5 and pH 6.0 but there is a loss of immunogenicity at lower pH *

Mice were donated 20 days after immunization and serum tested at 1:10 dilution in EII A gel gel purified gp160. Similar results were obtained using a commercial ELISA assay for HIV-1 proteins at a serum dilution of 1: 400.

Seroxverted mouse probe (P) to total test number (M).

YES? 11 [Alulogelnst Follow /

Hmg the tenderness of the hives absorbed rn160 / EC ^ A serum from a site important to test vaccine to induce its dangling syllable property. The following experiments were performed to confirm that the alum had fermented the rn16C vaccine to be immunogenic in sewing and to confirm that the adjuvant Banta strain had this immunogenicity.

On day 0, mice (groups x 10) were injected with a single dose

/ 0.5 micrograms, 1 micrograms or 5 micrograms / self © gp160, gp 160 adsorbed from alum or gp1bO in gllen 4ι4 · ι 's / adjuvant / PFY /. At day 28, blood was collected from mice and serum was checked by ELPA (1:10 dilution) for antibodies to rp160.

The results of the serum extracted on day 28 are indicated in the table below / Table 5 / .. In all / groups. more © 50% of mice show seroconversion. At all doses, the number of> seroconversions and the mean absorption / RR at a dilution of 1:10 in the ELISA were tried / eq higher. a poorer gp1bO absorbed than that obtained in mice immunized with gp1bO alone.

These results indicate that alum adjuvant significantly increases the immunogenicity of the γp 160 antigen.

Table 5- 28 days after injection ___

O.b ^ dose 1.0 ^ dose B.O ^^ dose medium medium © medium

p / i ⁴ 0D ⁵ p / lm oh P / N Or gp160 9/10 .407 7/10 .699 7/10 .430 gp160 / alum / 9/10 .547 8/10 .797 10/1 ⁿ 1,347 GP160 / P5L / 10/10 1.130 10/10 1.967 10/10 1,317 rP ^T 6 ^f EP 12

Determining whether a method is accepted

Neutralizing data

HIV-1 Neutralization Trials Infection with an antibody preparation slows down HIV-1 viruses in susceptible human LC cell cultures. Yaityserum et zinc immunized with rp16C ^was tested in the HIV-1 neutralization experiment and the results are summarized in the table below (Table 6).

The number of mice that are servo-inverted / P / compared to the total number of tests / N / at day 26 after immunization by 0.5

H, micrograms, 1.0 micrograms or 5 micrograms of Vaxjyn HIV-1.

The mean absorption (OD ^ q) of mice that are seroverted, as measured by the EII A method against rp160 at a serum dilution of 1:10.

Table 6

An animal Identification Immunogen / adjuvant Microgr.6 Neutralizing titer Result C55 ' gp120 ^ alum 16/8/8 1: 80-1: 160 Result H55 gp120 $ ounce 16/8/8 1: 80-1: 160 Result I 55 gp160 / alum 16/8/8 «= 1:80 Mice Pool 3 gp120 / .25 / .25 / .25 1: 40-1: 80 Mice Pool 8 gp160 / .1 / .1 / .1 1: 40-1: 80 Θ.Ρ Purified rn! bo $ 10/10/10 1: 320

(L _h S (juiWW / Guinea pigs), rabbits and Rhesus monkeys were also immunized with rnloC (using alum or ilk advantage). Immunization of these animals produced a good antibody response to HIV-1 envelope proteins.

EXAMPLE 13

Chimpanzee chunks

Genetically Shimnansets is the closest animal model to human injection

HIV-1 is a safe and unhealthy experience with tr xMc / pancreas, where a microgram or 80 micrograms rnlcO khhdehhxxx vaccine. Each animal received immunization (booster immunization) at 4 weeks with 40 micrograms and 80 micrograms gp160, respectively. A control animal was vaccinated at the same time with 1 ml of saline. The weekly serum samples of each chimpanzee are humiliated with the стиί№ ··· ΒΗΗΐ ό ό ¹ ',' Fi ^τ ^ ^π r ^ j. »» -.1 «l !. v'y I ¹ lM. G '·' ¹¹ · '.''

