PT98382A - PROCESS FOR THE PREPARATION OF A VACCINE CONTAINING A CALISSE II PROTEIN FROM THE OUTER MEMBRANE OF NEISSERIA MENINGITIDIS - Google Patents

Description

BACKGROUND OF THE INVENTION Meisserla meningitidis outer membrane protein COMPC) is used as an immunological vehicle in vaccines for use in humans. OMPC consists of vesicles containing a variety of proteins as well as membrane-lipids, including lipopolysaccharides CLPS or endotoxin OMPC has the property of immune enhancement and when an antigen is chemically coupled thereto results in an increased antibody response to the antigen. OMPC is currently used in vaccines for human neonates against infectious agents such as Haemophilus influenzae, and makes the newborns capable of producing an IgS and an immune memory response to polyribosyl ribalt phosphate (PRP5 of H. influenzae, when PRP is chemically linked to OMPC * OMPC is a mixture of a variety of proteins and lipids5 and it has not been known which component or components of OMPC conferring the beneficial effect of intense However, some of the potentially negative aspects of the use of OMPC in human vaccines include LPS-related reactions such as fever, entotopic shock, hypotension, neutropenia, alternative complement pathway activation, intravascular coagulation and possible death,

In addition, the OriPC-antigen conjugates are quite heterogeneous since the antigen may become conjugated to any of the portions of the protein constituting the OMPC '

PB3ECTIVES OF THE INVENTION

It is an object of the present invention to provide

Subunitly pure Class II protein, the major immunoenhancing protein < MIEP > derived directly from the outer membrane of Meisseria meningitidis. free from other outer membrane components of Meisseria meninqitidis. It is another object of the present invention to provide substantially pure recombinant MIEP of the outer membrane of Heisseria meninititis, produced in a recombinant host cell. completely free of all other Meisseria meningitldis proteins. A further object of the present invention is to provide an effective immunoblotting protein for the enhancement of an antigen immune response comprising either purified WEP directly from the outer membrane of Meisseria meningitidis. and recombinant Meisseria meninitidis produced in a recombinant host cell. Another object of the present invention is to provide a protein having immunogenic mitogenic activity, comprising either MIEP purified directly from the outer membrane of Meisseria meningitiriis. want MIE? recombinant Meisseria meningitidis produs in a recombinant host cell. It is a further object of the present invention to provide vaccine compositions containing either recombinant Μ.ΕΕ, or purified M1EP directly from the outer membrane of Meisseria meniogitidis. These and other objectives will become apparent from. following description. The present invention relates to the major Class II ClILEP immunostimulating protein of the outer membrane of Meisseria meninitidis. in substantially pure form, free from other outer membrane proteins of Meisseria girlitidis cont aminantss and L? S = A MIE? of the present invention, either purified directly from the outer cell membrane of

Meisseria meninqitidis, whether derived from a recombining host cell producing MIEP recombinant Mysteridine mutans, has immunological vehicle activity and mitogenic activity. The MIEP of the present invention, when coupled to an antigen, is immunostained as the response of the antibody to the coupled antigen is increased or the antigen is transformed into a ϊ -degree antigen which assures production of immunoglobulins of the IgB class. Antigens that can be coupled to the MIEP of the present invention include viral proteins, bacterial proteins and polysaccharides, synthetic peptides, other immunogenic antigens, and non-immunogenic or weak antigens.

Figure 1 - Responses from adoptive transfer recipient antibodies receiving spleen cells inoculated with PRP-DT and MIEP, or OMPC, or ΣΑΑ-OMPC were measured by ELISA on blood samples taken on the days indicated after immunization with PRP-OMPC.

Figure 2 - Lymphocyte proliferation assay for the initogamic activity of MIEP, in vibro, is absent from Î ± -H-thymidine incorporation into the cellular DMA was measured following exposure of the cells to bovine serum albumin CBSA ), PRP-OMPC, OMPC, MIEP or CNBr "

Figure 3 - PRP-MIEP conjugates were tested for immunogenicity in mice as well as in newborn Rhssus monkeys. Antibody responses were measured by ELISA and RIA,

It is noted that certain substances which by themselves induce an immune response consisting only of IgI class antibodies and no memory can be transformed into fully immunogenic antigens which induce IgG and mA antibodies as well as memory by chemical coupling to a strongly aniigenic substance (T-cell dependent). This immunological phenomenon is termed "vehicle effect", while the non-immunogenic or weak moiety and the antigenic substance are termed "hapten" and "carrier". The injection of the hapten-vehicle complex into an animal will result in the formation of antibodies by B lymphocytes, some of which are specific for and will bind to the hapenone, others that will be specific for and bind A further aspect of the vehicle effect is that upon subsequent exposure to the hapten-vehicle complex, a strong antibody response to the hapten will occur. This is termed a memory or anamnestic response to the vehicle effect seems to involve functions mediated by certain T-lymphocytes called "helper T-lymphocytes." the vehicle molecule stimulates the helper -influence to assist in some way the formation of 1-antigen-1 producing antibodies and a memory response.

Auxiliary T-lymphocytes are normally involved in the production of B-lymphocytes of antibodies specific for a certain type of antigens called "T-dependent" antigens, but not for other antigens called T-indepen- dent antigens, A carrier molecule may convert a weak or non-immunoaemic independent hapten to a strongly antigenic T-dependent molecule. In addition, a memory response will occur at a subsequent exposure to the hapten complex The utility of the carrier molecules is not limited to use with independent T antigens but may also be used with antigens. T-dependent. The antibody response to a T-dependent antigen can be enhanced by coupling the antigen to a carrier, even though the antigen can itself induce an antibody response.

Some other molecules have the ability to stimulate, generally the entire immune system. These molecules are called " mitogens " s and / or B lymphocytes, can greatly enhance many aspects of the immune response including increased phagocytes, increased resistance to infection, increased tumor immunity, and production of increased antibodies »

Many causative agents of infectious diseases can by themselves induce protective antibodies that can bind to killing, render them harmless, or may cause death or render harmless the causative agent of their secondary products. Recovery of these diseases results " usually in long-term immunity by virtue of the protective antibodies generated against the antigenic components of the infectious agent "

Protective antibodies are part of the natural defense mechanism of humans and many other animals, and are

found in blood as well as other tissues in body fluids, is the main function of most vaccines. inducing protective antibodies against inflexible agents and / or their side products without causing disease. N. meningitidis OilPC has been successfully used to induce antibody responses in humans when the MFC is chemically coupled to cell-independent antigens, including bacterial polysaccharides. The QsIPC contains several bacterial outer membrane proteins thus forming bacterial lipids. In addition, the OMPC has a three dimensional liposome structure. The efficacy of OMPC as an immunological vehicle was thought to depend on one or more of the bacterial membrane proteins. bacterial lipids, the three-dimensional liposomal structure, or a combination of bacterial proteins, lipids, and liposomal structure. Applicants have discovered that one of the proteins, MIEP, possesses the immunological carrier and immuno-sensing properties of QHPC vesicles, and is effective in the purified form, free from other membrane proteins of N.meningitidis membranes, and in a non- dimensional. Applicants have also found that MIEP, when chemically coupled to a bactsriane polysaccharide, functions as well as GMPC in inducing antibody response to the polysaccharide. Applicants have further discovered that εEP is the Class II protein of the outer membrane of M. meningitidis. Class II protein of N. meningitidis is a porcine protein EMurâkami, K., et al. (1989), Irrfection And Immunity, 57, pp. 2318-233. The porins are found on the outer membrane of all of the bacteria.

Although the present invention is exemplified by the ϊϊΡΡ NΡ men men men men men men men men,,,,,,,, it is readily apparent to those skilled in the art.

It is understood that any outer membrane protein of any Sram-negative bacterium having an immunological carrier is an immunostimulatory activity is encompassed by the present invention. Examples of Sram-negative bacteria include but are not limited to species of the genera Nsisseria, Escherichta, Eseudomonas. s Haemapifilus »SalmonslIa, SMaMlâ» iS2rdet ^ LlaP

Klebsiel et al., Serratia, Ysrsinia, Vibrio, and Enterobacter. MIEP may be employed to enhance the antibody response to highly antigenic, weakly antigenic materials? and not antigenic. The terms " antigen " and " antigenic material " which are used interchangeably herein include one or more non-viable, immunogenic, immunogenic, non-immunogenic, or antiallergic (desensitizing) agents of bacteria, viruses or other sources. The antigenic component may consist of a 5 ' in an aqueous phase such as an aqueous solution, or in an aqueous suspension and the like, including mixtures thereof, containing a non-viable, immunogenic, weakly immunogenic, non-immunogenic, or desensitising agent or agent. The aqueous phase may conveniently be comprised of the antigenic material For example, the aqueous phase may be in the form of a vaccine in which the antigen is dissolved in a balanced salt solution, physiological saline, phosphate buffered saline, tissue culture fluids, or the aqueous phase may also contain preservatives or substances conventionally incorporated into vaccine preparations. Adjuvant emulsions containing an. genome conjugated to MIEP can be prepared using techniques well known in the art.

may be in the form of purified or partially purified antigen including, but not limited to, antigens derived from bacteria, viruses, fungi, rickettsiaii or the antigen may be an allergen including, but not limited to , pollen, dust, volcanic ash, or excretes thereof; or the antigen may be in the form of a tonic or a poison including, but not limited to, poisons or poisons derived from reptiles or poisonous insects. The antigen may also be in the form of a synthetic peptide, or a fragment of a larger polypeptide, or any subportion of a molecule or component derived from bacteria, mammalian cell, fungus, virus, rickettsia, toxic allergen or poison ,. In all cases, the antigens will be in the form in which their virulent toxic properties are reduced or destroyed, that when introduced into a suitable host they will either induce active immunity by producing therein antibodies against specific proteins, peptides, microorganisms, extracts, or products of microorganisms used in the preparation of the antigen, toxins, poisons or, in the case of allergens, will assist in alleviating the allergy symptoms due to the specific allergen.

Antigens may be used either alone or in combination, for example, multiple bacterial antigens, multiple viral antigens, multiple mycoplasma antigens, multiple rickettsia antigens, multiple bacterial or viral toxoids, multiple allergens, multiple proteins, multiple peptides or combinations of any of the foregoing products may be conjugated to the MIEP "

Particularly important antigens are derived from bacteria including, but not limited to, B. pertussis, Yâ "† â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ â € ‡ S. paratyphi A and B, C-C-C-C-C-C-C _-C _-C-C-C-C-C-C-C-C t

s other bacteria of gas gangrene, ti anthracis, Y.__ pestis,

PiL-JMtotocid: V, ..... choiaraa, Neisseria jeninaitidis, Wgpnp.rrheag. Fig. X ^ gonsffia ..... gallitiiuffi, and the like; of mammalian cells including; but are not limited to, tumor cells, virus infected cells, genetically engineered cells, cultured cells, cell or tissue entries, and the like? of viruses including, but not limited to human T lymphotrophic virus Multiple Codes), human immunodeficiency virus (multiple variant types), poliovirus (multiple types), adenovirus (multiple types), parainfluenza virus (multiple types), measles , mumps, respiratory syncytial virus, inflammatory virus (various types), typhus (SF ">, West German encephalomyelitis virus, Japanese encephalomyelitis B, Russian spring summer encephalomyelitis, swine fever virus, Me, & castle, varicella, rabies, canine and feline synostosis, and similar viruses of rickettsiae including, but not limited to epidemic and endemic typhus or other members of the macuiosa fever (cerebrospinal meningitis) group of various venoms from spider to snake or of any of the known allergens, including but not limited to those of Santiago de Compostela, foot of house, pelican extrusions, grass pollen, and the like.

The polysaccharides of this invention may be any acidic bacterial polysaccharides, but are not intended to be limited to any particular types. Examples of such bacterial polysaccharides include polysaccharides from Streptococcus pneumoniae (pneumococci) of types 6A, , 1A, 18C, 19A, 19f, 22®, 22F, and 23Fs Streptococcus Brupo B of the polysaccharides types Ia, 1b, II and III? A, B, C, X, Y, W135 and 29E = polysaccharides of Escherichia coli KI, K12, K13, K29 and Κ1ΘΘ. However,

particularly preferred are capsular polysaccharides which are isolated from the group consisting of H, influenzae and sarotipo b polysaccharides, as described in Rosenberg et al., Biol. Chem. , 236, 2845-2849 (1961) and Zamenhof et al. The. Biol. Chem., 29, 695-744 (1995), the type 6A or type 68 polysaccharide of Streptococcus pneumoniae), as described in Robbins et al., Infection and Immunity, 26, NS 3, 1116-1122 (December, 1979), 19F-type pneumococcal polysaccharide, as described in C, J. Lee et al., Reviews of Infectious Diseas, 3, No. 2, 323-331 (1989)), 23F-type polysaccharide polysaccharide, as described in 0 Larm et al., Adv. Carbohyd Chem and Biochem., 33, 295-321, Ri, 3 ,; Tipson st, ed., Academic Press 1976 "MIEP can be purified from OMPC derived from cultures of N 'mertingitidis growing in the usual manner as described in U.S. Patent No. 4459286, in U.S. Patent No. 4,118,198. No. 4830852. Purification of the OMPC can be carried out in accordance with the procedure described in US Pat. U.S. Patent No. 4,171,447, U.S.A.A. = U.S.A. 4271147, U.S.A. 4459286, and U.S. 4,830,852. MIEP may also be obtained from DNA, constructed in host cells by expression of DMA reeamhinanie encoding MIEP = Q DMA encoding MIEP. be obtained from cells of N, Meninaitidis EMurafcami, K., et al., 1989), Infection And Immunity, 57, pgs. 23183, or the DMA can be produced synthetically using standard ÂμH synthesis techniques. DMA encoding MIEP may be expressed in recombinant host cells including, but not limited to, cells from bacteria, yeast, insects, mammals or other animals, giving recombinant MIEP. Preferred methods of the present invention for obtaining MIEP are purification of MIEP from OMPC and expression of recombinant DMA of DMA encoding MIEP derived from M. aeninoitidis.

Purified MIEP. prepared by partitioning with DMPC VSTO films by lysing with sodium dodecyl 5TF sodium BD5 / vesicles followed by SDS-polyacrylamide gel electrophoresis (ε). Λ V * i MIEP was eluted from i. 5 dialysate. High pH and concentrated pH. Standard polyacrylamide electrophoresis processes may be used to purify the OHPC materials. The procedures are described in. Mole: cu lar s A Laboratory Manua1, Sambrook, J, et al., ≪ 19 89), Cold

Spring Harijar Lafaaratory Press5 Mew York, and Current Protocols In Molecular Biology, (1987) Ausubel F. et al., Eds. Wiley and Sonsn New York.

Standard processes for elution of proteins from SDS-polyacrylamide gels are described in Bunkapi1ler, M. and Lujan, E., < 1986), Purification of Microamines, L & uxdw Ge · I otein Mi cr oc har SC teri (0d x T ton N., J "^ Irf Cu ΟΓ - t Proto col. 5> U DS X Fn M", et 1 ' in addition to those of mammalian, rickettsiae, gondii, protease, peptides,

Electraphoresis, in the 11th Edition, J. Shively > Humanna Press, in MoItology, 1987) Wiley and Sons, NcW VO rt :: mans-ira is readily suited for derivatives of bacteria, allergens, toxins or viruses, or any other Recombinant can be prepared by expression of the DMA of genomic meniniticidal DNA encoding ΜΪΕΡ in yeast bacteria, for example, S.S. i.

not what cod; Utr! Ns meninqxtidi p prepared for randomization of the DMA of 018V -Ξ-. Mani's technique c \ t i s s! =, SC al. For example, the DMA is extracted with molecular sieves followed by pellet (1)

Cell, 15, pg = 687¾, or by cleavage with a restriction sndonuclease by the method of Snsithiss et al., 1978); Science, 202, pg, 1248 =, the genomic DNA is then incorporated into an appropriate cloning vector, For example, the polymerase chain reaction (PCR) technique (Alternatively, the polymerase chain reaction (PCR) technique (see, for example, Lambda phage Ever Bainbroak, JL et al., 1998), Molecular Cloning, A Laboratory Manual, Cold I Spring Harbor Press, Psrkin Elmer) can be used to amplify specific DMA sequences in the DMA genomic CRoux, et al., (1989), Biotechniques, 8, pg = 483 = 0

The PCR treatment requires a DMA oligonucleotide which can hybridize to the specific DMA sequences in the AMD. The DMA sequence of the DMA oligonucleotides that can hybridize to MIEP DMA in the genomic DMA of N. meningitidis can be determined from the amino acid sequence of MIEP or by reference to the DMA sequence determined for the major Class II membrane protein Infection and Immunity, §7, pp. 23183 = Recombinant MIEP can be separated from other cellular proteins by the use of an affinity column made with monoclonal or polyclonal antibodies specific for the MIEP. These affinity columns are made by addition of antibodies to Affigyl®, a gel carrier which is preactivated with M-hydroxysuccinimide sterols so that the antibodies form covalent bonds with the support of agarose gel beads. The antibodies are then coupled to the gel via amide bonds with the splicing arm. The remaining activated esters are then destroyed with The column is then washed with water followed by glycine HCl (23 M H 2 H 2) to remove any unconjugated antibody or foreign protein. The column is then equilibrated in phosphate buffered saline (pH ), And cell culture overhangs or cell extracts containing MIEP are slowly passed through the column.

The column is then washed with phosphate buffered saline until the optical density drops to the background, then the protein is eluted with glycine-HCl 0 S23 Μ ipH2, 6). The protein is then dialysed against phosphate buffered saline.

The conjugates of the present invention may be any conjugates. polysaccharide-stable MIEPs coupled through broadeners containing a thioether group and a primary amine which form hydrolytically labile covalent bonds with the polysaccharide and the polysaccharide. However, preferred conjugates according to this invention are those which may be represented by the compounds Fs-A-E-SB-Pro or Ps-A'-S-E'-B'-Pro, wherein Ps represents a polysaccharide Pro represents the bacterial protein MIEPs and AE-S-B and A'- S-E'-B 'constitute bigeneric mops containing hydrolytically stable covalent thioether bonds and which form covalent bonds with hydrolytically labile amide bonds or ester bonds with the roacromolecules, Pro and Ps, S is S is sulfur, E is the thiol group which has been reacted with a thiol group, and is represented by

Wherein R 1 is H or CH 2, and p is 1 to 3; A is m -cAcch_.) CH3) m (n)

. wherein W is 0 or NH 5 m is Θ a 45 π is ω. -5? ts V è CK-? S, NR or UHLO-Η n where C * * " Λ 2 \ GT H or. aq. OR ϋ, -. OK! that if Y fo! " CH.sub.c so that n r * cU-i can be equal to Ξ5Π35 and if v is 0a. S, then m ώ greater than i and n is greater than 15 and 3 is -

JI. Dr Z C £ fjf | C] J C [2, Z is NH, NHCR ', COOH,

□ u ti f ono rc O | - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Then in the panzer, A " S-E "'B' 5 S is sulfur A:

W-1 '= and K' -CNHCH-)

is -OH where p is 0 or 0, and W 5 and R 'are as defentions bcx plus P - · ώ d the formation product of a thiophyl group which was reacted with a thiol group, and #

represented by -uH-, wherein R 2 is as defined above, and E 2 J

0, S

or

Ν Ο Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β Β And are quantitatively determinable with this identification reflecting the vicinity of the binding of the conjugate by bonding the side of the sulfur dioxide which originates from the cavity-modified polysaccharide with the side of the spacer which originates from the functional group.