W ^{! 24} were assayed for antibodies to rp160 and to HIV-1 viral antigens using three immunological experiments,. ESHA shp® against purified GP160 developed by ^l and commercial BIV-1 EC ') A experience. The results of these yamalis are written. below.

A. ESA / M6 ^with HTV 160 study /

EC.5A method, KG.S ^? <? ^ HIV 160, a trademark of Meriden, Connecticut, USA is an immunosorbent method against GP160 described in the patent application serial IP920197 / se · © IP IP 585,266 /.

Serum samples taken prior to immunization and during the eleven weeks following initial immunization were diluted in a ratio of 1:10 to 1: 100,000 and then incubated with nitrocellulose strips at sites containing 100 purified gp160. The titer dilution end point is the highest dilution at which the test is positive for the anti-rn1b0 antibody as determined by the goat anti-human ^^ - alkaline phosphate ia conjugate,

The serum samples of the control animal and the preimmune serum of the immunized animal were negative. The chimpanzee that received 80 micrograms dose was positively diluted 1: 100 by the second week, and the chimpanzee received the 40 microgram dose positive by the fourth week at 1:10 dilution. Ato antibody titers to rn! 60 continued to increase over the fifth week, at which time the endpoint of the titer dilutions was approximately 1: 100,000 and 1: 2,000,000. The antibody titer in both animals decreased slightly during the 6 -11 weeks.

This type of response is the same qualitatively and quantitatively as the antibody responses co-observed with chimpanzees, vaccines-

human hepatitis B virus vaccine.

F. Commercial test. It is clear from the> 44b / g NP16O, Sh5A, and serum of immunized with bh / yy / chimpanzees that they are seroconverted and have antibodies against recombinant dp1bO. To determine whether they also produce an anti-HIV antibody that recognizes native viral envelope proteins,

, Seattle, Washington. Living Vaxerum and serum from first to eleventh weeks are a shrinking legitimate commercial ELI5A test kit, the LAV EIA ™ d'C test kit. and &

sintered © with 80 micrograms gp160 is positive by the second week at a 1: 100 dilution and continues to show an increase in antibody levels by the sixth week. The animal immunized with 40 micrograms is positive at a dilution of 1: 100 by the sixth week.

EXAMPLE 14

Distribution of antibodies between gp160 and gp41

It is important to determine whether the antibody responses against gp160 in the vaccinated animal are directed against gp41, gp120 or both. Multiple immunoassay radioimmunoprecipitation (HT1), immunofluorescence (IF), Wl (, - / c27t ί & ά assays / K / B) and quantitative EI1SA against three different recombinant envelope antigens are used to determine and measure the distribution of 7 antibodies against different regions of IV -1 enveloping proteins.

Figure 6 summarizes the immune reactivity of three different recombinant genes: (ART) / TAV / (1) rp120-delta / stem rocombin nant HIV-1 rp120 with about 40 amino acids missing from the C-terminal of the molecule /; (ART) / TAV / (2) rn12C / full-length recombinant HIV-1 rn120; and / ART / / TAB / / 3 / gp160.

Human serum from 50 patients with Positive HIV-1 antibodies and human derived sera are highly reactive with rp1bO, medium reactive with rp120 and few or reactive with rh

120. The stem gp120, which represents more than 90 ^ of HIV-1 external gp, is likely to contain protective determinants. The observation that the human X-positive serum has several antibodies to this region of the envelope is compounded by the fact that the immune response to the viral infection is not fully protected and that the human positive serum simple © shows a low level of in vitro neutralizing activity.

In contrast, rhesus monkeys immunized with either gp160 immunogen ilisas stem gp120 have antibodies that react violently with © § ^ stem §9yay1 ^{degree of} 'HIV-1 envelope. This difference in the distribution of the recognition sites on the viral envelope and the more bizarre tigers observed in monkeys may explain the fact that monkey serum has a high neutralizing titer.

A quantitative assessment of immunoreactivity and the three human and immune rhesus serum recombinant envelope antigens is presented in Figure 7. The test serum of all monkeys is a high titre antibody to the trunk / truncated / gl220 antigen / gp120-delta / including animals of animals immunized with gp160.

These results indicate that recombinant rp160 elicits a spike response in rhesus monkeys that is different from that which occurs frequently during natural infection. N / A epitopes in the gp120-delta region of gp160;

160 for the prevention and treatment of HIV infection.

for the prevention of HIV-1 and an important property of recombinant gp

Therapeutic administration of vaccine '; Mr

Clinical experience with 3OHIV-seropositive patients was performed to determine the effects of the cloned H1U gp160 vaccine / produced in the bacillus © virus system as described above / on Nazimites.

Vaccination with recombinant rp1b0 resulted in an increase in rp160 of HIV-specific humoral and cellular immune responses in 19 of 30/63% / NP-seropositive / volunteers. 14 out of 15/93% / volunteers received 6 doses of the vaccine show an increase in the total gp160 antibody, why, recombinant HIV proteins (prp41, prp120, prp1bO and mixtures added) can be favorably used in the treatment patients infected with HIV.

The effective amounts of HIV protein used in the application of the invention can be determined according to techniques well known in the art, such as those presented below. In general, such effective amounts can range between about 1 microgram and about 100 micrograms per kg of human skeleton. The frequency of administration can also be determined by known means. The preferred administration is parenteral injection, i.e., intravenous, intraperitoneal, iitramuscular, intradermal, etc.

A. Selection of volunteers

3 volunteers with HIV infection were recruited. Only seropositives with an early stage of HI -; .n _A excI.i are defined as

Stage 1 gli 2 / the listed 0¾ cells are not ps-malx 400 for more than 3 months, with; or no lymphadenopathy / have the necessary study characteristics. , and xM & d · 314:

131-132 (1985). Additional criterion restricting well-being 'is the age between 1s and 5s, with icJiTJiiio.-Thermal blood count ·

IIpjpT / 7 wV ^ attr ^ ba • · .. * / In 'IvU ma organs ;, - «units. 'Ί I l' - 1 'team, with no evidence of alcohol and drug abuse in the last 12 months, or those who have not taken amt-retroviruses. or immunomodulatory drugs. All patients underwent two months of baseline randomization study in treatment; groups. Volunteers do not receive any anti-retroviral or immunomodulatory drugs during this process.

Twenty-six of the + 0 volunteers are men; 4 are women. 14 are white, 13 are red. and 3 mulattoes. The average age is 29 / between 18H 49 /. Acceptance © 8 volunteers are Stage ί and 22 are in Stage / Stage / 2. The starting mean CI cell count is 668 / between 388 and 1639 /. The average interval between the initial diagnosis and the study was 24 months (between 3 and 49 months).

B. Vaccine Product and Immunization Table • As described here®, the test vaccine includes the meinfecting subunit glycoprotein derived from rp160 as a baculovirus expressive recombinant protein. The protein protein is produced in zG-γ insects

cells biologically purified for the final formulated wax / axis / at

Use tridozes of micrograms per milliliter and gp160:

micrograms per milliliter, 160 320 micrograms / ml. The injection volume for both 40 and 160 microgram doses is 1 ml; 2 ml cm 3 320 micrograms per 1 ml were used to obtain 640 micrograms and injection dose.