Then the conjugates,! A-E-E-B-Pro, according to this invention may contain spacers whose components include derivatives of, inter alia, carbon dioxide, 1,4-butanadiafflin, and S-carboxymethyl-N-acetyl-homocysteine? carbon dioxide, 1,5-pentanediamine, and S-carboxymethyl-N-acetyl- carbon dioxide, 3-oxa-5,5-pentanediamine, and S-carboxymethyl-N-acetyl-hocnokstrins carbon dioxide, 1,4-hutanodoline, and S-glucosymethyl-N-acylsilyl- carbon, 1,3-propane diamine, and S-carboxymethyl-N-propyl D-homocysteine; carbon dioxide, 3-aza-1, S-penetin diamine, and S-carbo- -; imine 1-N-acetylisocyanate and carbon dioxide, 1,2-stanediamine, glycine, and S- <succin-2-yl) -N-acetyl-homocysteine = conjugates, Ps-A In accordance with this invention may contain compounds whose components include derivatives of, inter alia; carbon dioxide and S-carboxymethylcysteamine carbon dioxide and 8-carboxylyl> cysteamine carbon dioxide and

S-carbocyclic-methyl-hondiocysaminap carbon dioxide, S-isuccin-2-yl-cysteamins, and glycine and carbon dioxide are S-carboximetylcysteine.

In the process of the present invention, the polysaccharide is covalently modified by (a) solubilization in a non-hydrophilic organic solvent, then <b> activation of a bifunctional reagent, (c) reacting this activated polysaccharide with a bis-nucleoside, and finally, if necessary, (d) using said modified polysaccharide either by reaction, (i) with a reactant generating (i) with a thiolyl group generating reagent, (i) with a thiol group generating reagent, (ii) with a thiolyl group generating reagent, then the covalently modified polysaccharide and the functionalized protein are reacted together to form the stable, covalently bound conjugate, and the final blend is purified to remove unreacted polysaccharides and proteins. The process of this invention also includes the preparation of a micrococcus or bis-nucleoside which will react with the activated polysaccharide to form a covalently modified polysaccharide with pendant electrophilic sites or thiol groups pending, thus preventing the need to further functionalize the bis-nucleoside polysaccharide. was modified prior to reacting the covalently modified polysaccharide with the covalently modified protein. Functionalization of the protein for any one of the portion forms may also be performed in more than one step according to the selection of reagents in these steps.

In the first step in order to covalently modify the polysaccharide, the solid polysaccharide must be solubilized.

In one embodiment, the novel hydrophilic alcoholic groups of a polysaccharide can not compete chemically for sterolophilic reactants with the hydroxyls of the water in an aqueous solution, the golisaccharide should be dissolved in non-aqueous & non-hydrosolic solvents. Suitable solvents include dimethyl 1-formamide, dimethylsulfoxide, dimethylacetamide, formamide, N, N'-trimethyl imidacol idinone and other aprotic solvents, similarly polar, preferably dimethylformamide,

In addition, the use of these solvents, ammonium, 1-terminiccloC 2, 2, 3, 4, conversion of the polysaccharides (for example, H, influenzae type F capsular polysaccharides, which are rhodium-ribitoyl phosphate polymers, , such as mono- and diesters of phosphoric acid, in an appropriate salt form, causes the polysaccharides to readily become soluble in the above solvents.The acidic hydrogens in these macromolecules can be replaced by large hydrophobic cations such as tri- or tetra C1-4 alkyl, C1-4 alkoxy, C1-4 alkyl, C1-4 alkoxy, C1-4 alkoxy, C1-4 alkoxy, C1-4 alkoxy, C1-4 alkoxy, C1-4 alkoxy, or the resulting tetraalkylammonium salts of phosphorylated polysaccharides readily dissolved in the above solvents at about 17 DEG-5 DEG C., while stirring for from one minute to one hour, the H, influenzae, serotype B polysaccharide, partially hydrolyzed The product was converted to the tetrabutylammonium salt, then dissolved in dimethylsulfoxide (Egan et al., J. Chem., 104, 2898 (1982)), but this product is no longer antigenic and therefore is useless for the preparation of vaccines. In contrast, the Applicants will effect the solubilization of an intact, unhydrolyzed polysaccharide by passage of the polysaccharide through a strong acid cation exchange resin in

tetraalkylammonium formate, or by careful neutralization of the polysaccharide with tetrahydroalkylammonium hydroxide, preferably by the above procedure, and thereby preserving the viability of the polysaccharide for use in the immunogenic vaccine.

Subsequent steps are then directed to overcome the other significant limitation of physicochemistry for making covalent bonds to polysaccharides, which is the lack of functional clusters on the polysaccharides, other than non-hydrogen groups which are sufficiently reactive with reagents used normally or practically for the functionalization of units with which the connection is desired. Activation of the polysaccharide to form an activated polysaccharide, reaction with his-nucleophiles to form a functionalized nucleoside polysaccharide, functionalization with reagents generating either the electrophilic sites or the thiol groups are all directed to covalently modify the polysaccharide and to develop functional groups in the polysaccharide in preparation for conjugation.

In the next step, the solubilized polysaccharide is activated by reaction with a Pifunctional reagent at about 0 DEG-50 DEG C. while stirring for ten minutes at 1 hour with the critical weight ratio of activator agent to polysaccharide in the range of 1: 1 to 1: 12, In the past this activation was carried out by reaction of the polysaccharide with cyanogen bromide. However, derivatives activated with cyanogen bromide, which have a &quot; proclivity &quot; for neighboring dies, showed transient stability during dialysis against a phosphate buffer. Therefore, although activation with cyanogen bromide is still possible according to the present invention, this reagent is a fraction used for the activation of polysaccharides and is not

preferred. On the other hand, preferred bifunctional reagents for polysaccharide activation include 0-acid derivatives

. &quot; carbonic acid, H-C-K &quot;, where R 2 and R 3 may be independently azolyl, such as imidazolyl; .halites; or phenyl esters, such as p-nitrophenyl, or poly-nalophenyl, such as a particularly preferred reagent, will react with the hydroxyl groups to form imidazoleurethanes of the polysaccharide, and the aryl chloroformates including, for example, nitrophenyl chioroformate will produce mixed polysaccharide carbonates. In each case the resulting activated polysaccharide is very susceptible to nucleophilic reagents such as amines and is therefore transformed into the respective urethanes. done

In the next step, the activated polysaccharide is reacted with a nucleophilic reagent, such as an amine.

Η H

particularly diamines, for example, in which m is 4 to 4, n is 0 to 3 S and Y is CHâ,ƒâ,,, OR ', CHCOâ,,H, where Râ, . '' - t, · »« jr κπ cao ΠΙ a u; wherein if r is CFIv- or a C1-C6 alkyl or 0 may be equal to zero, if Y is 0 or 3, then m is greater than 1 is greater than 1, in a large The reaction is maintained in an ice bath for 15 minutes to one hour then held for 15 minutes to one hour at about 17 ° to 40 ° C,

An activated polysaccharide, when reacted with an amine, for example 1,4-butanediamine, will result in a polysaccharide in the ursine form with pendant amines, which may then be further functionalized by isolation. The mixed carbonates also readily react with diamines to result in pendant amine groups. Alternatively, the activated parent can be reacted with a nucleophile, such as a mono- haloacetamide of a diaminoalkane, for example 4-bromoacetamidabutylamine (see W B. Lawson et al., Tioppe SeylsrJs Z., Physisch Cham., 549, 228 (1908)) to generate a polysaccharide modified with pendant electrophilic sites. Alternatively, the activated polysaccharide may be reacted with a aminothiol, such as cis -camine, caminostanethiol), and examples, examples; ds derivatives; which are well known in the art of peptide synthesis, to produce a pendant thiol group polysaccharide. In both cases, no further functionalisation is required prior to the coupling of the covalently modified polysaccharide to the modified "baccarin" vehicle protein, the last step in the preparation of the polysaccharide, further functionality, if required, of the polysaccharide may take the form of a reaction of the functional nucleoside polysaccharide with either a reagent to generate electrophilic (i.e. thiophyl) sites or a reagent to generate thiol groups

Reagents suitable for use in the production of electrophilic sites include, for example, those for α-haloacetyl or s-halopropionyl acylation, derivatives such as OR (where R is H or CH-. X 'is nitrophenoxy, dinitrophenoxy, pentachlorophenoxy, pentafluoromenoxyl, halide, O-N-hydroxysuccinimidyl) or acid), in particular chloroacetic acid; Î ± -romopropionic acid, with the reaction running at a pH of 8 to 11 (maintained therein by the addition of base, if necessary) and at a temperature of about

of Φ a at 35øC for 5 minutes. An amino derivative polyssaccharide can be isolated with maleimido amino acids (see, 0. Keller et al., Helv. Chim. Acta., 58, 531 (1975)) to produce maleimido groups.

O

O

-CCCH2) PN JJ

Wherein p is 1 to 3 p with a 2-haloaceticating agent, such as p-nitrophenyl bromoacetate or an α-haloketone derivative of the carboxylic acid, for example, (Ber, 67, 12). (4) (1934)) so as to produce suitably functional polysaccharides susceptible to. replacement uncle. Suitable reagents for use in the production of thiol groups include, for example, additive reagents, such as thiolactones, for example, NH4 OH-O, CH2) p. In which R 1 is C 1 to C 4 alkyl or mono- or bicyclic aryl, such as MHCORT CH-COX-, and C.H-. â € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒwherein m is 4, R4 is Câ, Followed by treatment with HSCHâ ¶ CHOOH or IMHCORâ † 'CHâ,, - CHâ,, - CHâ,, CHâ,,) wherein X, Râ, and Xâ, These reactions are carried out in a nitrogen atmosphere at about 5 ° to 35 ° C and at a pH of B to ii (with the base added, when necessary, to maintain the pH within this range) for one to twenty four hours. For example, an amino-polysaccharide derivative may be reacted with

to produce an appropriately functionalized polysaccharide. These polysaccharide steps are then produced covalently raodified of the forms, Ps-ft-E- or Ps-A'-SH ™, wherein EI is -CCHX or

THE*

-CH (CH 2) p NR 5 and R 5 are as defined above. The separate function of the protein to be coupled to polysaccharide involves the reaction of the protein with one or more reagents to generate a thiol group, or the reaction of the protein with one or more reagents to generate an electrophilic center, thiophthalic acid) -

In preparation for conjugation with a functionalized electrophilic polysaccharide, the protein is reacted in one or two. two steps with one or more reagents to generate thiol groups. such as those acylating reagents used to generate thiol groups on polysaccharides, as discussed on pages 15-17 above. Thiolated proteins can also be prepared by aminating activated carbOKi proteins, such as those shown in Atassi et al., Bigcil et al., Biog. A preferred embodiment of this step of the process involves the direct acylation of the pendant amino groups (ie, lysyl groups) of the protein with IM-acetyl-amyocysteinathiolactone (IAS), with aminothioles, to create the protein. at about 0 ° to -35 ° C and pH 8-11 for five minutes to two hours, using equivalent weights of "

When the conditions for the preparation of the functional protein are as discussed above for the preparation of the polysaccharide complemented by reaction with activated maimido acids.

In preparation for conjugation to a bacterial polysaccharide covalently modified with pendant thiol groups, the protein is acylated with a reagent generating an erythrolelic center, including such agents as CH3. And XCri-CX '. in which K and X 'are the acylating acylesters, for example, X, X, X' as defined in claim 1 and wherein X ''; Suitable proteins with electrophilic centers also include, for example, those prepared by acylation of the pendant lysylamino groups with a reagent, such as activated maleimido acids, for example, or

Λ ° NOCCCH2) nNV V ο Ο

or by reaction of the carboxy-activated protein with mono-haloacetyl derivatives of diamines. In both the preparation reactions the temperature is at 35 ° C for five minutes to one hour and pH is 8 to 11 = formation of the conjugate is therefore a matter of reacting any one of the covalently modified polysaccharides having pendant elstophylic centers with the bacterial HIEP protein having pendant thiol groups at a pH of 7 to 9 in reason of approximate equivalent weights in an atmosphere of nitrogen, for six to twenty-four hours at about 17 ° to 40 ° C, to give a covalent conjugate. Examples of such reactions include

NHCOCH. ÒHCO-P OH

Ps-CCH3 CH3 CH3, CH3 NHCCH3 Br + HSCH3 CH3 OH;

In which a pharmaceutically acceptable salt thereof. . '. . . reacted with 4-bromoacetamidecylbutylamine is reacted with a protein which is reacted with M-acetyl-? -ocysteine-ethanedione to form a conjugate, and

OHIII PsCNY &quot;

-NHCCH3 HSCH2CH2NHCCH2CH2CPro

Hello

PsCNHT NHCCH2 -N

CH, CH2 NHCCH2CH2CPro

OII '(and Y is an (C1 -C4) alkyl radical); wherein an amino oolysaccharide derivative which has been reacted with activated maleimido acids is reacted with a carbohydrate protein which has been aminated with an amino- to form a conjugate *

Similarly any of the polysaccharides covalently modified with thio groups; can be reacted with the bacterial protein MIEP having pendant elliptic centers to give a conjugate covsins. An example of such a reaction is

0 0 0 H ϋ EE Et j

NHCOCH-Br ------------ &gt; &lt; / RTI &gt; OH,

Wherein an activated polysaccharide which has been reacted with an aminothiol is reacted with an acyloxy-activated protein which is reacted with mono-haloacetyl derivatives of a diamine to form a conjugate.

As the electrophilic activity of a haloacetyl group needs to be eliminated, the reaction of the conjugate with a low molecular weight thiol such as n-acetocystamine will accomplish this purpose. n-aestil-cysteamine, also allows the confirmation count of the haloacetyl moieties used (see Section D), because the S-carboxymethylstyrenamine which is formed can only be detected by the Spae: kman! lloore and Stein »

These conjugates are then centrifuged at about 1 ΦΘΘ ΦΘΘ g using a fixed-angle rotor for about two hours at about 1 ° to 20 ° C, or are subjected to any of a variety of other purification procedures, including gel permeation , tonic exclusion chromatography, gradient centrifugation, or other differential adsorption chromatography to remove non-covalently linked proteins and polysaccharides using the covalence assay for the organic spacer (see below) as a biological activity tracking process Further separation of the reactants may be carried out by size exclusion chromatography on a column or in the case of very large non-soluble proteins, separation can be performed by ultracentrifugation. Analysis of the conjugate to confirm the covalency, and consequently the stability of the conjugate, carried out by hydrolysis of the conjugate (preferably with 6 N HCl at 11 ° C for 2 hours), then quantitative analysis for the amino acid of the hydrolytically stable spacing containing the thioether linker and amino acids constituting the protein. The contribution of the amino acids of the protein can be removed if necessary by comparison with the appropriate amino acid standard for -a protein

wherein the amount of the remaining amino acid in the conjugate of the conjugate or the amino acid of the splicer can be designed to appear outside the standard amino acid of the protein in the assay. The covalence assay is also useful for monitoring the purification procedures to label the concentration of the biologically active components. In the examples

OH 0

MHCOCtL above, the hydrolysis of PsC1H2 CH3 CH3 CH3 CH3 CH3 CH3 CH3 CH3 leads to the release of S-carboxymethyl homocysteine, NH2

And the hydrolysis of a compound of the formula:

0 ° C C H H NHCCH 2 N

oo

II H 2 CH 2 NHCCH 2 CH 2 CO 2 results in the release of the aminodicarboxylic acid, and the hydrolysis of .alpha. , £ * £. <£. £. * *

HO, C

The compounds of formula (I) may be prepared by cleavage of the compound of formula (II): ## STR1 ## by cleavage of The peptogenic processes, such as those of Spackman, Lioore, and Stein, can then be conveniently applied and the ratio of amino acid constituents determined. T S-carboxymethylcysteamine

Optimal production of IqS antibodies may result in the collaboration of ϊε B lymphocytes with specificity for the antigen of interest. T lymphocytes are unable to recognize polysaccharides but may provide support for anti-polysaccharide IgG antibody responses if the polysaccharide is tightly bound to a protein that the T cell is able to recognize.

In mice this requirement exists for secondary, as well as primary, antibody responses and is vehicle specific, i.e., a secondary antibody response occurs only if the helper T cells have been previously sensitized with carrier protein used for the secondary immunization. Accordingly, the ability of a mouse to produce secondary antibody response to a PRP-protein conjugate is dependent on the presence of T lymphocytes inoculated with specificity for the carrier protein. Demonstration of the ability of MIEP to provide a vehicle inoculating anti-PRP antibody responses was done in mice that were inoculated with PRP covalently attached to a heterologous vehicle, diphtheria toxoid (DTK). Adoptive transfer was used in order to determine whether the administration of lymphocytes inoculated with MIEP alone was sufficient to generate helper T cell actives effective for the formation of anti-PRP antibody in response to PRP-0. iPC. Similar anti-PRP antibody responses were induced by PRP- DnPC when lymphocytes inoculated with MIEP or OMPC were transferred, thus indicating células cell recognition of OMPC residues in the MIEP · portion,

The PRP-MIEP conjugates were tested for immuno-genotype in mice as well as in newborn Rhesus monkeys. The immune response leaves both 'animal models sharing' with newborn humans, a deficiency in their ability to generate responses of anti-T-antigen antigens such as bacterial polysaccharides. These animals are standards used as templates for determining the immune response of newborn humans to various antigens.

In a similar manner, the peptidyl peptide conjugates can be prepared. for example where the peptide is a major HIV-neutralizing determinant (PND) peptide. A process for producing such conjugates includes the formation of a largeeneric spacing between the activated rlIEP and the activated HVV PND peptides as described and specified, The ligand may include a polysaccharide moiety as described in USSN 555 558 and Merck et al., 1988. The novel conjugate of this invention comprises MIEr, the major protein immunostimulatory protein of outer membrane COliPC) from Meisseris meningiiidis b, covalently bound to peptides PND of HIv =

The conjugates are prepared by coupling the activated peptide covalent to an activated protein. The peptide and sodium protein components are separately pooled to exhibit either pendant or nucleophilic groups such that the covalent bonds will form between the peptide and the protein upon contact »

Covalent conjugated ifflunogens resulting from the racemic combinations described above may conveniently be considered as a conjugate in which the multiple functionalities of the peptide are constructed following a MIEP foundation.