The thirty volunteers are divided into six groups of five Lists each. Two imaging tables were studied; Table A,

0, 30 and 120 dc; ι; and tab. F, where vaccination is 120, 150, and 180 days. In each immunization. or 57 have three groups, each receiving different doses of vaccine / table. 7 /. All vaccinations are vaccination on 0, 30, 60 tables / A peacocks. yiyamam ^ very short pellet. i. ·, νηκν.π. The Complicity of

'V », \ ·.

months ·. V · 's trial is 10 months; two months baseline study and '' l '* evaluation after initial vaccination. >

—Tabs & _7r_Shortsup <list

Application quantities gp160 // //

Day 0. 30 60 120 150 180 List_A_ Group 1 40 40 40 Group 3 160 160. 160 Group 5 640 640 640 List B Group 2 40 40 40 160 160 160 Group 4 160 16Q 160 640 640 640 Group b 640 640 640 640 640 640 C. Safety assessment and toxicity Every volunteer was interviewed and reviewed at 0, 1, 2, 3, 15 and 30 days after each injection. They are volunteers interviewed about tres-

ka, cold, nausea, vomiting, arthralgia / joint pain /, '- • ialgia / muscle aches /, malaise, hives / rashes /, shortness of breath, wheezing or headache. The local response to injection such as erythema, edema, itching, bleeding, darkening of the regional lymph nodes, change in the fusion of the injected limb, subcutaneous nodular formations on the side and injection are also evaluated. We also perform monthly blood tests, chemical analysis of the serum, coagula / iGiicix nre ^^! urinalysis.

^{The in} vitro cellular immune function was assessed by Ί-cell phenotype (general lympheditis, CP _z and CPp cellular (enzyme), as described in Ridcmccft et al., &lt; RTI ID = 0.0 &gt; ClC &lt; / RTI &gt;

to p. JnmiLLhc

85-95,1983; B'Z.X

4: 188-196, 1991 /. The T-cell proliferative response is a myth · t

tion is evaluated by determining the hypersensitivity of say antigen / mumps control, tetanus toxin control,

Viral cultures of peripheral blood mononuclear cells LKPC / and plasma were quantified as described © in 1159-1167,1991. DOA Polymer Chain Reaction / Wc ^ & S, et al. Doci, 33: 58-63,1991 / and the serum p24 antigen level determined mine, monitor in vivo HIV viral load.

No systemic toxicity was observed, but local reactivity was noted in 87% of patients (13 in each vaccinated group). Local response includes induction, soreness, transient nodular subcutaneous lesions at the injection site; there is rarely an increase in regional lymph nodes. No patient refused a booster injection. There was no difference in the frequency of local reaction · at the first injection or at booster / dosing.

There is also no evidence of adverse effects on the immune system as measured in vitro by mitogen and antigen specific proliferative responses, or vivo by determining the hypersensitivity of skin test responses, or by acceleration and quantitative CD 4 cell depletion. The basic number of 0¾ cells was 716 and 605 respectively for the vaccine and vaccine responses. screaming. The mean number of CD 4 cells during the study days 180-240 was 714 and 561, respectively, for the vaccine and vaccine responders. Pa. time aa study of a 240 day experience

the final change in the mean amount of cells for the vaccine responders is minus 0.2%, whereas in the vaccine response media the amount of CD 4 cells tends to 7.3% / FIG. 11 /. Vaccine-inducing HIV immunogenicity has been linked to the CD 4 slope acceleration data in some patients throughout the experience.

To evaluate the possibility of increasing HIV replication and viral load in patients as a result of vaccination, their viral activity was measured by the amount of plasma and ICPC BIWSKft CULTURE SCRC VDA polymerase chain reaction and serum levels of p24 antigen. Quantities of crops and DHA per

the limerase chain reaction does not show changes during the © pie. Serum p24 antigen is unnoticeable in patients. '

C. Determination of immunogenicity

Antibodies saturated against all HIV proteins were measured by using both recombinantly produced viral genius products gp16C, slave, p24, and whole HIV lysate of the prototype HIV staple. Technically for used A&M as described οι et al., ROS fteed. Sc <'. VSQ

76: 4350-4354 (1979). Antibody responses to specific envelope epitopes were also measured / see FIG. 7 /.