When the peptide components of the conjugate are capable of eliciting neutral immune responses prior to HIV? the conjugates of this invention can be administered to mammals in relatively effective amounts. with or without additional antiviral immunomodulators, or antibactific components? s are useful for inducing mammalian immune responses against the peptidyl portion of the conjugates? to induce HIV neutralizing lysing antibodies in mammals, or to produce vaccines for administration to humans to prevent contraction of infection or HIV diseases by inducing AIDS, or for administration to infected humans or HIV diseases including AIDS *

In a preferred embodiment &quot; the conjugate of the invention has the general structure

j < PEP-A-i-MIEP or its pharmaceutically acceptable salts? in which PEP is a HIV PND peptide? or a peptide capable of creating immune responses in mammals that recognize the PHD of HIV = F1IEP is a protein of the COMPO outer membrane protein complex of Neisseria meningitidis h either recombinantly produced or purified from DMPCq-A- is a covalent bond? preferably a largeeneric spacer j is the bulk percentage of the peptide in the co-conjugate? and is preferably between 1% and 5% of the total protein mass in the conjugate. The conjugate of the invention may be prepared by any of the common procedures known in the art for the preparation of peptide- such as? for example? the bigeneric chemistry described in U.S. Patent 4 695 624 and Marburg st. J.A.CxS. 1, S, 528 (1986); and in U.S. Patent Applications 362

In one preferred embodiment, a process is utilized which utilizes available nucleophilic functionalities found in proteins such as the amino group of lysins, the imidazole group of histidine, or the hydroxyl groups of serine, ireonine, or tyrosine. In practical terms, the number of available protein nucleophilic sites can be determined by an appropriate assay which may comprise thiolation with N-acetyl-homocysteine thiolactone 5 followed by Ellman CEllman, 8.L., Arch, Biocbem, Biophys, 82, 70, 959) for determination of free sulfhydryl groups and / or by alkylation with a bromoacetyl amino acid assayed by amino acid analysis. The preferred process may be carried out in various ways in which the sequence, and reaction of the protein and peptide groups may be varied, the process may comprise steps dss

Process 1; reacting the protein nucleophilic groups with a reagent, for example, with N-acetylhomocysteinathiolactone, which generates thiol groups in the proteins; s, reaction of the product of step a. with peptides hereinbefore derived so as to join an electrophilic group preferably comprising maleimide, to the peptide. A preferred embodiment of this invention, which may be prepared according to this process, has the structures

ο ΜΙΕΡ - (NH-C-R-ν II Ο

Ν-R-C-N-PEP) Ο

II I Ο Η ου. of the pharmaceutically acceptable salts thereof, in which PEP, MIP, P, and j are as defined above: (a) lower alkyl, (f) substituted lower alkyl; -cyclohexyl, - (d) substituted cycloalkyl-, e) phenyl-R 3 is 5-haloformate, b) lower alkyl, or L- Likewise, a preferred embodiment of the invention has the following structures:

Wherein all variables are as defined above can be prepared by the 2S method which comprises the steps of reacting the protein hydrophilic groups with a faifunctional electrophilic reagent such as ester, succinimide of malsimidoalkanoic acid in order to generate an electrophilic protein 2b resolving the product of step 2a with a peptide containing a nucleophile such as a thiol group

A highly preferred embodiment of the process 1s is described below in detail and in Scheme A, D according to the scheme &quot; Scheme &quot; the immunogenic protein is the Class II protein of the outer membrane protein complex COMPC) of IMeisseria meningitidis b3 either purified from the bacterial membrane or produced by recombinant means. The process comprises the steps of reacting the MIEPI ) P having nucleophilic groups including free amino groups due to the presence of lysins or amino-terminal proteinP with a thiolating agent, preferably N-acetyl-homoocysteinathiolactone; to generate M1EP C11 &gt; having!, m &quot; moles of the available sulfhydryl groups for reaction with a

thiophlag a = ii 'quantification of the number of available sulfhydryl compounds appended to MxEP in step 1, to determine the KmK value, preferably by Ellman CEllman assay, 6 = 1. Arch = Biochem., Biochem., Biophys. 82, 76 <1959) 3p and contacting the product of step (a) with excess, <5> of a HXV PMD that had previously been derived so as to form an electrophilic group, preferably with A maleimidoalkanoic acid is most preferably maleimidopropionic acid. The derivatization is achieved by N-profection of all amino groups in the peptide which should not be derived from the reaction of the free amino groups of peptide with a bifunctional reagent, preferably maleirate Alkanoyloxysuccinimide, and more preferably maleimitritepropionylsuccinimide) to generate the conjugate of this invention. The conjugate can be purified, for example, by dialysis in a buffer having an ionic strength of from 4 to 5% and more preferably from 4 to 5% more preferably in an aqueous medium having an ionic strength between 10 and 10, and a pH between 6 and 10,

I. MCEP

II. MEEP-C (h-c- or H) m NH-COCH3

III. MIEP-

Scheme A may be modified so that MIEP is derived so as to be covalently attached to a polyphl, such as a maleimide derivative, while the peptide is so linked covalently to free sulfhydryls. This further process would naturally fall within the scope of this specification including variations of this process. such as variations in the reaction sequence of activated species, or reagent ratios. The process for producing the conjugates of this invention may be employed to produce any conjugate in which a peptide-protein conjugate is desired and is particularly significant where increased immunogenicity of the peptide is required,

The conjugates described herein may be included in compositions containing an inert carrier and are useful when suitably formulated as a vaccine. This may include first the adsorption on alumina or combination with emulsifiers or adjuvants known in the art of vaccine formulations. The methods which employ the covalent conjugate immunogens of this invention include &lt; a &gt; Use as an immunogen to achieve ΗϊV neutralizing antibodies in a mammal whose antibodies can be isolated and administered to a human in order to prevent infestation by HIV5 or to limit the proliferation of βIV infestation, or to treat humans affected by infestation or. Î "V diseases including AIDS, (c) use as a vaccine to immunize humans against H1V infestation or to treat human post-infection or to assist an immune neutralizing immune response in a human infected with AIDS, or H1V diseases including

As a laboratory tool, the conjugate is useful when administered to mammals in an immunologically effective amount to generate an anti-PND peptide, anti-HIV immune or neutralizing immune responses, the mammal may be enhanced with additional conjugate to raise the immune response, the antiserum is obtained from such a mammal by bleeding the mammal, centrifuging the blood to separate the cellular component from the serum, and isolating the antibody proteins from the serum if necessary, according to the procedures known in the art. Such antiserum or antibody preparations may be used to characterize the efficacy of an HIV PMD peptide in a conjugate for. create anti-peptide PMD, anti-HXV antibodies or mammalian HIFV neutralizers in a mammal. ELISAs using the unconjugated peptide are antisera are useful in in vitro tests to measure the induction of anti-peptide antibodies . An in vitro assay for measuring HIV neutralizing capacity of the antiserum comprises incubating a living HXV preparation with an antiserum preparation, then incubating the antiserum treated HIV preparation with CD4 receptor bearing cells, measuring the extent in. protection provided by the antiserum. These assays for the characteristics of the antiserum produced by a given conjugate can be used to study the structure-function relationship of the peptide PHB. The conjugate is useful for inducing mammalian antibody responses as described in the previous paragraph, such antibodies may be used for passively immunize humans to prevent HIV infection, or to limit post-infection HIV proliferation, or to treat affected humans with infection or HIV disease including AIDS, cs

The conjugate is useful for a vaccine which can be administered to humans to prevent the infestation or proliferation of HIV or humans suffering from HIV disease from HIV infection, including AIDS and related complexes, or to humans with seropositive test for the HIV virus. The conjugate may be administered in conjunction with other anti-HIV compounds, such as AZT, or more generally anti-viral compounds, or in conjunction with other vaccines, antibiotics, or immunomodulators (see Table 1 below). A single molecular species of the antigenic conjugate III will generally be useful as a useful adjuvant for the prevention or treatment of HIV disease including AIDS or ARC. Other antigens in the form of the mixtures are also advantageous and consist of a mixture of conjugates differing, for example, in the ratio of peptide mass to total protein = conventionally, the conjugates in a mixture may differ in the amino acid sequence of the PMD '

A vector, carrier or immunological adjuvant may be added as an immunological vehicle in accordance with conventional immunological tests or practice '

Adjuvants may or may not be added during the preparation of the vaccines of this invention. Aluminum is the typical and preferred adjuvant in human vaccines, especially in the form of a thixotropic, viscous and homogeneous aluminum hydroxide gel. For example, one embodiment of the present invention is the prophylactic vaccination of patients with a suspension of alum adjuvant as carrier and a mixture of conjugates as the selected set of immunogens or antigens'

The vaccines of this invention can be administered efficiently either in pre-exposure or post-exposure beads, in combination with effective amounts of the anti-viral agents. immunomodulatory antibiotics or AIDS vaccines from Table I Cfonte5 Market Letter "Nov 30 = 1987? p 26-27; Genetic

Engineerinq News .. uan "198a" Goes «p» 26 «3

i

Warm of the drug AL-721

TABLE I

Producer Ethigsn

Indicaglo ARC, PGL BETASERON &lt; interferon beta)

Triton Biosciences AIDS, ARC, Ktí

CIPRRYSYN)

Carrington Labs

ARC CYTOVENE (ganclovir)

Bynlex

JMV DDC &lt; dideoxystyridine)

Hoffmann-La Roehe

AIDS, ARC FOSCARMET &lt; trisodium phosphonoformate &gt;

Astra At inf HIV, CHv retinitis

Rbone-Poulenc Sante infection by

HIV Abbreviations? AIDS (Acquired Immunodeficiency Syndrome)? ARC (AIDS Related Complex) p CMV (Cytomegalovirus, which causes an opportunistic infection resulting in blindness or death in AIDS patients) 5 HIV Human Immunodeficiency Virus, formerly known as LAV, HTLV-III or ARV) k KS (Sarcoma of Karposiís, PCP (Pneumonia Pneumonocystis carinii, an opportunistic infection> PSL (Persistent generalized lymphadenopathy), v 5

Nojg ...... da, Uroqa Producer Indications

ORWIDYL Merrell Dom PCP <eflornitins)

PEPTIDE T Peninsula Labs AIDS (Occurrence of octopus /

RETICUL03E Advanced Virus AIDS, ARC (nucleophosphoproline) Rssssrc h

IR Bu.rrouohs welieoms AIDS, ARC

(zidovudine ΑΖΪ), pediatric AIDS, KS, ΗIV a s s i n i ", ΗIV less acute, neurological involvement.

aJ

J

RIFABUTIN ans am am ic ic ic ic ic ic ic ic ic ic ic ic ic ic L L L L L L L L L L ΑΦ. int0r1'5v &quot; Ãu al f

Ad

U.- L * HJ

Warner-Lam

HKL -! LI-

Ueno l-iofcf C-hssis h f LA AHC Industry iCfSl jci Air

Burrouans KS? HXV? sfn comtainscSa com Β I jsunompd »1 adores

Proliferation of Bacteriolytic Protein (PPR) (Bropyrimine) Upi ohn AIDS Advanced k: s AMPLIBEN CRNA poorly paired) DuPont HEli Research ARC, PSL (Antibody Intrinsic Antibody) Advanced Biotherapy Concepts AIDS, ARC, KS Stimulating Factor Santyocene Senetics AIDS, ARC, Colony HI (CSF) Institute KS CL246738 CCL246738) American Cynamid AIDS IMREB-1 AIDS, ARC, PSL, KS IHRE6-2 Imreq AIDS, ARC , PBL, KS IMUTHYOL (diethyl carbamate) nerieux Institute AIDS, ARC IL-2 (interlaucine-2) Cetus H i ο Ko

Morna da Drooa P fi-d U t ϊ_! Γ Indications L L-2 &lt; interleukin 2 &gt; Hoffmann-La Rache Immune KIDDEN; S! INTRON-A i n t r f r S a 1 f a) Sc heri ng- &quot; P 1 oug h K8 ISORRIMOSINE < inosine drugs) Newport Pharmaceuticals ARC = PSL, HIV-infected patients (methionine sncafaline) INI Pharmaceuticals AIDS ARC rlTP-PE &lt; 1, 2, 3, 4, 5 & Ciba-Geigy ΤΗΥπΟΡΕΝΤIN (TP-S) (thymic compound) Ortho Pharmacsuticals HIV infection by ROFERON CintsrfsrSo alpha) Hoffmann-La Roche KS (erythropoietin recommander) Orthopharmaceuticals severe anemia associated with AIDS & therapy of RETROVIR TREXAM Cnaltrexone) OuPon ™ AIDS, ARC 5f Sr

iWF tumor necrosis factor)

UjeSíf ϊ i! i

H-1: 5 K :: ffi cation with interferon qam

C. ANIMAL

LyphoMed

PCF PENTAM 3 PC

Pentanylidene Cisstethionate)

AIDS, ARC

Sag

It should be understood that the scope of the vaccine combinations of this invention with antivirals, immunity, antibiotics or vaccines against AIDS is not limited to the list in the Table above, but in principle includes any combination with any pharmaceutical composition useful for the The AIDS or HIV vaccines of this invention include vaccines to be used pre- or post-exposure to prevent or treat infection or disease by ΗIV, and are capable of producing a specific immune response to the immunogen.

The conjugates of this invention, when used as a vaccine, are to be administered in immunologically effective amounts. Dosages of between 1 pg and 5æm conjugated protein, preferably between 50æg and 3æg conjugated protein are to be administered to a mammal to induce anti-peptide, anti-HIV, or neutral is immune responses prior to HIV. Approximately two weeks after the initial administration, a dose of the booster may be administered, and then again whenever the serum antibody titers decrease. The conjugate should be administered intramuscularly or by any other convenient or efficacious route at a concentration between 100æg / ml and mg / ml, preferably between 50æg to 5æg / ml, in a volume sufficient to total required for immunological efficacy. The conjugate can be preadsorbed on aluminum hydroxide gel or in RLBI adjuvant &lt; GB 222021IA, priority document US 212 919 required at 29 / Θ6 / Ι988) and suspended in brine physiological prior to injection. The protein moiety should behave as a desirable immune inhibitor in the choice of protein to avoid those resulting in non-specific activation of the recipient immune response toresorbicity. In US Patent 4 695 624, H &amp; rburg et al. Used the outer membrane protein (OMPC) complex derived from Meisseria meninaltidis to prepare polysaccharide-protein conjugates. The OMPC proved to be suitable, Other immunogenic proteins may be used. The present invention utilizes the major immunosynchronized protein (a) &lt; MIEP &gt; OMPC Class II purification methods Various purification processes have been devised for OMPC from Gram-negative bacteria EFrasch et al., J. Med. Chem., 140, 87 (1974). Med. 147, 629 &lt; 1978) Zollinger et al U.S. Patent 4,707,543 (1987), Hsiting et al., Acta Path Microbiol. Scand. Sect. C, 89, 69 (1981) Helting et al. , U.S. Patent 4,271,133. OMPC may be used herein essentially in accordance with the Helping procedure, from which MIEP can be further purified by LMurakami, et al., Infection and Immunity, 57 , 2318 (1989), to provide an immunostimulation necessary to induce immune responses in mammals to HIV PND peptides, H1EP can be derived by dissociation of the isolated OMPC, or. alternatively, produced by the recombining expression of the desired immunogenic portions of OMPC. The methods of preparing and using an OMPC subunit are described in US Patent Application Ser.

USA co-pending Series Nos. »555 d29p 55o 9 / 8.5 ε 555 2 & 4 (Merck Cases # 18159, 1811Θ, and 1818ΘΡ respectively)

HIV PMD peptides which may be used to prepare species of the conjugate of this invention may be linear or cyclic peptides. Linear peptides may be prepared by the known solid phase synthetic peptide chemistry, expressing reminiscent of DMA encoding the peptide sequences or by fragmentation of the isolated HIV proteins. Cyclic HIV PHD peptides may be prepared by cyclization of linear peptides, for example (a) by oxidation of peptides containing at least two cysteines to generate linked cycles by dissolving the peptides. by forming an amide-linked cycle (c) by forming a thioether-linked ring. The processes for the preparation of such peptides are described herein, but this description should be construed as exhaustive or limiting. The conjugates of this invention are useful whenever a component peptide is an HIV PMD or is capable of inoculating immune responses in mammals that recognize HIV PMDs'

The peptides PMD5 are those known in the art and the novel compounds described herein and separately claimed in commonly assigned U.S. Patent Application Serial Nos. 555-112 and 555,227 (Merck Mos Cases, 18149 and 1815) and Merck Nos. 18 (1681 B) are defined as peptidyl sequences capable of inducing an HIV-neutralizing immune response in a mammal, including the production of HIV neutralizing antibodies »

A major problem overcome by the present invention is that of the variability of the HIV inhibitory sequence. For example, in PND occurring in the third hypervariable region of gpi2 (see abaiKo), although certain amino acids have been shown to occur in isolates, there is no strictly preserved piece of primary sequence. This difficulty is overcome by this invention because it allows the conjugation of a mixture of peptides having PND sequences from as many HXV isolates as necessary to obtain a broad A mixture of highly protective conjugates may be prepared by admixing conjugates, each of which is prepared separately with a portion of the peptide which provides pro-ionization against a single or several isolates of HIv =

Amino acids found near or between the amino acids 296 and 341 of gp122e have been shown to meet the criteria that define a PND = Mo isolated HIV IIIB a sequence of 41 amino acids has been described as follows &lt; tb &gt; -He Asn Cys Thr Arg Pro Asn Asn Asn Thr Arq Lys Ser Ile Arg He Oln Arg 6 Iy Pro Oly Arg Ala Phe Go Thr Ile Gly Lys Ils Gly Asn Met Arq Bln Ala His Cys Asn Ile Ser --- 5 being the two cysteines disulfide bonded to one another to form a loop. The trimer-Bly Pro Bi and is exposed at the end of the LDL loop. Peptides from different HIV isolates from the same gp120 region form isolated specific neuiralizing antibodies when presented to systems (Fissurella) hemocyanin conjugates. The major neutralizing epitope in the 41 mer sequence shown above is comprised of the eight amino acids that surround and include the trimer Gly Pro Gly- The following table shows a number of linear peptides of different length and composition that can be used to prepare the conjugates of this invention, given the name of the isolate which contains a peptide having the tabulated peptide sequence, together with a name hereby assigned to that

10 'C and peptide for ease of reference. of each of the peptide peptides possessing a unigenic iim protein? 'V * jP; As a result of the binding of the peptide, the amino acid sequence of the peptide is substantially equal to that of the peptide. ΜΣΕΡ in that position »• such as norleucines • PMtMga ..... PHD dg HIV .Lineares

Isolation of peptides Name HIV SEQ ID No MN R-Tyr Asn Lys Arg Lys Arg PND 2 2 Ile His I 1b Sly Pro Sly Arg Ala Phe Tyr Thr Thr Lys Asn He Ile Bly Thr SC r-Asn Asn Thr Thr Arg Ser PND- SC 3 Ile His Ile Sly Pro Sly Arg Ala Phe Tyr Ala Thr Gly Asp Ile Ile Sly Asp Ile IΙΪΒ r-ft-sn Asn Thr Arg Lys Ser Ile PND13S 4 Arg Ile Bln Arg Sly Pro Sly Arg Ala Phe Vai Thr Ile Gly Lys lys Gly Asn IIIB r-Arg Ile Gin Arg Gly Pro Gly PND135-1S 5 Arg Ala Phe Vai Thr Ile Sly Lys Ile Gly Asn IIIB MN Arg-Arg Arg Sly Pro Sly PND135-12 6 Arg Phe Vai Thr-His Ils Sly Pro Sly Arg Wing PND-MNS 7 Phe

q. - MN R - G I and Pr o lly Arg A 1 PND-MNó 8 LftV-1 r-Ile Sln Arg Ala Phe B1 and Pvn. Arg-Phe-LAV-1 hf SF2 -I le Tyr 11a Ala Phe Sly Pr d Slyn and PND-SF2 Ί C'i NY5 r-Ile Ala Ils Thr Leu SI v Pro B1 and Arg PN / J -117 CDC4 Go Trp Gly Pro J v Arg PMD-CDC4 12 RF r-Ile Thr Lys Go Ile G1 and Pro Gly Arq PMD-RF ·? T. ELI r-Thr Pro Ils Ser Leu! Ri 1 and Leu SI v Bln PND-ELI 14 *.-Ϊ. (7t r-Thr Pro Ils Ala Leu 61 and Leu Sly Gin I-1, r-1, r-1, r-Ile His Phe A1 a Leu G1 and Pro Sly Sln PND-liAL 16 7 '' '' '' '' '' '' '' '' '' '' '' '' '' '' '' '' '' '' '' '' However, there is an illustrative guide for the purposes of the suggestion or the instructions of the authors. PMD as described

Pd can form the

R

immunogen of this invention are any of the tabulated peptides or immunologically equivalent variants of the subject suggested by these peptidyl sequences. The nature of the variations is considered hereinafter. The primary sequence of this HIV PND appears to have a conserved core amino acid sequence comprised of the tetrameric sequence -Bly Pro 81 and Arg-, CSEQ IDs i8s) with a divergence increasing on both sides of this sequence between the HXV isolates Some isolates have sequences that diverge even in the tetramer 5 having nucleotide sequences -8, and Pro 81 and Lys ™ (BEQ and 19S), -Bly Pro Bly 8Ln- (8EQ IBs 2), and even -Bly Leu Gly Sln-CSEQ IDs 21s). All of these possible sequences are within the scope of this disclosure as peptide sequences which are advantageous for conjugation according to this invention. The length of the peptide is a significant factor in promoting chronic immune responses. That is, an immune response raised against a given peptidyl epitope may recognize similar epitopes of the same or different HIV isolates based on the number of amino acids in the above-mentioned antibody over the critical neutralization epitope. Additionally, the length of the peptide is also responsible for determining the likelihood of immune system imputation of the determinant responsible for the production of a neutralizing HIV response.

In order to minimize the probability of presentation of important epitopes. the chemistry has been developed whereby the PND peptides can be closed in a given three-dimensional configuration. The PND of 4 amino acids of the 11β isolate of HIvf is known to be. represented above, is formed as a loop by the presence of the cysteine-to-cysteine disulfide bond. However, the disulfide compounds may be present under certain conditions by

The peptides may allow the loop to open ε if the peptide exists in a linear fashion. Accordingly, in addition to the linear peptides, the cyclic peptides attached to disulfide and the novel HIV PMB peptides having cyclic non-lipid structures disclosed herein but separately claimed as free peptides are disclosed in U.S. Patent Application Ser. )? 555 112 and 555 227, may all be used as the PEP component in the formation of the conjugate of this invention.

Peptides that can be used in the formation of these conjugates can be derived as fragments of the natural proteins (for example), by recombinant expression of their moieties or by chemical synthesis according to methods known in the art. In addition, the novel cyclic PNDs may be synthetically prepared according to the processes described herein. The sequences may contain either L-amino acids or unusual u-amino acids or amino acids. Additionally, the chemistry of the conjugation is sufficiently flexible so that the appropriate choice of peptide derivatizing reagents allows for successful conjugation.

Synthetic peptides have been prepared by a number of strategies conducted either in solution or on solid supports. The excellent texts that cover the principles and techniques, basic, sound Principles of Peptide Synthesis. Bodanszky. M., Springer-Verlag &lt; 19841 § Solld ....... Phase ...... Peptide. Synthesis,

Stewari J, M., Young, J.D., Pierce Chemical Corapany (21 ed. 1984) and The Psptides. Sross, E., Meisnhofsr, J., Academic Press, Inc., &lt; 1979). However, the processes described herein are not limited to the description of these texts.

Synthetic cyclic peptides may be prepared in two steps. First, the linear peptide can be synthesized on a Hilligen peptide synthesizer 9Θ5Θ or an ABI 431A using the chemistry of 9-fluorenylmethylcycloalkylcarbonyl (Fmoc) and pentafluorophenyl esters of amin protected FmacGas which are known reagents or using Wang derivative derivative, PmGC chemistry and symmetric amino acid anhydrides

Fmocs protected in the side chain, prepared in___situ, as

Second, the linear peptide can be cyclized either in solution or with the peptide still bound to the solid phase resin. Cyclization may be performed by any technique known in the art, which may comprise, for example a) the incorporation of cysteine residues in the linear peptide at either end of the sequence which will form the ε-loop allows the formation of the disulfide bond under oxidative conditions known in the art; the preparation of a cysteine-containing peptide as in (a) but maintaining the cysteines as free sulfhydries Cou as mA-protected thiols which are deprotected to free sulfhydryl) and treatment of the peptide with oxyethylene dibromide or a similar reagent, such as diiodide, dichloride or a di-halogenated straight or branched chain lower alkyl having from two to eight carbon atoms; such reactants react with the sulfur atoms of the cysteines to form a cyclic structure containing two non-benzene or alkyl-labile thioether bonds; c) allowing a free group on one side of the looped amino acids to be linked by amide to the free carboxyl group on the other side of the looped amino acids through the formation of peptide bonds mediated by DPPA, BOP or similar reagents. of these strategies is given in more detail below, upon presentation of a generalized description of the cyclic peptides produced by these processes.

Thus, without limiting the invention of conjugates of the following peptides or production sites, the following peptides

Which may be useful after appropriate grouping of peptides when necessary according to this invention include those represented by the PEP structure which includes the linear peptides of Table 1 above and the cyclic peptides: • r1-n-r8-c- r6

Pro-Gly /? Gly ArgR 2 W? -R 5 7 wherein: r is the position of 1a bond between PEP and optionally a marker amino acid, if any. d α m arqueros t ***. n is 1 a) a bond, 1 b) a polypeptide of 1 to 5 amino acids; A labeled aminocarbon which migrates. in a preferred embodiment of formula (I) which is isolated from amino acids which occur essentially in the range of amino to furoxy. } 5-alanine or ornithine

it's

a) or a bond or a peptide up to '7' amino acids if R &quot; is a peptidD of at least 2 amino acids, or b &gt; a peptide of 2 to 1.7 amino acids, if R3 is a bond, or a linkage or a peptide of up to 17 amino acids if R3 is a peptide of at least 2 amino acids. or b) a peptide of 2 to 17 amino acids, if R &quot; is a bond or a) -NH-CH-CO-, with K is attached to the merino carnon, if R '= -7; to Ι-Γ iZT.  € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒâ € ƒR ', if R "is carbonyl or - ) MHCO-? FC a? a peptide of one to five amino acids, optionally including a marker sminoacid, b) -OH, -COOH. d) -CONH, or e) -NH2, f)

R a) a side chain of amino acids selected from

from the side chain of any of the common D-amino acids, (see Definitions and Abbreviations table), if optional link 7 (...................) R 3 is absent; b) -R d-BS-, or if R 1 is CF 2, or c) -RW-HH- if R 'is -C -CeOs 0 CONHL · b) -C = 05 or έοΝΚ.

R R &quot; a bond or lower alkyl of one to eight carbons; RV is a)

or b) xllylene

a) lower alkyl, or b) -CHO-O-CHâ,, - Z is. When a peptide is synthesized with a protected amino terminal amino acid, the amino terminal protista group such as henzyloxycarbonyl (Z) to protect amines, or artamidomethyl (Ccm) to protecting the sulfhydryl groups can be removed according to the processes known in the art and herein as shown. The deprotected group thus disclosed can be used in the formation of covalent bonds, via the β-immunogenic protein linker then the amino acids R '-81 and Pro 01 and Arg-K' which form the nucleus of the PND peptides and contribute to the formation of the loop of a cyclic peptide will be referred to as loop amino acids or micelles' However, when the optional 6 * 7 bond between Ru and R &quot; is missing, the structure, PEP. is linear and includes all linear peptides of Table II.

Whether the peptide is linear or cyclic, the β-amino acid sequences comprising PEP E and K 'may be any combination of amino acids, including the sequences surrounding the tetramer -Bly Pro Bly Arg- any of the sequences of the Table # Thus, the amino acids of the nucleus represented by -R '-Gly Pro Sly Arg -K- - may be further defined as having the amino acid structure of ~ W2 ~ Siy Pro nuclei Β1Υ ftrg-K3X4Kfl |

What is the meaning of the term? lesson & ck ctes a / ss? . ΧΠ c &gt; srginin d) -histidine, a) cit. a (r i π a si cu f) trsoninas

VcnstituintB ds R '"

. b) arginine, d) raionothionine; X is a constituent of the formula: ## STR1 ## is a constituent of R 1 selected from: a) alanine. b) arginine, mc) valines ΚΆ is a constituent of ΚΆ is selected from (a) phenylene, (b) isoleucine, (c) valine, or d) leucinap is a constituent of R ' s is a linkage of a peptide to up to 15 amino acids.

Cyclic peptides may be disulfide-bonded or single-bonded structures formed through a non-labile linkage or structure. The term &quot; non-alkyl bond &quot; means a covalent bond, other than a disulfide bond. Examples of such non-aliphatic linkages are thioether amide linkages as described in co-pending U.S. Patent Applications 555 112 and 555 257. These covalent linkages may be through a bridging structure 5 such as xylylene, via a lower alkyl, through -CH 2 -O-CH 2 or via a bridged linker. By changing the bridged structure and / or the combination and the combination of the smino-acids included in the peptide, the structure of the loop structure of the cycle can be optimized, allowing the good attunement of the β-spitoto presented to the immune system . For example, the use of an oxy-lithene bridge generates a &quot; stretcher &quot; loop structure &quot; different from when it is used, for example, an eight-carbon straight chain lower alkyl, such as the bridge. Thus, the conjugates of this invention are useful as either reagents for analyzing the c ratio

the structure-function of the PMD epitaphy in the generation of anti-peptide immune responses, anti-HIV, neutralizing antigens and anti-BIDA to mammals, or as components for the formulation of anti-disease and viral vaccines including AIDS

The synthetic products obtained can be characterized by mass spectrometry with fast bombardment of EFAB-MSj atoms? Reverse phase HPLC, amino acid analysis or nuclear magnetic resonance spectroscopy (CRHN). Cysteines bound by ..... Disulfides

Peptides containing cysteine residues on either side of the looped amino acids can be cyclized under oxidizing conditions to the disulfide-linked cycles. Methods for achieving dissulfide bonding are known in the art. An example of the disulfide bonded peptides useful in this invention is given below in Example 1, where cPMD4 is produced and in Example 1 where PND33 is produced. a process is used which utilizes the cysteine derivative mAb to generate disulfide-linked cPNDs, but other processes are also applicable. In Example 18, the peptide containing two sulfhydryls is oxidized to dilute acid. Disulfide-linked peptides are preferred in present invention.

Thus, in a preferred embodiment of this invention, the peptide has the structure C8EQ IDs 18;) s

Pr ο Η Η O Gly r-R1 -N-C-C-R2 & -σίγ

Arg-R-N-C-C-R-

-R ου. of its pharmaceutically acceptable salts, in the absence of hydrogen. i where and where rr? i f -O

O

0

CH

U

~ "Ί ί ί ί ί ί ί ί ί ί ί ί ί + ΙΛΙΛ Á. .1 \

OR

HS G

R 1 is

a) the binding, or optionally including b- a 5-amino acid peptide, in particular a marker-amino acid; A peptide of 3 to 1 amino acids 3 K &quot; is a peptide of 1 to 5 amino acids a) -QH, a) a peptide of 1 to 5 amino acids, optionally including a roar amino acid, or R 1 &quot; is lower alkyl of one to eight carbons. The lower alkyls consist of straight or branched chain alkyls having one to eight carbons, unless otherwise specified. Then the amino acids

Pro Bly Arg-R'- ', which contribute to the formation of the loop of a cyclic peptide, will be referred to as looped amino acids.

In one embodiment of the invention, the cyclic peptide having the structure &lt; SEQ IDs.

Η Η Ο H-Nle-NCCX Χ 2 D Ργο -Gly / G Gly Arg Ή Κ Η Η Η Η Η Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ Γ H H H H H H H H 8

I SH

Prg -Clyl Argxnx, x2 x3xtxm-

N-C-C-R 5 SH m which is as a constituent of the formula: 1 a) serine, b) proline? c) droinine. d &gt; histidine, e) g 1 lysine 5, which is r-. •? or

f) threonines

Xr is a constituent of R &quot; selected from the group consisting of a polypeptide selected from the group consisting of polypeptides, polypeptides, polypeptides, polypeptides and polypeptides. vinsins, or d) methionines

X 1 is a constituent of R 1 - and is an amino acid. a peptide up to 8 amino acids; Xâ, ... is a constituent of R "selected from; a) alanine, b) arginine, = or c) valines X 'is a constituent of R' 'and is selected from H' (a) and (ii). b) isolsucin; c) valine, or ri) 1 isuccinyl is a constituent of -C and is an amino acid or a peptide up to "asino acids" is preferably isoleucine.

The novel cyclic disulfide-linked peptides used in this invention are separately claimed in the case

Merck co-requisite 18.68%, USSN ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________ protected in the chain, side as reagents or using the Wang derivative derived? Fmoc chemistry and symmetrical Fmoc amino acid anhydrides protected side chain, prepared in situ as reagents.

Second, the linear peptide is cyclized either in solution or with the peptide still bound to the solid phase resin by incorporation of cysteine residues into the linear peptide at either end of the sequence that will form the loop the oxidation of these to the disulfide . In one preferred embodiment, the cyclization is performed by exposing the peptide to <a) H, Cu, <b) atmospheric oxygen, (c) aqueous CA-UCN contains about 0.5% TF A or Cd) ferricyanide about θ, π. The preferred process is exposure to atmospheric oxygen.

The products obtained can be characterized by mass spectrometry, with fast bombardment of EFAB-MS3 atoms, reverse phase HPLC, amino acid analysis or nuclear magnetic resonance spectroscopy (NMR).

As used herein, the peptides useful in this invention may be prepared as further described below in Scheme 1, and (ii) the peptide of the peptide in the peptide. 1 _7

A linear peptide containing C * and U * &quot; on either side of the 5'-amino acids in the loop, wherein the two cisterns or another amino acid containing free sulfonyl groups on the side chain, is prepared according to known synthetic procedures (see discussion above), Mo cyclic PWD In addition, the sulfhydryl side chains (-RU-SH &gt;) contribute to the preparation of the -R-S- groups of the completed cyclic HIV HMD structure shown above. The amino acids to be incorporated having reactive side chains R &gt; groups are used in a suitably protected form in the group R. For example, the histidines are triphenylmethyl- or Boc-protected and arginine is 4-methoxy-2,3, 6-trimethylsulfonyl- (Mfc) protected,

Preferably, a resin is obtained in its protected form by mAb bound to the resin, for example, Fmoc- "CysCAcm resin" "Wang, cysteine incorporated into the asynine terminal side of the loop amino acids, C % may also be the 17 derived mAb, either C * or C ** may be linked to additional amino acids, R * or FT respectively, which may be used in the formation of conjugates with carrier molecules or may serve as amino acid labels for subsequent analysis of amino acids, such as when norleucine or ornithine is used, the sulfate of the acatamidomethylated cistaines is reacted at room temperature for about 15 hours in a resin compatible resin such as a concentration of 1-50% of an organic acid , preferably about 10% L-acetic acid in anhydrous dimethylphosphamide (DMF), with a molar excess of

(OAc &gt; n2), for each of the resulting heavy metal thioether group, for example, the mercuric acetate thioether of the peptide &lt; RTI ID = 0.0 &gt; PEP &lt; / RTI &gt; S-HgOAc The addition of the excess hydrogen sulphide in DMF yields the insoluble metal sulfide, for example mercuric sulfur (CH3 O), to the peptide with free sulfhydryl groups. The free sulfhydryls are then oxidized by one of the above processes In addition, the amine-protected thiols can be converted directly to the cyclic disulfide by treatment with methanol / DMF solvent.

It essentially applies to the same process described above for solid state cyclization with two major variants and the peptide is cleaved 95% TFA / 4% ethanouithiol / 1% thioanisoyl) from a pepsin KA resin. Also, if the CystAc protection is used, the cleavage of mercaptoacetate / hydrogen sulphide is required for the group - 8H, as an independent procedure, with the linear peptide either on the resin or off the resin.

However, a process &amp; the use of protection

Cys (Acm) and Sasrina or Pepsin KH resin, and cleavage of the linear peptide tc-protected from the resin with 0.2% TFA / NaCl. The mercuric acetate / hydrogen sulphide then selectively converts to Cys 2 C ) in the free -SH group and cycling is carried out in the otherwise protected peptide. At this point, the peptide may be maleimidized in situ, selectively at the N-terminus. The acid labile side chain probe groups are

cleaved with 98% TFA / 2% iananisole and the cyclic palladium is isolated by HPLC. However, the preferred process is to cleave the peptide of the resin to allow cycling by one of the above-mentioned processes. to allow oxidation to air for about one to fifty hours between 40 and 40 ° C.

Thus, in one particularly preferred embodiment of this invention, a peptide &lt; CPMMD 33) having the structure CSEQ (Ds 22s); H-Nle Cys Tyr Asn Lys Arg Lys Arg Ils His lie Bly Pro} {81y Arg Ala Phs Tyr Thr Thr Lys Asn

Is conjugated to ΜΪΕΡ either through the amino terminus Mie or of one of the internal lysines to generate a or a mixture of all • -υ structures

The compound of formula (I) is prepared by reacting a compound of formula

Η 1 C-H-H-1-Cj-C !- ieoc «cyi

Tyr Mn Ly · Arg Ly »Arg 22 · Kl · li · Oly Fre

(A) Mg e-2) c-Mg ty-Mg XL · · U · &quot; -CH2 -CH2 -CH2 -CH2 -CH2 -CH2 -CH2 -CH

C-K- (CII) 4-β-K-Mn Tyr γ-M-γ · x1 · r1 · Mn Lye · N1 · COOH

I Η H

Tyr Thr is -CH3 -CH2 -CH2 -CH2 -CH (X-tCX, J, -CN, -CCl,) 4

OiC-Mg il · κι · II · Oly Pr o 01 and Aro Alt ptw Tyr Thr O-N-Xrj Ly · tan Tyr ey .- * - * - ey · Oly II · II · | Un Ly ·

R 2, R 3 and R 4 are as defined in claim 1, wherein R 1 and R 2 are independently selected from the group consisting of hydrogen, C 1 -C 4 alkyl, HHCOCH, 0.05

, Wherein R 1 is the weight percent of isomers in the range from 21 to 5% by weight, % of the peptide in the conjugate and total mass of the protein in the conjugate.

to- Peptides dc. A linear peptide 1 contains C and CP &quot; C &quot; and &quot; C &quot; and &quot; C &quot; on either side of the loop amine acids, where 1 ... o C and ι: * &quot; are both oistein or another sulfhydryl-containing amino acid is prepared in accordance with the known synthetic procedures (see discussion above). In the completed cyclic PMD, € 1 to 7% are part of the R "and R" groups ; of the PEP structure shown above. The amino acids to be incorporated having reactive side chains (R groups) are used in a suitably protected form in the group R = For example, histidine is protected triphenylmethyl (Trt), arginine may 4-fluoro-1,2,3,6-trimethylphenylsulfonyl- (Mtr) protected tritylols of____Peptide

Synthesis »Bodanszky» M », Springsr-vsriag C984) f Solid____Phase

Peptide Synthesls, Stewart 3, M, Young, D, Pierce Chemical Company &lt; 2d ed 1984) | s The Feptides, Gross, E., Meienhofer, J, Academic Press, Inc, (1979), 2

Preferably, a resin is obtained with C in its protected form by mAb already bound to the resin, for example, Fmoc-L "Cys (Acm) -0 ™ Wang resin. The cysteine incorporated into the amino-terminal side of looped amino acids, C% may also be oio derivative Acm. Wusr C "or ΙΓ 'may be attached to additional amino acids, κ, or R' &quot; respectively, which may be used in the formation of conjugates with carrier molecules or may serve as marker amino acids for subsequent amino acid analysis, such as when using norleucine or ornithine, the sulfur of the acetamidomethylated cysteines is reacted at room temperature for about 15 minutes. hours in a solvent compatible with the resin, such as 1% acetic acid in anhydrous dimethylformamide (DMF), with a molar ratio of

approximately four times greater than that of the heavy metal salt, such a mercuric acetate CH ((OAc), for each resulting heavy metal amide group, for example the acetate thioether of the peptide-S H-H₂OAc,, is The addition of excess hydrogen sulphide in DMF yields the insoluble metal sulfide, for example sulfuric sulfur (HaS), and the peptide having free sulfhydryl groups, a mixture of about equimolar amount , when compared to the peptide, dibrorostat or α-xylylene dichloride, a dihromed or dichlorinated lower alkyl, -CH-O-CH-1,3-dihalogenated or a similar reagent which will provide a desirable chain length a large excess of tertiary amines 3 is preferably added, preferably triethylamine (NE 4) in DMF. The reaction with the bis-sulfhydryl peptide portion is allowed to proceed for about sixteen hours at room temperature The deprotection of the acid-sensitive side chain protecting groups and cleavage from the resin are carried out by treatment with 95% trifluoroacetic acid (TFA) in the presence of of 4% 1,2-ethanediitrile and 1% thioanisole. The dissolved cyclic peptide can then be separated from the resin by filtration, the filtrate is evaporated and the crude product is purified by high pressure liquid chromatography (CHCl 3) with known procedures, for example by reverse phase HPLC, LLP, LLP, LLP,

The same process described above for the solid state cyclization with two major variants applies; If the peptide is cleaved (95% TFA / 4% ethanediol / 1% thioanisoyl) from a pepsin KA resin, they are

also removed; the acid labile side chain protecting groups including CysRt) which provides the required free SH function. However, if the Cys 2 A moiety is used, the cleavage of mercuric acetate / hydrogen sulphide is required for the free -SH group 3 as an independent procedure, with the linear peptide either on the resin or off the resin.