Ia ^g nti 7 are selected epithelium 66 / amino acids 86-96 in. nil 20 / and 4460 / agnilo acids 446-514 in rn12 (>), because antibodies that are detected against these regions appear to be associated with the early stage of NG. ^T infection.

Epithelium 106 (amino acids 106-121 in gp120), 241 (amino acids 241-272), 254 (amino acids 254-272), 300 (amino acids 3 ^P -34O), 308 (amino acids 308-322), 422 (amino acids 422)

454 (and 735) (amino acids 735-752) were selected because of their predetermined functional importance. gshtstp-te 1i6 and 4g <sa 1> kl

compulsory in CD 4; epitopes 241, 254, 735 are included in the specific neutralization group; and epitopes 300 and 308 are included in type-specific neutralization.

Epitope 582 (amino acids 582-602) was selected as a control because it represents the immunodominant envelope region of a natural HIV infection. Additional epitopes including 49 / amino acids 49-128 / 'are being studied; and 342 (amino acids 342405).

In Figure 7, the shaded section represents a documented change in the HIV envelope - direct immune response. Shaded by / = / indicates a primary humoral response. Shaded with / + / represents a secondary humoral response; / - / stands for negative, anti-EGF antibodies. the specific epitope pre and postimmunization; and by (*) denotes positive antibodies to a specific epitope before hypostimulation, but without quantitative change. Shading with /./ represents a new T-cell proliferative response to rn1b following immunization. Only /./ no cellular response to gp160; / | $ means »A ^ A chava» Ήΰί dcvCc

T-OPrP (not interpreted); search / know-how is not done. »

Neutralization activity was measured against three prototypes, isolated (HIV-IIIE, KR and? III) in the scene inhibiting syncytium as described in fl'fa 1Cc _f t-Cy 333: 469-470 / 1988 / _G ^T specific cellular responses were measured by known lymphocyte proliferation, determined technically by the use of rp160, p24, and a baculovirus expression protein control system / β'Ζ Υ, /.

E. Raxin-digesting and non-responsive epitopes are combined with the vaccination series (Fig, 7).

Patients were identified as vaccine-responding only if the reproductive. Selective increase in both cellular and * HIV response-specific

^ι ?. *

A humoral immunity-inducing vaccine is defined as a serocon version of HIV-wrapping specific epitopes and / or a secondary booster immune response to 'wrapping-specific epitopes'. Vaccine inducing cellular immunity is defined as the development of a new, reproductive rp160.

vaccine-related, proliferative, and response to Patients who do not develop either a humoral or cellular proliferative response, or who develop only a humoral or cell-only peripheral response to the gp160 epitope or HIV envelope are classified as non-responsive.

F. Vaccine and humoral response

According to instant 7, 19 out of 30 patients (63%) showed a vaccine-inducing increase and a specific humoral and cellular immune response. These 19 patients were classified as "vaccine responders". Four of the 11 »» responders »» develop saloo hu :. oral gl cellular immune response. All 7 xsit patients failed to demonstrate any detectable c-thcbcdr aberrant vaccines receiving only 3 doses (List A). They are not: ι altschEvlshtasAs related to E.IY polymerase / dew /,

r, whether scrup ^ p [... . ........ .. r ... T ^t T * TG ~ SALLEY / POP / lip PRODUCTS, / OR LEIS. Che- .QlV FM l— trolls δ.ίΐΒΓ 011 Ί U T SD 1 j / C · U 'IJxiiOl · - lied i.lfjOi'lTI'j j. l / iilCc- lc. .W Cv. . »1 i'xJ pulse virus dCpt Cellular Kissurslen Melt Breakdown with 11 ITALS.

ixi body /B1J.SC/ cc ^ catching at 13

A-L ί - • ν'ΙόιΡΟίχ'ίΙνΙ ': ... _Ί 0 Jll'i J. Ϊ D? .LrU C Cl 1 L1 *

Psioi des

In contrast, of the 10 patients who lost their seroconversion to each envelope specific epithelium, none showed an increase in envelope antibody via Weskitt Md. The remaining seven patients who were seroconverted to specific envelope ¹ epitopes showed no change in the entire viral envelope antibody through No changes in the antibody directed against the non-enveloped HIV protein in any patients.