However, the preferred process is the use of the Cys (A) protection of Sasrina or Pspsina KH resin, and cleavage of the fully protected linear peptide from the resin with TFA at 0 ° C / CH 2 Cl 2. The mercuric acetate / hydrogen sulfide then converts to Cys (A) into the free -SH group and cyclization is carried out on the otherwise protected peptide. The acid-side side-chain protecting groups are cleaved with 95% Removal of excess reagents such as xylylene dibromide which does not precipitate prior to acid cleavage of the protecting groups is conveniently carried out, for example, as described above. by a discontinuous gradient reverse phase HPLC operation prior to more salient gradient elution.

Cyclic PND peptides prepared according to this subsection include, but are not limited to, the sample cPMDs shown below. The procedures of this subsection are applicable to small peptides and particularly applicable to peptides between 5 and 3 amino acids An optimal ring size may include from 5 to 10 amino acids, including the Gly-Pro-Gly-3 trimer, and this ring size is readily maintained by production of linear peptide rings having the number the amino acid combination =

S 9

Representative peptides resulting from the process described in this subsection b) parts Ci) and (ii) are described in U.S. Patent Application Ser. the invention of conjugates should not be construed as limited to the use of those particular embodiments of HIV cyclic PND peptides. Other linear HIV PND peptide sequences may be essentially cyclized in the same manner as to provide those peptides. Serials could be generated of peptides having divergent primary sequences and would be beneficial in this invention as long as they continued to induce an anti-peptide, anti-HXV or neutralizing immune response of HIV and Cyclization via Afflicted Formation

The novel amide-linked cyclic HIV PND peptides can be prepared by essentially two-stage conjugation. First, the linear peptide is prepared, for example in an ABI-43IA peptide synthesizer, by the synthetic chemistry of the solid phase peptides, for example using Fmoc chemistry and the amino acids appropriately protected in the side chain, as reactants =

Second, the linear peptide is cleaved from the resin and cyclized in solution allowing the free amino terminus of the peptide, the amino group to be free of an amino terminal isoglutamine, or a β-amine or β-amino group free of a lysine on one side of the loop amino acids, is amide-linked to a free carboxyl group on the carboxy-terminal side of the looped amino acids through DPPA-mediated peptide bonding? BOP or similar reagent u.ik '

The obtained products can be characterized by mass spectrometry of the fast bombardment of atoms

ChAB-ray, reverse phase HPLC, amino acid analysis or nuclear magnetic resonance spectroscopy; »

Thus, the presently preferred embodiments of this invention are conjugated having covalent linkages of HIEP to an amide-linked cyclic HIV PND prepared as described above. Where the PMD is from a predominant isolate, such as the isolated HIV or HIV MN, a conjugate vaccine or a mixture of such conjugate vaccines is highly advantageous for prophylaxis or treatment of AIDS or ARC. Examples of such preferred embodiments having the structures a) are: a)

O

(C-C-His-Ile-Gly-Pro-Cly-Arg-Ala-Pho C-1)

1 H-N-C (2 H) 2 CH

-N-C (O)

H-NMR (DMSO-d6):?

0 H

Η II

N-N-C-C-Gln-Arg-Gly-Pro-Cly-Arg-C

(E) n-C-H S

Ala. -Phe J or pharmaceutically acceptable salts thereof in the percentage by weight of peptide in the conjugate and is preferably between 1% and 5% of the total protein mass in the conjugate and is allowed to induce anti- peptide in mammals to induce HIV neutralizing antibodies in mammals,

to formulate vaccines to prevent disease or infection by HIV or to treat humans afflicted with the disease or infection of HIV, including AIDS or ARC,

One or more of the conjugate vaccines of this invention may be used in mammalian species either for active or passive protection prophylactically or therapeutically against infectious agents such as, in the preferred embodiment of this invention, Haemophilus insf luenzae serotype B, or diseases caused by Human Immunodeficiency Virus Active protection may be carried out by the injection of an effective amount capable of producing measurable amounts of antibodies <i.e., about a microgram at about 50 Âμg, depending on the antigen) of an antigen (for example PRP, peptides PND from HIV) in the form of rILP conjugate of each of the conjugates to be administered per dose. The use of an adjuvant (eg, alum) is also intended to be within the scope of this invention. Passive protection may be accomplished by injection of total antiserum obtained from animals previously dosed with ΜϊΕΡ-conjugated or conjugated, or to gi obulin or other antibody-containing fractions of said antisera, with or without a pharmaceutically acceptable carrier, such as sterile saline. Such gulbulin is obtained from the total antiserum by chromatography, fractionating in alcohol or salt or by electrophoresis , passive protection may be accomplished by standard monoclonal antibody procedures or by immunization of suitable mammalian hosts.

In a preferred embodiment of this invention, the conjugate is used for the active immunogenic vaccination of humans, especially newborns and children, immunocompromised individuals. For additional stability, these conjugates may also be lyophilized in the presence of lactose (e.g., 20æg / ml PRP / 4mg / ml lactose) prior to use. s c ε

A level of UIP of the MIEP -conjugados 'H Γ'? Ι! The color of the film is between 5 and 6 g. J ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^ pc =? or PND peptide of the present invention is the amount of each derivative thereof to be administered. 2 and 1 μg of PRP, or peptide-like peptides in the form of essential EPlP, may also be additional to the MIEP-binding of HIV-1, HIV-1, in a single administration »If you take one or two doses or your pain? in a similar dosage of the invention, the following examples are set forth by reference, which are intended to be illustrative and not

Preparation of OKPC of Msisssria meningitidis Bli serotype 2 A. Fermentation I. Nsisseria meningitidis group BI 1

A tube containing the lyophilized culture of Nsisseria meningitidis &lt; obtained from the Dr = M, firtstsinsins Walter Esed Array Institute of Research (WRAIR) Washington, D = C.) was opened and Eugonhroth (BBL) was added. The culture was scribed on flinier Hinton agar slides incubated at 37Â ° C with 5% CO2 for 36 hours at which time the growth was collected in 10% skim milk medium and the aliquots were washed with water. frozen at ≥70 ° C. Body identity was confirmed by specific antiserum agglutination provided by WRA1R, and standard serum supplied by Difco.

One vial of the second pass culture was thawed for 1 Âμl Columbia Bheep Blood Agar plates (CBAB-BBL). Plates were incubated at 37Â ° C with 5% CO2 for 18 hours. the growth was collected in 10 ml of 10% skimmed milk medium, the aliquots were taken out in amounts of 5 ml and frozen at -70 DEG C. The organism was positively identified by agglutination with specific antiserum ; fermentation in sugar and Oranu coloration

One vial of the culture from this passage was thawed, diluted with Mueller-Hinton Broth and scratched on 4 plates of Mueller-Hinton agar plates. The plates were incubated at 37 ° C with 6% ν for 18 hours after which growth was collected in 17 mL of 3

of skim milk at 3% were withdrawn aliquots in amounts of 3 ml. and frozen at -7Â ° C, the organism was positively identified in staining with specific antiserum agglutination and DKidase test. Fermentation and collection of cell paste was performed in a frozen flask of meningitidis of Group B, II-B above (passage 4). Ten slopes of Muelle-Hinton agar were inoculated and six were harvested later 18 hours later as inoculated into 3 25 ml vials of dialysed yeast medium. Gotschiich at pH 6.35. The TQ1 was adjusted to λ, 18 and incubated to 0 = 0. 1.3 ml of this culture was used to inoculate each of 5 ul. The Erlenmeyer flasks (each containing 1 liter of medium) were incubated at 37øC on a 2-liter shaker. Θ Θ rpm, AD, foi foi foi foi foi foi foi foi foi foi foi foi foi foi foi foi foi foi foi foi foi foi foi foi foi was monitored at one hour intervals following inoculation.

Fermentation of 7-Liter Seeds Approximately 4 liters of seed culture were used to inoculate a sterile 7-liter fermenter containing about 4 liters of complete production medium (see section). The conditions for the fermentation of 7- liters included 37øC, 185 rpm with air spraying of 10 gallons / minute and constant pH control at about pH 750 for about 2 hours. For this batch, the final 0.0 Âμg 3,, 732 after 2 hours,

Bw-Litross Production Facility

Approximately 4 liters of seed culture were used to inoculate a sterile B: M4 · liter fermenter

Ci

containing 568.2 liters of complete production medium (see below). This batch was incubated at 37 ° C, rpm with air spray of 6 liters / minute and constant pH control at pH 7, for this portion. final was 5.58 thirteen hours after inoculation. 3 = Complete Medium for Erlenmeyer flasks and 7 Âμl and 8 Âμl fermenters.

EoesiiLJl. L-glutlímic acid 1,5 g / liter MaC 1 6.6 g / liter NaoHP04, anhydrous 2.5 g / liter ΝΗλ01 1,25 g / liter KOI ® ® ® 9 g / liter L 'cystine HC1 ®, ®2 g / liter

Frsc§So B (Dialect of Sotschlichlí Yeast

Difco Yeast Extract (128 g) was dissolved in 6.4 liters of distilled water. The solution was dialyzed into 2 dialysis units of Amicon DC-3® camphor loadings. and the total volume brought to 15 liters with distilled water. The pH was adjusted to 7.4 with NaOH, sterilized through a 22Â ° filter, and transferred to the fermenter containing Fraction A Â · 4

For Erleruneyer flasks Fraction A and 25 mL of Fraction B were added to the flask and the pH was adjusted to 7.0-7.2æC NaGHn

For the 70 liters fermer 41.8 liters of Fraction A and 9.0 ml of Fraction B were added and the pH was adjusted to 7.0-7.2 with NaOL-L

For the 8ΘΦ liters fertilizer 553 liters of Fraction A and 5 5, mL mL of Fraction B were added and the pH was adjusted to 75 Φ-7 cd 2 cd NaOH »Harvest and Inactivations

After the fermentation was complete, phenol was added to a separate vessel, to which the cell broth was then transferred, yielding a final phenol concentration of about 0.5L. The material was kept at room temperature with gentle stirring until the culture is no longer feasible (about 24 hours). I love you

After about 24 hours at 4 ° C, the 614.4 liters of inactivated culture fluid were centrifuged through Sharples continuous flow centrifuges, the weight of the cell paste after phenol treatment was 3.075 kg "

Isolation of ΟΗΐ-'ϋ

Step 1, Concentration and diafiltration The phenol-inactivated culture was concentrated to about 30 liters diafiltered in sterile distilled water using λ., 2 μ CENKA) fiber filters.

Step 2 »Extraction

An equal volume of 2X TED buffer Θ TRIS buffer, Ι M EDTA Θ 1.0M, pH 8.5, with 5% sodium deoxycholate at, 5% was added to the concentrated diafiltered cells. The suspension was transferred to a tank regulated temperature for extraction of the OMPC at 56 ° C with stirring for 30 minutes. The extract was centrifuged at about 18βββ rpm in a Sharples continuous flow centrifuge at a flow rate of about 80 mL / min at about 4 ° C The viscous supernatants were then collected and stored at 4 ° C. The pellets of extracted cells were reextracted in TED buffer as described above. Supernatants were pooled and stored at 4 ° C.

Step 3: Concentration by UItrafiltration The pooled extract was transferred to a temperature-controlled vessel connected to polysulfone filters of Θ micron AB-Tech. The temperature of the extract was maintained at 25 ° C in the vessel throughout the concentration process. The sample was concentrated ten times at a mean transmembrane pressure between 75.79 and 165.30 kPa.

Step 4: Collection and Lavaqem of the UMPC The retained of the Pi / ssq 3 was cer 's X. VX s ϋ.ϋΐΓ ^ Ο w: * í ΐ y »·» P d Qp * 1600Θ0 K and (; Λ3? · ΤΚ at about 70Â ° C in a flow centrifuge continuously at a temperature of 100Â ° C at 50Â ° C / minute, and over-flowing at 0Â ° C The UMPC pellet was suspended in TED buffer (1 x 10 mL buffer 20 mL / g peptone) and Step 2 and evaporated to dryness twice.

This recovery of the OMPC ODUHCS aqueous product was a 4: 1 process.

The washed pellets from Step 4 were suspended in 1 Θ mL of distilled water with a glass rod and a homogenizer to ensure complete suspension. The suspension of OMPC was evaporated to dryness. SC.X i -B.ÚS. A bifurcation of a concave ω-1 and a concave b

Pr s p a r l. to the <PRP> lysis of the chaos of the impurities.

A sample is given in Table 1.

SBluC oohi 1

(s) received from the State in t ml of inoculum medium) and this b5-like suspension (cultivated from the University of New York) was suspended from sterile HaemoTiO4 (see Abai et al.

scattered on 9 slopes of CBBL Chocolate Agar.) The pH of the inoculum medium was adjusted to 7.2 +/- 1 (a peak value was pH 7.23) and the stock solution was sterilized prior to use in self- at 20 ° C for 23 minutes. After 20 hours incubation at 37 ° C in a candle pot, growth of each plate was resuspended in 1-2 ml. of Baemoohilus inoculum medium, and were pooled with hemolytic slopes of Hasmohilus g / Liter Peptone of soybeans 10 NaC 15 HaHnP04 3.1 Na2HP04 13.7 κ ,, ηρο, ώ. H * 2.5 distilled water to volume

The resuspended cells from each pair of slopes were inoculated into three 25 ml Erlenmeyer flasks containing about 1 ml of Haemophilus Production and Seed medium. The 256 ml vials were incubated at 37øC for about 3 hours to be A D0. of about 1 = 3. These cultures were either used to inoculate the 2 liter vials CabaiKo) = containing

Phase Bs Erlenmeyer flasks (Baffled) 2 liters - 5 mL of "A55 Phase" culture (above) was used to inoculate each of the five two-liter flasks.

HM

A typical value at the end of the incubation period is about Ι, β liter of the production medium and complete Hasmophilus seeds (see below). The vials were then incubated at 37 ° C on a rotary shaker at about 2 ° rpm for about 3 hours at about 1.0 ° C.

Means of Production and Seed of Complete Hagmoophyllus

Per liter

Mal-UPO ^ 3.1 g / L

to the

NaHCO3 13.7 g / L

Soy Peptone 10 g / L

Diafiltration of yeast exirate (1) 10 g / L

K? HP04 2.5 g / L

5.0 g / L MaCl

Glucose (2) 5.0 g / L

Adeninadynucleolide Nicotinamide

(WAD) (3) Ξ mg / L

Hemin &lt; 4 &gt; 5 mg / L

The soya bean peptone salts were dissolved in small volumes of the pyrogen-free, hot water and brought to the correct final volume with additional warm pyrogen-free water. The ferrocellers or vials were then autoclaved for about 25 minutes at 121 ° C (1), glucose C2>, NAD (3), and Hemin (4) were added aseptically to the vials or fermenters prior to inoculation.

Ci) Yeast extract diafiltrate were dissolved 10 g of brewer's yeast extract (Ainher) in 1

liter of distilled water and ultrafiltrates using a unit of Amicon DC ™ 3 "congeal fiber with a cutoff of 50 kd. The filtrate was collected and sterilized by passage through a filter (2> The glucose was prepared as a 25% sterile solution in distilled water. &lt; 3 &gt; A stored NAD solution containing 20 mg / ml was sterilized by passage through a (9.22 μ filter ) and added aseptically until inoculation. (4) A concentrated solution of 3% Hemin was prepared by dissolving 2Φ & mg in 1Θ mL NaOH Θ Ι M and the volume adjusted with stripped water to distilled to 10 © mL The solution was sterilized for 20 minutes at 21 ° C and added to the final medium prior to inoculation.

Phase C Seed Fermenter 7 Âμl Three milliliter of the Phase B product were used to inoculate a fermenter containing about 46 l of Complete Production and Haemoohilus Seed Medium (prepared as described above) and 17 mL of UCON B625 antifoam agent. The pH at inoculation was 7.4.

Production Fertilizer Phase 8--liter-Approximately 4Θ liters of the product of the "Phase CH" product were used to inoculate an 8 liter fermenter containing 57 litros liters of Production Medium and Haemophilus Seed (prepared as described above ) were measured to the required volume and 72æl of UCON LB625 antifoam agent was added

The fermentation was maintained at 37Â ° C. Stirring at 1Â ° rpm5 with OD2C and pH levels of 6Âμl measured approx. Every 2 hours until OD2 stabilized for a period of two hours, after which time the fermentation was terminated. Typical final Cuma was about 1.2 after about 2 hours).

cQLHEim, ... E_iMAmmíM

Approximately 6 liters of the batch per harvest were inactivated in a "death tank" containing 12 liters of 1%

CLARIFICATION

After IS hours of inactivation at 4øC, the batch was centrifuged in a Sharples continuous flow centrifuge with a 10 Âμm dish at a flow rate adjusted to maintain product clarity (variable between 1Â ° 3 and 3, 6 liters per minute), the supernatant obtained after centrifugation <15Φ6Θ rpm) was used to recover the product,

ISOLATION AND CC

The supernatant from two production fermentations was pooled and concentrated at 2 to 8 ° C in an ultrafiltration unit

Eoiiiicon Xrl ~ 50 with twenty concave fiber cangas with a cut v

(Membrane area 4 = 5 m 2 air flow 2.0 Lpm s 137.8 kPa). The concentration was such that after about 4.5 hours, about 19 l of l had been concentrated to 57, 4 liters. The filtrate was discarded.

PRECIPITATION WITH ETANQL ..... 48%

53 liters of 95% ethanol were added dropwise to the 57.4 liters of the ultrafiltration retained for 4 hours with stirring at 4 ° C to a final concentration of 48% by volume ethanol. stirred for an additional two hours at 4Â ° C to ensure complete precipitation, and the supernatants were collected following passages through a single 10,16 cm Sharples continuous flow centrifuge at 15Â ° C &amp; rpm at a flow rate of about 0.4 liters per minute. The pellet was discarded so the clarified fluid was brought to 82% ethanol with the addition of 40.7 liters of 95% ethanol over a period of one hour. The mixture was stirred for three additional hours to ensure complete precipitation. The precipitate was insoluble in 82% ethanol and the resulting 48% ethanol was collected by centrifugation in a 10. 16 cm Sharples continuous flow centrifuge at 15000 rpm with a speed ds f luxury of about,, 24 liters per minute & the supernatant was discarded in 82% ethanol. The crude product yield was about 1.4 kg wet slurry.

The 1.4 kg of 82% ethanol-insoluble material was mixed in a Daymax 2 ° -8 ° C dispersion vessel with 24.3 liters of cold distilled water. To this mixture was added 24.3 liters of cold CaClâ,ƒâ,ƒâ, € â,,â,,â,,Oâ,, and the mixture was incubated at 4 ° C for 15 minutes. The vessel was then washed with 2 liter of 1M CaCl2 .SH2 O, resulting in about 5% volume of final volume.

The 5 Âμl of the CaCl extract was brought to 25% stanol by dropwise addition of 16.7 liters of 95% stanol with stirring at 4Â ° C for 3 minutes. After further stirring for 17 hours, the mixture was collected by passage through a Sharples continuous flow centrifuge at 40Â ° C. The pellets were collected and the pellet was discarded. PASTA ....... ΡΕ_ΡΒ0 PAS

The 25% solubles in 25% ethanol were brought to 38% ethanol by the dropwise addition of 13.9 liters of 95% stanol with stirring over a period of 3 minutes. The mixture was then allowed to rise with stirring for one hour, then without stirring for 14 hours, to ensure complete precipitation. The resulting mixture was then centrifuged in a 10,16 cm Sharples continuous flow centrifuge at 15ΦΘΘ rpm & flow rate of 2 liters / minute) to collect the crude H. influenzae polysaccharide precipitate. The spin pellet was transferred to a 4.545 liter Waring mixer containing 2 to 3 liters of the absolute ethanol and mixed for 3 seconds at the highest rate. The mixture was continued for 3 seconds on Con), and 3 segundos seconds off, until a hard white ρ6 resulted. The slurry was collected on a Buchsr funnel with a filter dial. washed sequentially, in situ, with two 1/4 liter portions of absolute stanol and 2 liter portions of acetone. The material was then dried in vacuo at 4 ° C for 24 hours, resulting in about 337 g (dry weight)

About 168 grams of the dry material from the crushing step (about half of the total) were resuspended in 12 liters of 488M sodium acetate, pH6.9, with the aid of a vessel of the Dsymas-i * A dispersion sodium acetate solution was immediately stripped with 4.48 liters of a fresh aqueous phenol solution made as follows 590 mL of sodium acetate ®, 488æ, pH 6.9, were added to each of eight bottles of 1.5 kg of phenol (crystalline Mallinckrodt) in a 220 liter pressure vessel and mixed in the suspension. Each phenol etch was centrifuged for 2.5 hours at 3000 rpm at 4Â ° C in the ultracentrifugation K2 Eelectronucleics. The aqueous effluent was three additional times entrapped with fresh aqueous phenol solution as described above. The phenol phases were rejected *

ULTRAFILTRACSQ The aqueous phase of the first phenol-phenol (12.2 liters) was diluted with 3.0 litros liters of the fresh distilled water and diafiltered at 4øC in an Amicon BC-30 ultrafiltration apparatus using 3 cutoff loads of θθ kd , To remove the transport phenol. The Amicon unit was washed and the wash added to the retentate, such that the final volume was 31.5 liters. The residue was discarded. ..ETFTNOL A 67%

0.81 liter of 2.0M CaCl2 was added to the 31.5 liter of dialysate from the previous step (the concentration of CaCl2)

The final cDNA was from ®, and the solution was brought to 82% EtOAc by dropwise addition and rapid stirring for one hour, 4855 liters of 95% ethanol. After 4 hours of stirring, after being maintained for 12 hours at 4 ° C, the supernatant was siphoned and precipitate was collected by centrifugation in a 5-1 / 2 inch Sharplss continuous flow centrifuge (158ørpm). 4 ° C for 45 minutes. The resulting polysaccharide pellet was ground in a 4.545 liter Waring blender using vibrations of 3 seconds with 2 liters of absolute ethanol collected on a Buchner funnel equipped with a Teflon filter disc , washed in situ with four portions of absolute ethanol followed by two portions of 1 liter of acetone. The sample was then dried on a heavy plate in vacuo at 4 ° C for 24 hours. of about 1.2 grams of dry powder, the yield of all substitutes with phenol was pooled resulting in a total of 212.6 grams of dry powder.

?????????????????????????

LAST

The 212.6 grams of the above dried powder were dissolved in 82% liters of distilled water, to which were added 2.13 liters of 0.501%, 211%, 2%, 5% CCaCl2O5 M &gt; , 2.5 mg of polysaccharide / mL &gt; the mixture was brought to 29% ethanoi with dropwise addition of 29.86 liters of 95% ethanoi for 3 minutes. The mixture was clarified immediately by centrifugation in a centrifuge (<2 containing a beaker of titanium <3) and a Noryl kernel (150 rpm s of flow rate) at 4Â ° C. The pellet was discarded and the supernatant was added to 38% ethanoi by the addition of 17.22 liters of 95% ethanoi for 3 minutes with stirring. After stirring for an additional 3 minutes the mixture was allowed to stand without stirring at 4øC for 17 hours and the precipitate was collected using a centrifuge

"Sharp Continuous Fluid K? Of 10.16 cm to 15? rpm (45 minutes required). The resulting slurry was transferred to a 4.545 liter Waring blender containing 2 liters of absolute ethanol and mixed at the highest speed for 4 or 5 cycles of 30 seconds on and off until a powder hard white. This p6 was collected on a Buchner funnel equipped with a Zitex teflon disk and washed sequentially, in situ, with two Φ.5 liter portions, fresh and one liter portion of absolute ethanol? with two portions of X liter of acetone. Was the product removed from the funnel and transferred to a heavy dish for drying? in vacuo at 4 ° C (for 25 hours). The final yield of the product was 79.1 grams dry weight.

Cloning of the genomic line coding for lilEP

About 150 μg of phenol -induced N-meniniticidal cells (see Example 1) were placed in a fresh tube. Phenol-inactivated cells were resuspended in 567 μΐ. of TE ETRIS-HC1 æm mM buffer? 1 mM EDTA, pH 8? 3. The resuspended cells were added 3 μg SDS-1 and 3 μg of 2 μg / ml. proteinase K (Sigma) cells. The cells were mixed and incubated at 37 ° C for about 1 hour, after which 1 μg of 5 M NaCl was added and vigorously stirred. 8Θ μl of cetyltrimethylammonium bromide (CTAB) at 1¾ in 0.7 M NaCl was then added? mixed vigorously? and incubated at -65 ° C for 1 minute. An equal volume (about Θ 57 to 0.8 mL) of chloroform / isoamyl alcohol (at a rate of 24 æl, respectively) was added, vigorously mixed and centrifuged at about 10,000 κ g for about 5 minutes. The aqueous phase (upper)

was transferred to a new tube and the organic phase was discarded. An equivalent volume of isopropyl alcohol / chloroform / isoamyl alcohol was added to the aqueous phase at 25 ° C, sparingly, vigorously, and centrifuged at ΙβΘβ κ g for about 5 minutes. The (upper) aqueous phase was transferred to a new tube and volumes (about 42Θ μΙ) of isopropyl alcohol, vigorously mixed, and precipitated DNA was centrifuged at ΙΘΘΘκ g for 1 minutos minutes. The overflow was discarded, and the pellet was washed with 1% to 7% strength. The DMA pellet was dried and sparged in 10æl of buffer TE, and represented the DMA genomic of N, meningitidls »

Two DMA oligonucleotides corresponding to the 5 'end of the MIEP gene were synthesized at the 3' end of the MIEP gene EMurakami, E 'C, et al. "Infection and Immunology, 57: 2318-233, The DNA sequence specific DMA oligonucleotide for the 5 'terminus of the MIEP gene was: 5'-ACTAGTT8CAAT8AAAhAATCCCT8-3' | s for terminal 3 &quot; of the MIEP gene were 5'-GPIATTCASATTAGGAATTTGTT-3 '. These DMA oligonucleotides were used as primers for the amplification of the polymerase chain reaction (PCR) of the flIEP gene using 1 nanograms of M genomic DMA, meningitidis, PCR amplification step was performed according to procedures provided by the manufacturer CPerkin Elmer), the amplified MIEP DMA was then digested with the restriction endonucleases Spel and EcoRI, the 1.3 kilobase B DNA fragment (kb &gt; the complete coding region of MIEP was isolated by electrophoresis on a 1.5% agarose gel and recovered from oss by electrochemical protocols (Molecular Biology, (1987), Ausufael, R, M., Brerit, R., Kingston, RE, Moore, D = D, Smith, Seidman, S., and Struhl, K., eds., Breene Publishing Assoc., PUC-19 plasmid vector was digested with Spel and EcoRI. Gel-purified HIEP Spel-EcoRI DNA was ligated to the vector pUC-19 Spel-EcoRI® was used to transform the E. coli strain DH5. Transformants containing the pUC-19 vector with the β3-kpb HIEP DMA were identified by mapping a restriction endonuclease map and the DMA of rilEP was sequenced to ensure its identity '

The GalÎ ± promoter was isolated from the plasmid YEp52 EBroach, et al. (1983)) in Experiment Nanipulation of Sene Expression Inoue, M ( Et al., Academic Press pp. 83-1173 by purification in gsl of the βg kg fragment from the Ckβbb bases) obtained after cleavage with Sau 3A and Hind III. The Î »DIN was isolated from the pGAP vector. to 1 ", &lt; i98a &gt; &gt;

Gene, 46, pg. 135-1412 by gel purification of the γ 35 kpb fragment obtained by cleavage with Hind III and Spel. The two fragments were ligated with the .DMA ligase 14 to the purified pUC1Deltarfind1I1 vector in the HindIII site and purified by digestion of the pUC1S with Hind III, cohesive termination with the Klsnow fragment of E. coli DNA polymerase I; and ligation with DMA ligase 14) which had been digested with BamHI and Sohl to create the vector

This has a unique HindIII cloning site at the SalIOP site. The unique HindIII cloning site of HindIII was changed to a single BamHI cloning site by digestion of HindIII with HindIII, gel purification of the cut DNA. and ligation using T4 DNA ligase; to the following ligand Hind III: 5'-ASCTCBSATCCS-3 '3' -BCCTA66CTCBA-5 ', the resulting plasmid, pSali (B) tADHi3 deleted the Hind III site and generated a unique BamHI cloning site. The Gall@p.tADH1 fragment was isolated from pBAL0 <B) tftDH1 by digestion with SmaI to Sohl "terminated cohesively with DMA polymerase T4? and gel purified. The shuttle yeast pCl / i EBrake et al. (1984). Proc.

Nat'1. Acad. Know. USA, 8æ5 pg. 4642 --- 4e4e3 was digested with Sph.I. terminated with T4 DNA polymerase, unpurified.

This fragment was ligated to vsctor with DNA ligase. The ligation reaction mixture was then used to transform E. coli HB + cells to smp-resistant cells3 and the transformants were annealed by hybridization in a 3 ' of the Hxd 111 BamHI ligand labeled &quot; The new construct of the vector, pcil 1 .Oal 1 p (B &gt; ADH1) was confirmed by digestion with HindIII1 and lH2. EXAMPLE 5

Construction of a Vector of MgP pressure of Yeast MItP with MIEP Seqyfnciss of DNA primers Cleader)

A DNA fragment containing the complete MIEP coding region was generated by digestion of pUC19? MIEP # 7 with IFN? EcoRI, gel purification of MIEP DNA, terminated cohesively with T499 polymerase chain reaction DNA polymerase. of yeast pCI / 1. BalΙθρΐΒΙΑΟΗΙ foi was digested with dephosphorylated BttMHX π with calf intestinal alkaline phosphatase, and terminated cohesively with T4 DNA polymerase. DNA was gel purified to remove the uncut cutter.

The fragment was finally terminated, 11.1 kbp of MIEP was ligated to vsctor pCi / 1, Bal Bp (B) ADH1 terminated cohesively. The ligation mixture was used to transform DHS E. coli competent cells into ampicillin cells Transformants were analyzed by &quot; T-2 hioridase &quot; in a DNA ligand labeled AABCTCB6ATCCTASTTBCAATB, &lt; 3 &gt;, which was designed to be homologous with sequences overlapping the MIEP-vsctor junction. DNA preparations were made from the hybridization of positive and digested transformants to Sa11 to verify that the MIEP fragment was in the correct orientation for expression of the SalI promoter. Further confirmation of the DNA construct was obtained by dideoxy sequencing of the Gali0 promoter at the MIEP coding step. Expression of MIEP by the transformants was detected by Western blot analysis. The MIEP recombinants produced in the transformants co-migrated into polyacrylamide gels with MIEP purified from OnPC? and was immunologically reactive with antibodies specific for MIEP, -.λ

α V / * ·;

Construction of a Yeast MIEP Expression Vector with a 5 'Modified MIEP DNA Sequence

A DNA oligonucleotide containing a HindIII site, a Cleadsr primer) 5 &quot; of conserved yeast (NTL), a coding start of methionine (ATG) 3 the first 89 codons of mature ivyx (starting with Asp at position + 20) and a Konl site (at position +89) was generated using Polymerase chain reaction (PCR) technique == PCR was performed as specified by the manufacturer (Elmer Cetus Profiling) using the plasmid ρϋ019ΝΙΕΡ4Ξ # 7 as the template for the following DNA oligomers as primers: ΰ'CTAASCTTAACAAAA7SBACSTTACCTTGTAC80TACAATP * Ώ ACSeiACCGAA8CCGCCTTTCAA8 ' (I.e.

To remove the 5 'region of the IL-1 clone: the plasmid pUC19r1IEP42 # 7 was digested with KpnI and HindIII, and the 3S4 kbp vector fragment was purified on agarose gel. The 28Θbp PCR fragment was digested with KpnI and HindIII, purified on agarose gel 5 and ligated with the 3 P4 kpb vector fragment. Transformants were analyzed by hybridisation of DNA oligonucleotides to DNA of positive transformants by analysis with restriction enzyme digestion. To ensure that no mutations were introduced during the PCR step, the 28β-PCR PCR-generated DNA of the positive transformants was sequenced. The resulting plasmid contains a HindIII-EcoRI insert consisting of yeast NTL3 ATB codon, and the complete open reading frame MlbP-starting at the codon Asp-amino acid + 2®) =

The vectors of yeast rlXEP were constructed as follows. The vectors pBaliS / pcl / 1 and pBAP / pCl / 1 CVlasuk, B.P., et al. (1989), 284, pp. 121-123 were digested with BamHI terminated non-cohesively with the Klenow fragment of DNA polymerase I and dephosphorylated with calf intestinal alkaline phosphatase. These linear vectors were ligated with the treated KlenotAj and the gel purified HindIII --- EcoRI fragment described above containing the yeast NTL, AT8 and fPIEP ORF and forming pBal / pcl / HIEP and pBAP / pBAP / pCl / MIEP. The Saccharomyces strain is prepared as described in Example 1. was transformed with pSal plasmid pSal 1 / p / pI / pI IEP = The recombinant clones were isolated and examined for HIEP expression. The clones were grown at 37 ° C with shaking in synthetic medium &lt; containing 2% glucose / v) at a D = 0: about 6.0. 2% galactose (v / v) was then added to induce the expression of EPl from the SalI ® promoter-cells were grown for an additional 45 hours following challenge with galactose at a concentration of about 9%. Cells were then harvested by centrifugation. The pellet was washed with distilled water and frozen.

Western blotting for MIEP Recomfining

To determine if the yeast was expressing MIEP, Western blot analysis was performed using i.e. Movex Laxmlx gels at 15 wells, i.e. at twelve percent. Yeast cells were plated in H 2 D using pearls 2% sodium dodecylsulfate (BDS) can be used during the

The cell debris was removed by centrifugation for 1 minute at 1 ° C. The supernatant was mixed with sample development buffer as described for MIEP polyacrylamide gel purification. Samples were grown at 35 mA Using OMPC as a reference control? until the bromophenol dye label leaves the gel.

Proteins were transferred to paper of nitrocellulose-Sj pore size Φ Φ 45 μm using a transfer apparatus NOVEK = After transfer the nitrocellulose paper was blocked with 5% bovine serum albumin in phosphate buffered saline during i hour after which 15 ml were added. of a rabbit snti-rIIEP (generated with Μ1ΡΡ gel-purified dilution using standard procedures). After incubation overnight at room temperature, 15 mL of a Ιπ dilu dilution of goat anti-rabbit IgG conjugated to alkaline phosphatase was added. After 2 hours incubation the stain was developed using FA8T RED TR BALT (Sigma) and Naphthol- AS-HX (Sigma)

Recombinant HIEP bactsriana expression The ρϋ019-Μ1Ρ p piasmid containing the 1.3 kilogram base pair MIEP gene insert was digested with restriction endonuclease Spel and EcoRI. The 11 kb fragment was isolated and purified on a gel of aqarose using standard techniques known in the art »piasmide ρΤΑΒ3Σ> 5 containing the two-channel TAC promoter to a single ECORI site5 was digested with ECORI. Cohesive ends were formed in either the 1.3 kbp MIEP DMA

(Boehringer Mannheins) according to the manufacturer's instructions. The cohesively terminated i53 kpb MIEP DMA was ligated to the terminated vector cossivasses using DMA ligase T4 (Boehringsr ríannhsim) according to the invention. According to the manufacturer's instructions the bound DMA was used to transform the E. coli strain DHOalOMAX (BRL) according to the manufacturer's directions. The transformed cells were placed in agar plates containing 25æg cannabin / ml and 5æμ periicillin / ml, and incubated for about 15 hours at 37 ° C. A DMA oligonucleotide having a homologous sequence with SilEP was labeled with P s used to analyze nitrocellulose filters containing denatured colonies lysed from transformants plates using hybridization techniques of DMA standard. Colonies that were positive by hybridization were mapped using restriction microsomes to determine the gold The expression of HIEP by the transformants was detested by Western blot analysis. The recombinant HIEP produced in the transformants consigned to polyacrylamide gels with purified HIEP from OMPC vesicles and was immunogenically reactive with the antibodies specific for the HIEP »

: Hi R

Preparation of Purified HIEP from OMPC liposomes or from Recombinant Cells by Eletrophoresis on Polyacrylamide Gel

The acrylamide / BIQ gels (37.5%) were used, 14.5 mm thick. The agglomeration gel was 4% palladium-on-wet gel and the separation gel was X2% polyacrylamide. Approximately 5 μl of recombinant host cell protein or protein by gel. To 1 ml of OMPC snL sample buffer was added 4% Cg-glycerol and 3 μM DTT. TRIS 100 mM bromophenol blue to The mixture was heated at 105 ° C for 20 minutes and allowed to cool to room temperature before being loaded onto the gel. The gel was developed at 200-400 millimeters with cooling until the bromophenol blue reached the bottom of the gel. A vertical band of the gel (about 1-2 cm wide) was stained and stained with Coomassie / cupric acetate. The strip was bleached until the MIEP band (about 38! &Lt; d) became visible. The strip was then placed in its original gel position and the MIEP area was excised from the rest of the gel using a scalpel. The excised area was cut into cubes (about 5 mm) and eluted with TRIS buffer (pH 5) = 1. After 2 cycles of elution, the eluate was evaluated for purity by S-PHASE. combined with a common pool of eluates is dialyzed against low cost buffer (boric acid 0.5M, pH 11.5). After dialysis, the eluted protein was concentrated using an Amicon cell shaker with YM10 membranes (cutoff) molecular weight 10.0). The material was further purified by passage through a column of size PD1 (Pharmacia, Piscataway, NJ), and stored at room temperature in borate buffer.