15 patients / 93 // from List B Iccachazate / 6 doses / showed an increase in total gp160 antibody, against only 7 of 15 patients / 47 // from List A / 3 doses / / P = 0.01 T / SkzZ / / Figure 7 /.

As shown in FIG. 8, the pre-immunization to priming-EEE® post-vaccination prevalence of each rp160 specific epitope is, respectively, as follows: Epitope 49/27 to 70 //, Epitope 88/28 to 52 //, Epitope 106/50 to 87 //, Epithelium 214/0 to i 14 //, Epitope 254/0 to 13 //, Epitope 300/47 to 77 //, Epitope 308/42 to 69 //, Epitope 342/0 to 27 //, Epitope 422/3 ds10 //, Epitope 448C / 73 to 87 // and Epitope 735/17 to 33 //. It was noted that the vaccine induced seroversion at all specific epitopes except Jaa 582 (FIG. 7/). Antibodies / seroconversion / directed against Epitopes 241, 254 or 342 are only downstream of vaccination.

Essential immune responses were detected by the following epithelium: 88, 106, 300, 4480, and 582. The predominance of an antibody directed against epitope 582 was 100 / pre-vaccination and only 1 patient / 34 / showed secondary immune stsgr. The structure is. the vaccine caused. NG ^D antibody ktm enveloping epitopes is variable / FIG. 7 /

Gtrwitch antibody responds / seroconversion / at least one sleep-

List A / P = Lists A. Lists are Random Sam © & 5m 15 of 110/14? c / n · -.

* <ί .5 ' ¹ ί ·, *. · ',,' Tj »·; _with potential epithets to which they do not have antibodies. List B patients are seroconverters to 60 et 129/47% / /P<0.0001$X^& Λ Seroconversion to three or four epitopes occurs in 9 patients / 60% / randomized etic list E but alone © 2 patients / 13% / randomized from List A /P=0.02

Serum neutralizing activity against three separate clones (HIV-IIIB, MP and WG) was determined at 0, 90 and 195 days in 7 patients. 4 fl 5 vaccine non-responders indicate an increase in neutralizing activity to one or more fl isolates. Vaccine responders versus non-responders show an increase in the ability to inhibit syncytial formation.

L. Vaccine iodizing cellular response

Changes in the cellular immune response are based on a comparison of the major pre-vaccine vaccine (basal) and post-vaccine lymphocyymphocyte stimulating index (LSI).

30 patients / 70/7 develop left T-cell proliferation and response to rp1b0, psst immunization / FIG.

Figure 9 illustrates the preferential responses to rp16C, ₽ 24 and baculovirus control protein in 4 typically vaccine responders. For all patients, gp160 produced a proliferative increase in the median basal level of LSI from 3 to 1G (measured using 4 values following the last immunization). In contrast, no changes were observed in the proliferative responses interspersed with HIV p24 protein or the control baculovirus protein.

The vaccine elicited mean LSI values for all patients, for patients grouped by vaccine stsnor group, and for patients grouped by the inactivated list, which is illustrated in FIG. 10.

'As ί,

1.

There is a significant difference (synonym) in the first response to the rmHpHyodu response and non-response.

· />. '9,' * 'speakers. / OOOSH ^^' /.'ζΓπΙβΟ proliferativism 'replied

H> - · ''. . · '•' .- ν '? ····' «·:. ' . A. ·.,. · ',. · ·> ·· ». ·. · Triggered in List B / 6 doses / are greater than List B / 3 nozzles /. / «1-0, /.