MIEP Vehicle Activity in a Covalent PRP-DMPC Conjugate

Immunizations: Male C3H / male mice (Charles River, Wilmington, MA) were immunized intraperitoneally (IP) with PRP covalently bound to OriPC CPRP-QIiPC? Compounding 2.5 pg of PRPs and 17 pbw of PCV &gt; or PRP coupled to DT? PRP? DTn containing 2.5-2.7 pg of PRP and 1.8-5.4 pg of DT) Connaught Laboratories, Willeldale, DNT>, suspended in 0.5 ml of solution A second group of male C3H-HeN mice received either 17æg MIEP, 17æg OriPC, or 17æg OMPG-IAA βOMPC- derived with N-acetyl- homocysteine thiolactone, and closed-capped) with iodoactamamide). Cell donors for adoptive transfer experiments were immunized twice IP, with 21 days interval, and spleen cells were collected 1 days after the second immunization. transfer from male C3H / HeM mice giving gamma irradiation in the total body of 5ΘΘΡ from a 1 / C7 source

Cs and reconstituted by intravenous administration of spleen cells from each of two syngeneic donors inoculated with PRP-D7, and OMPC, MIEP, or OMPC-1A. v

Control mice received ax 1 ® spleen cells from a PRP-OHPC inoculated mouse and an equal number of spleen cells from an uninoculated donor mouse. ELISA ______ for anti-PRP antibody. purified by treatment

with carbonyldiimidazole and reaction with butanediamine as described by et al. U.S. Patent 4,882,237 = This PRP derivative was eromathographed on Sephadex G-25 in sodium bicarbonate buffer ®, 1.1 pH 8.0 = N-hydroxysuccinimidobiotirase CPistric Chemical, Rockfort, IL) was added in dimethyl sulfoxide was added to the eluate to a final concentration of 3.7 mg / mL and reacted in the dark for 4 hours at room temperature (about 25-28 ° C). The unreacted N -hydroxysuccinimidobiotin was removed by gel filtration on Ssphadex B-25 in PBS = Costar Polyvinyl Chloride ELISA plates (Cambridge, VIA) were coated with 5æg / well avidin (Piercs Chemical)

at 1 μmole / ml in sodium bicarbonate buffer (pH 4.05) overnight at room temperature and 100% humidity. The plates were washed 3 times with TRIS® 5 M * pH 8 * 5 ™ buffered saline containing 0.05% TMBS-20% CTBS-γ) and blocked with TBS-T plus gelatin at Θ 1 (blocking buffer) at room temperature and 100% humidity for 1 hour. Plates were stained without washing. 5æg / well of PRP-biotin in 15-4æg / ml PBS was added and plates were incubated for 1 hour. The plates were washed 3 times with TBS-T prior to addition of the sample. Samples were added in series of two dilutions in blocking buffer incubated for Ξ hours at room temperature and 1% moisture. The plates were then washed 3 times with TBS-T * and appropriate alkaline phosphatase conjugated anti-immunoglobulins diluted in blocking buffer. Antibodies used were Goat anti-mouse IgG Immunoresearch ™ West Srove * Ig8 ( Immunoresearch) * IgSl (gamma) (BRL * Saithersburg * MD) * IqS2a (gamma) (BRL) * IgS2fo (gamma) (Southern Biotechnology Associates * Birmingham * AL) * Ig83 (gamma) (Southern Biotechnology Associates) and Goat anti-rabbit IgS (Jackson Immunization). Plates were incubated for 2 hours at ambient temperature and humidity of 1% washed with blocking buffer. The development of the substrate was performed using p-n-triphenyl phosphate ií The dilutions were considered positive if the absorbance of the sample exceeded the mean absorbance plus 3 times the standard deviation of the white S reagents and if the difference in adhesion between The final titers were defined as the reciprocal of the highest dilution which gave a positive reaction in the ELISA as described above. The logarithms of the reciprocal titers were compared between the groups undergoing treatment by two-way analysis gives

variance CLindeman, K = H »st a1 =" i9Q ©}, Introduction to

Bivariate and Multivariate Analysis, Scott Foresman (pub. &Gt;, New York),

RIA for quantification of anti-PRP antibody

Experimental samples from the sera tested for the amount of anti-PRP antibodies were diluted to is2, 1:55 and 1hrs using calf fetal serum as the 25 μl dilutions. of each diluted serum sample were transferred in duplicate to 5555 ml RIA vials. (Sarstedt). A solution the I-labeled PRP was diluted to give between 3 ° and 8 ° C. minute counts (cpm) per 50 μl using phosphate buffered saline solution as the 5 μl diluent. diluted will be transferred to each RIA flask, mixed vigorously and incubated for about 15 hours at 4 ° C = 75 μl. of a saturated ammonium sulfate solution at 4 ° C were added to each RIA flask, mixed vigorously and incubated at 4 ° C for 1 hour. The RIA flasks were then centrifuged for 10 minutes at 100-60 x g, the supernatant was discarded and the cpm in the pellet were measured in a gamma counter <LKB &gt;

A standard curve consisting of series of two dilutions a and an antiserum containing a known amount of anti-PRP antibodies was prepared as described above and were assayed concomitantly with the experimental serum samples. between 14 pg / ml as the highest amount of antibodies and 0 = 56 pg / ml as the smallest amount of antibodies = all samples were grown in duplicate. The mean cpm of the duplicate samples was compared to the standard curve for calculating the amount of anti-PRP antibodies present in the experimental serum samples

Antibody Responses of Transfer Recipients is Mvgs!

Receptor spleen cells inoculated separately with PRP-DT, and either MIEP or OMPC or JAA-Qr1 PC * responded to PRP-OHPC Immunization by producing equivalent amounts of anti-PRP IgB1 and IoG2a serum antibody in 4 days (see Figure 1). Irradiated mice reconstituted with spleen cells that were inoculated with RIPI or OnPC or IftA-OMPC virus had significantly higher anti-PRP IgB1 antibody titers <Ρ50Θ1) and IgS2a (ρ <lt , θ3θ4> after immunization with PRP-OMPC than the control mice, giving spleen cells inoculated with PRP-DT but not with OMPC-inoculated spleen cells. Serum antibody responses are PRP-OHPC immunization in mice at which were given spleen cells inoculated separately with PRP-DT and either MIEP or OMPC or IAA-OMPC were comparable to those in which mice were given spleen cells inoculated with PRP-OMPC &lt; p &gt; 12 to IgB1 antibody on days 6-13, and p &gt; 0.5 for IgS2a antibody on days 9-13). MSo antibody response was observed when irradiated mice reconstituted with spleen cells inoculated with PRP-DT and inoculated spleen cells either with MIEP or with OMPC were immunized with PRP without a protein vehicle. Statistical analysis was done by analysis of two-way ANOVA) ELindeman, R «H. et al., Introduction to Bivariate and Multivariate Analysis, &lt; 198 * 3), Scott

Forestsn Cpub., &Lt; / RTI &gt;

1 Or-i

These results demonstrate that ILIF was effective in mice as well as OMPC to induce a vehicle helper T cell response to generate anti-PRP IgB antibodies. EXAMPLE:

Mitochondrial activity of HIEP HIEP purified from the OMPC of M1. mice were tested for mitogenic activity in a cell proliferation assay. Murine splenic lymphocytes were obtained from C3H / HeN5 C3H / FeJ mice; C3H / Het, or Baib / c. The mice either were either naturally or had previously been vaccinated with PRP-1i1PC = spleen cells were passed through a fine mesh sieve. sterile solution to remove sterile dentifrices suspended in CRPI1I 1640 (OIBCO) medium 10% fetal calf serum CArmourK Slutamine 2 mIBIBCO)? Hepes (mIBSIBCO), 10 μg / ml penicillin / 100 μg / ml streptomycin (GlBCO), and B-mercaptoethanol 5 μg / Biorad> 3 μl Following is pipetting to break groups of cells. suspension was centrifuged at 3ΘΘ κ g for 5 minutes; and the pellet was resuspended in a plug of red blood cells (SEQ ID NO: 9) at 95% CFS-5). TRIS-HCl @ 57 M at 10% (sigma) pH 7 = 23. right temperature; 51 ml of cells / ml of buffer for two minutes. Cells were stratified with 5 Âμl. of calf fetal serum and centrifuged at 40Â ° C for 30 minutes and then washed with medium. twice and resuspended in medium K to O x i 1 &quot; cells / ml. These cells were implanted in 9Âμl dishes (Âμg / well) together with 10 Âμg of protein or peptide sample in triplicate. Nitric Acid HIEP was purified as described above in Example 7. Control proteins include

serum albumin? bovine, PRP-OMPC and OMPC itself, lipopolysaccharide (endotoxin) = All samples were diluted in K medium at concentrations of 1, 6.5, 13, 26, 52, æ 5, ε 13 æg / ml after implantation into plates as described above such that their final conÂcontrations were inetade from their original concentrations. The triplicate wells were also incubated for each cell type suspended in K medium only to determine the cell line of cell proliferation *

On days 3, 5, or 7 of culture, the wells were

Mrp pulsed with 25 μΐ. The amines were collected 18 hours later in a Skatron column, and the counts per minute (CPM) were measured in a liquid scintillation counter * A exchange of the rsds in cpm was calculated by subtracting the mean number of cpm titered per well by cells only in K-medium, the mean of the cpm experimenting the stimulation index? was determined by dividing mean mean cpm by mean cpm to control wells

As shown in Figure 2, the ΙΊΪΕ vac vaccine as well as the DMPC and PRP-OfiPC vaccine resulted in the proliferation of lymphocytes from previously vaccinated mice. This mitogenic activity did not appear to be due to the lipop-oligosaccharide CLP3) since ΙΊΙΕΡ was free of detectable LPS as measured by rabbit pxrogenicity assays, and the proliferative effect was greater than that which could have been caused by LPS present in amounts below the level of detectability in silver-stained polyacriX qels *

Ί 1 .1 exampleji

Conjugation of the polysaccharide PRP ds H "influsnzae-b-type MIEP of H. meningitidis

The chemical conjugates were conducted according to the process described in U.S. Patent No. 4,882,317.

10 mg of ΗϊΞΡ in 3 æl of 0.1 M borate buffer, pH 11.5, was mixed with 1 mg of ethylenediaminetetraacetic acid disodium salt CEDIA, Sigma Chemicals) and 4 mg of dithiothreitol (Sigma Chemical). The solution of (1.25 mg) of M-acetyl-homo-cystathiothiolactone (Aldrich Chemicals) was added to MIEP solution, and the mixture was incubated at ambient temperature for 1 hour. It was then dialyzed twice under reflux for 2 hours. hr against 2 L of borate buffer 031 M pH 9.5 containing 4 mM EDTA for 24 hours at room temperature. The thiolated protein was then assayed for the thiol content by the Ellman reagent (Sigma Chemicals) and the concentration in protein was determined by Bradford CPierce Chemicals reagent). For the conjugation of the MIEP to the PRP, a 1.5 fold (w / w PRP assay of the influenza A virus and the bromoacetylated virus was added to the MIEP solution and the pH was adjusted to 9-9.5 with 1M NaOH The mixture was allowed to incubate under N2 for 6 to 5 hours at room temperature. At the end of the reaction time, 25 ul of N-acetylcysteamine (Chemicals Dynamics) was added to the mixture, then it was allowed to stand for 18 hours under N. .. the temperature

The conjugate solution was acidified to pH 3 to 4 with HCl and centrifuged at 1 κ g for 10 minutes. 1 æl of the supernatants were applied directly to a column of 6B FPLC (1.6 κ 50 cm · Pharmacia) was added and the conjugate was eluted with PBS of the void volume peak containing the conjugate of

polysaccharide-protein (PRP-HIEP) was pooled. The conjugate solution is then filtered through a 6.22 μ filter for EXI &amp; 0-12 sterilization

Demonstration of Immunogsicity of PHr-MIEP conjugates

Immunization Male Balloon / c mice (Charles River, Wilmingion, MA) were immunized with PRP covalently conjugated to MIEP as described in Example 11 using 2.5æg PRP in θ, 5 ml. The control mice were immunized with equivalent amounts of PRP given as PRP-CRM &lt; 2.5 μl PRP / è, 25 μl of CRMs 1/4 of the human dose), PRP-DT (2 5 pg PRP / 1.8 pg DT5 1/10 human dose), PRP-QHPC (2.5 pg PRP / 35 μg OMPC, 1/4 human dose)

Newborn Rhssus monkeys, 3-13.5 weeks old, were immunized with alum-absorbed PRP-MIEP conjugates. Each monkey received 0.25 mL of the conjugate at two different injection sites for a total dose of 0, 5 mL. Monkeys were immunized on days Φ, 23, and 5,, and blood samples were taken every two to four weeks.

Antibody responses were measured by the ELISA described in Example 9, which distinguishes the class and subclass of the immunoglobulin response. An RIA that quantifies the total anti-PEP antibody Cver Example 9) was also used to evaluate the response of the monkey. antibody conjugates of PRP-HIEP conjugate containers are shown in Figure 3 =

The results show that the PRP-MEPE conjugates are capable of generating an immune response in newborn Rhasus monkeys and mice consisting of anti-PRP IqG1 antibody in a memory response. This is in contrast to PRP-CRH and PRP-DTs which do not induce measurable anti-PRP antibody. Thus, MIEP functions as an immunological vehicle protein for PRP and is capable of producing a response of the anti-PRP antibody when conjugated covalently to PRP antigen. is therefore an effective immunological vehicle protein substituting the heterogenous DnPC in the construction of bacterial polysaccharide conjugate vaccines,

To 10.5 ml of a solution of MIEP &lt; 1585 mg / ml, 18.4 mg total) contained in a 50 ml vial, 2.6 ml of a borate buffer (II) The pH was adjusted to 1 &amp; VQ with 5N NaOH after addition of 37 mg of EDTA and 11 mg of dithiothreitol. Then, 346 mg of N-acetyl-hoiiiocysteinatholactone was added and the pH adjusted again with 5N NaOH was degasified? the air replaced with nitrogen and the solution aged for 23 hours under a nitrogen atmosphere. The sample was then diaiised against 4L borate pH 9f.Ξ containing 1 mL EDTA for 7 hp against new 4L for 22 h and diluent against a buffer of borate M pH 9.5 containing 1.9 mg of DTT for 16 h. This treatment gave a solution containing a total of 4.84 pmol of thiol (per Ellmani assay. This compares with 249 nanomoles of SH / mg protein)

A sample of the 1 mg of the most exemplified cPND1S from Example 13 was dissolved in 1 ml of H2 O at 5 Âμl of this was used for a reverse acid maleimide assay to give 5 = 4 umoles (total ) of maleimide. An aliquot of, 9 mL &lt; 4.88 pmol) of the solution was added to the thiolated MIEP solution (pH which was immediately turbid and after 3 h and ® minutes no thiol titre remained (per Eilman K assay). The solution (14 mL) was dialyzed twice versus 4 L of an inexpensive ® buffer, pH 9.5 for 27.5 s at 38 h respectively. A 1 Âμl pL assay for an amino acid composition gave the The following results were found: 15.9 13.7 48.8 in 10 Âμl showed ®, 95 1111, this translates to nanoffloles protein loading = 1 ml of norleucine β-alanine lysine samples

A Bradford mg / ml protein assay. Using a molecular weight of 176.7 pg / mL patella. Thus the peptide was 18.6%, EXErSPLb 14 PREPARATION OF MIEP-CPND31 CONTAINERS

6.5 ml of a solution of MIEP (1.7 mg / ml), 1.5 ml of a borate buffer, pH 4, pH adjusted to 11 with 5 Âμl of NaOH SN were added to 6.5 ml of a solution of MIEP (1.7 mg / ml). To this were added 21 mg of EDTA &amp; 6.5 mg of DTT was added and the solution was rotated for 15 min. Then 2.00 mg of N-acetyl-ho-mocysteinathiolactone, the deaerated solution and the substituted air were added

. After aging in the N0-box for 1.5 h, the pH was adjusted to 10.68 with 5 N NaOH, the repeated degassing process, the aging continued for 20.5 h. The solution was dialyzed versus 4 L of borate 0 , pH 9.5 containing EDTA δ, δtM for 6.5 h followed by 41æ of borate æl, pH 9.6, 1 mM EDTA containing 1 mg dithiothreitol for 17 h = An Ellman assay indicated 2.27 pmol (total) tallow which is equivalent to 205 nanomoles SH / mg protein. To this thioylated protein solution was added Θ 55 ml of maleimidated cPNDS1 from Example 14 (3.77 pmol / mg, reverse Eilman assay pair, 2, Θ7 pmoles total &gt; * A flash turbidity was noted * 5 mg of additional maleimidated cPMD31 were added, and the blend aged for 1 hour *

To remove unconjugated peptides, the mixture was dialyzed in dialysis tubes, having a molecular weight cut-off of 12ΘΘΘ-1400Θ, versus 4 de borate, pH 9.48 for 5.25 hours versus 4L of borate ΰH ΘΙH, pH 9.68 for 66 hours * A total of 8 ml of solution remained from which 200 μΙ were removed for amino acid analysis norleucine 22.8 nanoroles / 200 μ lysine 85.9 nanosoles / The solution was then dialyzed against 20 μl of 0.1% phosphate buffer pH 7.07 which was 5 M in urea to give a final volume of 6.5 ml. A Bradford protein assay revealed 1.26 mg of protein / ml (8.2 mg total). Thus, 0.912 pmoles of peptide (8 mL X 22.8 nanomoles / Θ, 2 mL) at a molecular weight of 1204 = 1.1 mg of peptide Thus, in this case, 13% of a peptide was obtained for a protein kelp *

Slntsss are Solid State of disulfide-linked cPND4

A linear PHD peptide was prepared on a wang resin using an ABI-43IA peptide synthesizer from Fmoc-L-CysCAcm resin -0-Wang (Θ, ΐΐ msq / qram). (in situ prepared 4KS excess) with reactants on a ®S2Smmole scale to generate 745 mg of the peptides from the Fmoc-Hys-Hys-Hys-Hi-11e-B1y-Pro-S1y-krg- A1a-P he-Cy s - 0- 'rt ÃCffl

Wang »

A solution of iodine in 5% methanol / anhydrous DMF (1 ml) was added to the Wang derivative, dried above, and stirred at room temperature for 4 hours. The resin was filtered, washed with anhydrous DMF (5 x 2 ml &gt; 3 & finally resuspended in DMF &lt; 2 ml). Two drops of a 0.1 M sodium thiosulphate solution in water were added and stirred for a few seconds. The resin was washed with 95% aqueous DMF (3 x 2 ml), anhydrous DMF (2 ml), methylene chloride (3 x 2 ml), ether (3 g, 2 ml) and dried (MgSO 4). removed by treatment with 24% piperithines in DMF for 2 minutes, and the resin was washed and dried. The resin was cleaved from the cyclic peptide disulfide bound by treatment with 95% TFA / 4% stanodithiol / thioanisole (100 ml) at room temperature for 6 hours. The residue was filtered, the resin washed with additional 3% TFA (3 x 2 ml) and the solution again dried. The preparative HPLC using two Vydac C18 2 * ΙΞ κ 25 cm reverse phase columns in series was a gradient of 2% CH 2 CN to 24% for 9 5 allowed permeation of a 36.66% eKacto elution peak under these conditions. Analytical HPLC yielded a single peak after c-cramatography of a cyclic standard peptide bound by disulfide with the product obtained from the HPLC preparation: FAB-1S gave a ¹³-β-13 'of: 1171, which is consistent with the cPMD4 cyclic structure attached to the disulfide to CSEQs.