21 patients who develop a proliferative response to rp160 also develop a humoral response (vaccine responders). the maximum mean IMF of the stimuli index (LSI) to rn1bO was observed for all 50: 1 vaccine responses. However, each vaccine response is variable (peak of LSI values is from 3 to 171), representing a temporary correlation of vaccination with the size and duration of cellular responses to rp160 (FIG. 9).

3. Discuss the results

Despite the limited sample size in this experiment, there are several factors that are associated with vaccine immunogenicity. 6 cm 15/40% / of patients in List A, compared to 13 of 15/87% / cm patients in List B are vaccine-responders /P=0.02 / 7sJiib / / Fig.7/. Of the 16 patients with a median baseline

CD 4 measured ps greater than 600 per milliliter, 13 (81 $) are vaccine-producing, against 6 of 14/43% / patients whose mean baseline CD 4 was less than 600 cells per milliliter /P=0.07T ? shu /. Eacts is summarized in Table 8, multiple immunizations improve immunogenicity, µCri in patients with a baseline ardic level of CD less than 600 cells per milliliter. For example, st b patients st list compared to only 1 sap A; Pm C.03Z; ^ £ _ d> 5 injections / cs vaccine-responding receiving 3 injections / Spi · / Table 8/ r / o x- /

CT 6, /

GP160 Vaccine Immune Response via, Baseline CD l _{w for} »ate 'x'> III ' ¹ I A' Level and Munitions List

CJ) 4 Level II Responding /% / ₍ Non-Responding /% /

List A 600 500-600 7 5 5/71% / 1/20% / 2/29% / 4/80% / • 500 3 0/0 ^ / 3/100% / sub- talent 15 6/40% / .9 / 60% / List E 600 9 8/89% / 1/10% / 500-600 2 2/100% / 0/0% 500 4 3/75% / ' 1/25% / sub- talent 15 13/87% / 2/13% / T! '·) 30 19/63% / 11/37% • Therapeutic use it's vaccines introduced by Pasteur in the XIX century for the treatment of the devil. The benefit from this attempt to that you drink ria other infections ί ie has been comprehensively studied. Repres- that there are other examples of post-infection © drop down modifications of you-

Rus- sian specific immunity (such as after hepatitis A or EI) has not been documented in humans for demonstrating the implementation of this attempt for a proven or chronic viral injection.

The invention provides a viral specific immune modification by active immunization after infection. The NG ^D envelope gene derived from the gp160 vaccine increases the virus-specific humor.

n answers at 19 from 30 o .HIV infected ^ pa ·> ·? 4 *. '''-C';'''. ·. „· ,,, / !. -,. yj, 4 ¹ ·

This; investigate quantitatively and qualitatively measures individual antibody responses to specific HIV epitopes at native j; Ν> $ 4 · /, · '''e - infection compared to postinfectious mumps. Due to the '* ³ i gad' ^1. An accurate determination of the ia vaccine induced humoral immunogenicity in already infected humans was noted in 70% of patients. For example, 20 patients / 19 vaccine-responders and 1 vaccine non-respondent / are seroconverted. to specific * enveloping epitopes. Vaccination-only seroconversion (epitopes Ά 241, 254, and 342) occurred in 10 patients.

In addition, variations in humoral responses to this vaccine characterized by epitope mapping allow for a prospective follow-up of the cause and effect of assays for specific antibody responses and allows only the possibility to characterize the potential immunoregulatory mechanisms not effected during the the natural infection. . ? ·

Although in vivo serum neutralizing activity is currently unknown at τ, the observation of increasing neutralizing! This activity against the various HIV maples / species / - / ESE, ^ F F * N / at 4 cm 5 vaccine responding suggests that post-infectious immunization causes changes in functional antibodies. The test vaccine causes an increase in serum neutralizing capacity against individual NGs / species and helps to identify a group of specific neutralizing epitopes.

In natural HF infection, a proliferative response to HJ7 enveloped proteins rarely occurs. After immunization with rn1bO

Tsa. specific cell proliferative responses at

PUM patients. The reason for this difference is not clear.

be cut against the volume

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It is possible that the new preferential response is the ciliated epithelium. epithelium / identical to the vaccine / as a result of a vaccine production methodology or alternative production of an in vivo antigen /. Alternatively, the protein used in the proliferative experience may not stimulate our proliferative responses against the homologous primary T-cell nbd'vLcL envelopes of the natural virus. Vaccination has been observed to enhance the cellular immune response of the body: selected vaccine responders show HIV-IIIB type cytotoxic specific T-cell responses following booster immunization.

The factors responsible for the vaccine immune response in HIV-infected individuals are still unclear. Even with early HIV infection, patients respond sub-optimally to different vaccines compared to controls. The decreased response is associated with early cell-cell dysregulation and T-cell dysfunction. Vaccine immune stevsr is associated with basal CD4 cell count, which is likely due to the body's immunological status and an important determinant for the vaccine response. The / undisciplined list of specific T-cell quantitative intervals also influences w of the vaccine response: List E / b injections / prior / Decreased vaccine salvage is observed in patients with low CDz cell count and / is improved with increasing numbers. vaccinations, xygos suggest further modifications in dosage, regimen, adjuvants, or syrules, which may enhance the immune response. There is no evidence of immune-specific toxicity in HIV-infected individuals. Quantitative cultures, ΠΙΑ pellimerase chain reaction, and serum antigen test showed an increase in HIV visa. The change in mean CX values for responders is -0.2% and-7.3% for non-responders. The data indicates that he has fasted an exhilarating, immune response

is not associated with CD4 upregulation and is associated with a decrease in NG7 vivo replication.

The results of the vaccination were also compared with the data from 10 infected, untreated patients, selected by age, ethnic group and basal 6D4 cell count. The average CD4 amount decreased to 8.7% in this reference group, decreased to 7.2% in patients © transplanted from List A, and increased to 0.6% in patients in ListB. These results indicate that post-infectious vaccination with recombinant HIV envelope protein is possible, and further research is encouraged with a view to the prophylactic use of such vaccines.

Claims (8)

PATENTS Claims 1. A method for preparing an HIV vaccine composition comprises alumina adjuvant and recombinant HIV enveloping proteome particles with a molecular weight of at least about 2,000,000. Said method includes the following steps: a / treating insect infected cells with recombinant baculovirus; said recombinant baculovirus having the HIV envelope protein coding gene for expressing the recombinant envelope protein; b) recovering said recombinant HIV envelope protein from said treated insect cells; c) isolating and collecting purified recombinant HIV envelope protein particles by separating said recombinant protein into an affinity matrix containing; lentil lectin immobilized in gel medium; and d / absorbed by said purified recombinant HIV vol Squirrel protein particles on said alum adjuvant. The process according to claim 1, characterized in that the rocombinite extracellular particles comprise about 757 consecutive amino acids of said HIV envelope protein and in fact reject about 40 consecutive terminal amino acids of said protein. 3. The method of claim 1, wherein the protein particles are. spherical and having a diameter of 30 to 100is. 4. The stsd according to claim 1 is characterized in that the bulb is that the magnitude of .t> 1l 1lb; bl is a vector / carrier / Ac304o. 5. etsdtt according pretsadil 1, characterized in that firstly in step d / isolated purified rekomopnanten pro tein cc subjected _d gave ".to. 6. The method of claim 1, wherein step d / is carried out at a pH of about 7.0 to 7.2. 7. The method of claim 1, wherein step b / comprises: (i) washing and homogenizing the cultures of infected insect cells; (ii) cell rupture; and / or recovery of recombinant protein from the cleaved ones - cells .. 8. The method of claim 1, wherein step in / includes: (i) applying the recovered protein to said affinity matrix; (ii) washing the matrix with the first lentin-yl lectin buffer; and / iii) separation of the protein from the matrix with the second lentile lectin buffer. according to claim 8, characterized in that