H-NIa-Cys-His-IIs-Bly-Pro-Sly-Arg-Ala-Phe-Cys-CQOH

EXAMPLE 16. The peptide of the linear peptide Cbz-Nle-LysCBoc &gt; -Brn-Arg-Protr.) Was synthesized following the solid phase procedures on a peptide synthesizer ABI 431A using 373 milligrams CO,! The preparation of the norleucine, which was obtained in the protected form of the benzyloxycarbonyl CChz), the L-amino acids used were the fluoromethylcycloalkyl derivatives (Fmoc), the Suitable acid-labile side chain probe groups The product of the derivatized polypeptide resin was transferred to a sedimentated glass funnel, washed with dichloromethane and dried to give 6.5 g of polypeptide resin product was cleaved from the resin by treatment with a 95 ml 2: 3 mixture of TFAsis2-stannadiisanisal for 16 hours. The reaction mixture was filtered through a funnel of /

the resin was washed with 10 ml of TFA and the filtrates combined. After concentration at about 2 ml of oil, the linear peptide was recovered by trituration with 40 ml of distilled ether in portions of 5% ml and filtration on a sedimented glass funnel. Dissolution with 10 ml of 1% TFA followed by lyophilization yielded 298 mg of the linear peptide. The peptide powder was dissolved in 1 ml of 1% neutralized with 42 ml of the diisopropylethylamine and treated with 77 ml of diphosphorylphosphonates. The solution was stirred in the dark for 7 hours at -4 ° C. ° C to allow formation of the cyclic lactam. After saturation by addition of 3 ml of glacial acetic acid the reaction mixture was concentrated to about 1 to 2 ml of oil, dissolved in 10% aqueous acetic acid and lyophilized. The cyclic peptide was purified by flash chromatography of size 8 -15 using 5% acetic acid as the mobile phase The fractions monitored by Uv5 dehiscation containing the peptide were pooled and lyophilized to give 135 mg dry cyclic peptide. All results obtained were consistent with the structure cPMD1Ss

which can also be represented by horns

Z-Nle-Lys-Gln-Arg-Gly-Pro-Gly-Arg-Ala-Ile 4-N-c = ° (ε)

Des p r c? tation of cPND15 for the form of Hydrogen?

Deprotection of ePNDs was θ. I c sn π by dissolution of the cyclic peptide. ml acetic acid: 1 to 3% &amp; ΙΊ .1. The reaction mixture was stirred at reflux for 1 hr. The mixture was stirred at reflux for 1 hr. The reaction mixture was stirred at reflux for 1 hr. ? A reversed-phase HFLC employs a semi-orep column. vydac C1S was prepared in a convenient manner, and all of the compounds of formula (I) were prepared by the following procedures: RTI ID = 0.0 &gt; FAB-MS &lt; / RTI &gt; * esulates u * C3. 1 to give carbonyl = to give activated ion or to be analytical HPLC and amino acid analysis and were consistent with the structure of cPNDISs

(A) CH2 -: - - - - - - - - - - - - - - - - - - - -

(CH2) n-Gln-Ar-Gly-Ply-Gly-Ar-Ala-Phe (CH2) -NC = O (ε). The Fmoc-Phe-Wang resin was loaded into an ABI synthesizer. Fmoc single-coupling protocols were used to add Fmoc-Hαα5 Fmoc- Fcoc-His, Boc-Lys (Fmoc) and Chz-Nls to produce 3-7 grams of linear peptide resin having the sequences BGc-Lys &lt;; N-3-N-cis) -Hydro-Tris-1-Gly-and-Pro-B1 and -Arg C0) -Aa-Phs. The peptide was cleaved from the resin by treatment with 95% TFA? 5% water for two hours. The resin was removed by filtration, the TFA removed from the filtrate by evaporation in vacuo and the residue triturated with distilled ether. The precipitate was recovered by filtration and dried to give 1.7 g of linear peptide The peptide was treated with Boc-iso-lutamine-QMp (Ar) (Ala-Phe-Ala-Phe-Phe-Gly-Pro-Gly-  € ƒâ € ƒâ € ƒ7.7 grams, 2 nmoles) and DIEA (¢ 3 =, 35 ns -1 32 nmol) in DHF C10 ml> overnight at room temperature. The BMF was evaporated to dryness.

The treated precipitate was collected by filtration and washed with ethyl acetate. The dry peptide (1 9.9 grams) was treated with TFA (10 ml) for 5 hours. The TFA was evaporated in vacuo. the resin triturated with distilled ether and the precipitate is recovered by filtration and dried. The peptide was desalted on Sepbadex G-10 in 10% acetic acid as the eluant. The peptide fractions were lyophilized to give 152 grams C57? (a) and (b), and (b), and (b) a compound of formula (I)

Two servings 55 gm. 33æmoles) of the peptide were removed separately in 10æl of ice-cold DMF and DIEA (δ ml) were added and the solutions were stirred overnight at room temperature. The DMF was evaporated in vacuo and the combined residues and solubilities in CHCl3 The organic fraction was washed with 5% aqueous citric acid. then dried over Na2 SO4 and evaporated to give 0.78 gm of crude cyclic peptide. This material was treated with liquid HF &lt; 10 mL) containing anisole 1 <1 m-L) for two hours at 0 ° C. The HF was evaporated and the residue was purified by stepwise elution in reverse phase HPLC (Vydac 0-18., CH 2 CN at -50% 5 s 5 min using 1% aqueous β-TFA as the buffer 5 to give 25Θ mg of pure cPMD31 (12Θ)) =.

His-Ile-Gly-Pro-Gly-Arg-Ala-Phe HoCHN-C = O EXILEIPLE

Preparation of Haleimidopropionyl-cPND15s

1 milligram of cDMPDT-trifluoroacetate salt dissolved in 0.8 ml of a mixture of 2N HCl GsiClN. The solution was cooled in an ice bath and then 10 æl of NaHCO 3 .beta. Β 345.5 solution followed by 3.5 mg of maleimidopropionic acid ester M-hydroxysuccinimide. The reaction was allowed to proceed with stirring for one hour followed by quenching with 3 μl of trifluoroacetic acid. The reaction mixture was filtered through a 0.2 micron filter and the filter was washed with 2 ml of water. The combined filtrates were injected into a reversed phase column of 2.times.15 X 25 cm 3 CiS. The column was eluted isocratically for 1 min at a flow rate of 1 ml / min to 25% conFNC / 051% TFA. followed by gradual annealing of rCCN Ξ 5 to 40% / 0.1% TFA for 2Θ minutes. The product eluting between 2Θ and 32min was concentrated to lyophilisate affording 7 mg of the maleic / Hydropypropionol-cPMD15 trifluoroacetate salt as a The amorphous powder of the FFTB-MS showed a major Î ± -H (H) ion at 12 &amp; 2. The titration to the maleic acid was determined by quenching Ellman to give a concentration of 0.054 pmoles per mg of the maleimidopropionol "C: PMD15B EXAMPLE 19

Preparation of Risks:

Following the procedure of Example 13, 37.0 mg of the cPMD3i trifluoroacetate salt were reacted with 8.3 mg of maleimidopropional ester of M-hydroxysuccinimides in 4 ml of a solution of NaHCO3. β, 322 M and 1.2 ml of isopropylamine, followed by quenching with i.sup.5, of TFA. HPLC of 3% CMS / isocratic TFA preparation for 1 minute followed by gradient elution of 3% -5% NaCl for 5 min gave a product elution peak of 18-25 min. The lyophilized product weighed 26 mg and the maleimide of the title was 0.57æl / mg FAB-nS gave a major ion (M-Hi) at 1356. Analysis of amino acids gave Nle-4α, β-alanine = 420 and Lys = 40 nmoles / m @ 2. NMR analysis gave a singlet at 6.93 ppm imimimido H). EXAMPLE 2A.

Protocol for Inoculation of Animals with the conjugates MIEP-cPND15 and MEPEP-cPND31 of this invention

Alum was used as an adjuvant during the inoculation series. The inoculum was prepared by dissolving the conjugate in physiological saline at a final conjugate concentration of 3 .mu.g / ml. Aluminum alloyed aluminum alloy was added to the solution at a final level of 50æg / g of aluminum. The conjugate was allowed to adsorb onto the slumen gel for two hours at room temperature. Following adsorption, the gel with the conjugate was washed twice with physiological saline and resuspended in saline at a protein concentration of 3æg / ml.

African green monkeys were inoculated individually with three doses of 3 μg / μl or tris doses of 10 μg of the adsorptive conjugate in alum. Each dose was injected intramuscularly. The doses were given one more interval (week β, 4 ., 8, 28) 'The animals were bled at intervals of two weeks. Serum samples were prepared from each bleed to assay the development of specific antibodies as described in the subsequent examples.

Serum Analysis for Antibodies IqB Anii-Peptides

Each serum sample is analyzed by enzyme-linked immunosorbent assay (ELISA). The polystyrene microtiter plates were coated with 5æg per well of the syntactic peptide (non-conjugated to HIEP) in phosphate buffered saline (PBS) at 4 ° C. Each well was then washed with TBSW-20 (PBS-T) -containing PBS, G-test serum, serially diluted in PBS-T, was added to the wells containing the peptide and allowed to react with the peptide sasorbous for one hour at 36 ° C. After washing with PBS-Ts the alkaline phosphatase-conjugated goat anti-human IgB was added to the test wells and allowed to react for one hour at 320 ° C. The wells outside And then washed extensively in PBS-T. Each well received 0.1% ρ-nitrophenyl phosphate in 10% diethanolamine, ρ 9.8 containing 1% NaCl, 0.5% NaOH, 5% was allowed to warm to room temperature for 3 minutes, at the same time as t ermined by the addition of NaDH 3,

The greater the intersection of the antibodies in the test serum with the peptide substrate, the greater the amount of

alkaline phosphatase bound to the cavity * Phosphodiester enzymes mediate the decomposition of p-nitrophenyl phosphate into a molecular substance that absorbs light at a wavelength of 485 nm. Consistently there is a direct relationship between the absorbance at 485 nm light at the end of the ELISA reaction and the amount of antibody bound to the peptide

All monkeys inoculated with the maleimino-propionyl-cPWD 15 -IEP and maleimidopropianyl-CPND31-MEPEP conjugates develop antibodies specifically capable of binding the peptide,

Analysis of Serums for Activids which specifically neutralizes H1N1V infection Viral neutralizing activity is determined by an assay described by Robertson et al. , J. Virol, Methads 28% 195-282 C889 * The assay measures the specific HIv-neutralizing activity in the test serum The assay is based on the observation that HT-4 cells, a human T-cell line , are readily susceptible to HIV infection and after a period of virus replication * are killed as a result of the infection. * The test serum is treated at 56 ° C for & minutes prior to the test. This treatment is required to eliminate non-specific inhibitors of HIV replication. Heat-treated serum diluted in series in culture medium of RPM 1-16485 cells is mixed with a dose of standard HIV infection. The dose is determined prior to the assay as containing the small amount of virus required to kill all MT-4 cells in the

test culture after a period of 8/8 days. The virus-virus mixture is then allowed to interact for one hour at 37 ° C. It is then added to 1Âμ) β1 cells not-4 suspended in growth medium RPMI-40 supplemented with 11% fetal bovine serum. Cultures were incubated at 37 ° C in an atmosphere of 100 to 5 ° C for 7 days. At the end of the incubation period a metabolic dye is added to each culture This dye is yellow in color after visual inspection. In the presence of live cells the dye is processed metabolically for molecular species which yield a visible color. The neutralized H1V can not replicate in the target MT-4 cells and therefore not kills the cells. Consequently, positive neutralization is determined by the development of the blue color following the addition of the metabolic dye. EXAMPLE 23

Preparation of a Cyclic Disulfide for Conjugations 1 Preparation of cPND33 (SEO IDs 22¾> H-Mle Cys Tyr Asn Lys Arg Lys Arg Ils His Ile and Pro Gly Arg Ala Phe Tyr Thr Thr Lys Asn Ile Ile Gly Cys-DH & lt , The weight of the formula = 3023, the 26mer's were assembled on the Milligen # 905 synthesizer starting from the partially racemized Fmoc-L-Cys-TRU-OPKA resin (Million portion B 090426, 0081 meq / g), using 2.47 (0.2-0.0 meq). The theoretical yield is 6.4 mmo. The resin

was mixed with a beaker volume of Sigma 150-212 μl beads). The mixture completely filled two columns of serially ligated æ cm 3. The ramagents were Fmac-Pfp esters (except for trsonine, which was OHBt ), using four times the molar ratio in M-methylpyrrolidine solvent. Side chain protection was; Tyr-heptyl) and Lys (Boc); Arg (litr) The protocol was modified to give &quot; A &quot; &quot; A &quot; Ile's 01v &quot; X Pro *; Βίν * ϊ "15 '. 17 18 19 20 * T, 23 * '24 "'ηγο ϋ Pne; Tyr 5 i hr § Thr | The times the reaction cycles were extended from 3 to 6 minutes for all units except for Blyiv and Ala's for 90 minutes for Al2 O2. &Quot; &lt; 2x &gt;;; The resin derivative was maintained as the free amine terminal was washed with water and dried in vacuo. The mixture of the dried derivative resin was dried over sodium sulfate, dried over sodium sulfate, and evaporated to dryness. the glass beads were suspended in 95% TFA, 4% ethanolic, 1% CH3 CH3 (30 ml) at 23 ° C in a sealed vial with gentle shaking on a 3 hour oscillation tray. The bright yellow mixture was then filtered and the insolubles were vigorously extracted with 10% TFA (3 x 20 mL). The dark orange filtrates were evaporated to give a pale brown oily gum. After trituration with ether (mL) this material was taken instantly in a colorless solid, which was transferred to a filter by trituration with additional ether (3 x 2 mL). After drying, the crude product was obtained as a fine colorless powder &lt; 583 mg).

Analytical reverse phase HPLC at -10% aqueous / 22% CTFA, λmax = 215 nm, A = ®, δ5.2 æm / min, J on a Vydac column of 0.46 κ 25.6 cm of about 5 μg of sample, dissolved in 50 μl of 0.1% aqueous TFA / CH2 Cl2. to 2%, injected 4 μl, revealed a major component (36.29%) and a secondary component of subsequent elution. These were collected separately after

injection of a 300 mg aliquot into another sample of 50 mg sample for two columns of preparation Vydac C., c &gt; of 2.21 κ 25, Θ cm in linear gradient series for 60 "; 0.02% TFA / CH 2 CN, 23-27%, lambda '215 ni', A * 3, 10 ', 10' L / min 3. A total of 35.2 mg of the above elution material (44.45 &gt; 8.2 mg of the subsequent elution material was recovered following lyophilization. FAB-MS of the major product gave a 1 H-H + 3022.1 and a CM + Na3 + ::: 3 → 44..2 which is consistent with the calculated mass .. EXAMPLE 2 Preparation of the compound of formula (I) Nys and Tyr Asn Lys Arg Lys Arg Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr Thr Lys Asn CH2

The linear 26-mer dithiol compound (35.0 mg) was dissolved in distilled water (38 mL) at 23 ° C to give the title compound as a white solid. 1H NMR (DMSO-d6):? a clear colorless solution at pH 2 = 73. The pH was adjusted to 8.5 with 0.1N NFLOH and the solution was covered with an astatine atmosphere. An aliquot of the material was run immediately on analytical reverse phase HPLC and it was found to be emitted as evidenced by the appearance of an anticipated peak *

It was added by means of a hypodermic syringe to a magnetic solution, a freshly prepared solution of N, N-dimethylformamide at 23øC under nitrogen. Analysis of a small, HPLC aliquot revealed the total conversion of the material

starting at a previous elution time. The reaction mixture was cooled to -78 ° C. was mixed with aqueous acetic acid and stirred at a given pH of 4.0. The solution was filtered to remove the insoluble material and the pale yellow solution was evaporated and then lyophilized to give about 9.9 mg of a pale yellow powder. The material was dissolved in 2% and 2% gradient-gradient TFA in CH2 Cl2 / CH2 Cl2 on preparative HPLC. A major prioritization peak at a subsequent elution peak C4 (i) were separately collected and lyophilized to give 6 = 1 mg of the above elution material and 1.5 mg of the latter. FAB-MS analysis of the above elution material; ΕΜΉ-Π '3019 * 7? rfi + Na-Γ 3042 * 5; FAS-MS analysis of the subsequent elution material CM-H- 3Θ2tH + Naj * former material uncle = 3041 * 5? which all correspond to the correct mass for cyclized PMD33, the subsequent elution material is the carboxy terminal diastereomer of D-cysteine. Amino acid analysis of the products gave the predicted amino acid compositions for the cyclic products: And confirmed that the subsequent elution material was the diastereomer containing D-cysteine,

B. The linear oxime prepared in (1) above &lt; 8.00 mg * 28.4 ppm) was dissolved in aqueous 2% TFA / acetonitrile (284 nsL) at 23 ° C and the solution was cooled to -78 ° C. left open in the air. Cyclization was monitored by reverse phase HPLC and the sample showed to be almost completely converted to the material in the previous elution with almost complete disappearance of the linear starting material for 24 hours. The clear, colorless solution was evaporated to ca. 8 mL where it was

added an additional i mg sample prepared in the same manner as 86 mg. The combined sample was evaporated to ca 9 ml. The cloudy colorless solution was subjected to HPLC separation in two separate runs on two columns C * g Vydac de 2.12 x 25.0 cm, in series. The two fractions were collected separately, one peak at the previous elution and one elution peak. Each peak was separately evaporated and lyophilized to give 33.0 mg and 9.7 mg of the foregoing and subsequent materials respectively. The above elution material was combined with other preparations of the above cyclized elution material to give a total of 47 , 5 mg of a very light colored powdered powder. Analytical HPLC of this material gave a single peak. PREPARATION OF THE ANTIRIDE, ..... PP ... ÂCi..l &gt; Q, _ .. 3 &quot; MftLElllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll The 3 "-malimidopropionic acid (226 mg) was overlaid with 5 ml of anhydride and the mixture was heated at 113 ° C for 3.75 h and then aged overnight at ambient temperature. The solution was concentrated to an oil and the NMR spectrum (CDCl3) indicated a mixture of the homoanhydride and the mixed anhydride of acetic and maleimidopropionic acids. The starting acid shows the methylene adjacent to the carbonyl as a tripeptide centered at 2.68 ppm whereas in the anhydride these resonances appear at 2.81 ppm. Purification was by fractional sublimating, first at 7β and 2 mM and then at 12 DEG C. The subsequent fraction was removed from the apparatus by dissolution in CDCl 3 to afford 34 mg of pure homoanhydride after evaporation of the solvent. This was recrystallized from CDCl3 and cyclohexane yielding the melting material at 43-147Â ° C.

Calc'd for C, 52.51. H, 3.78; N, 8.75.

Found: C, 51.73; H, 3.67; N, 8.16, RHN at 20 MHz (CDCl3): 2.83 (2H, t), 3.84 &amp; 73.2 (2H, s)

CPND33 (22.5 mcn peptide estimated to be 7Θ% is equivalent to 15.75 inq or 5.212 Âμmoles) was dissolved in  ± 2.0 ml of a morpholinestane acid buffer Analysis of this solution on the progress of the reaction were followed by HPLC on a 25 cm DBS column using 25% aqueous acetonitrile and 1% trifluoroacetic acid (1%) as a white solid, as an eluent. The maleimidopropionic acid anhydride (2.20 g, 25 micromoles) was dissolved in dry tetrahydrofuran (60 ml) and this solution (corresponding to 3.2 micromoles of anhydride) was added to the solution. After 3 minutes, an aliquot of 7 ml / liter was removed and evaluated by HPLC. This assay was repeated at 25 DEG-50 DEG, 1.25 DEG, 2.25 DEG C. After 3, The solution was lyophilized. The lyophilisate was dissolved in 2 mL of 2% aqueous acetanitrile, filtered through a 2 micron filter and preparably chromatographed in three, © © mL-1 operations. column C-18 Zorfaax of 21.2 mm κ 25 cm The following flow rate-1 ml / nsinp isocratic elution was used with 25% aqueous acetonitrile / 10% aq (12 min) (8 min) The tail fractions were isolated by concentration and lyophilization to give 8.9 mg of recovered starting material (penultimate fraction) and 9.0 g , 6 mg of a product having a mass spectrum (FAB) indicating a molecular weight of 3172 (ΐ), the nono-maleimidopropionyl derivative of cPND33) The product was subsequently screened by sequence analysis for the absence of iisine (absence of any sequence would imply acylation at the amino terminus). The results indicate that most but not all portions

Most of the lysine is bound to the carboxyl terminal.

Although the foregoing specification teaches the principles of the present invention, with examples given for the purpose of illustration, it will be understood that the practice of the invention encompasses all variations * modifications or variations which are within the scope of the following claims and their equivalents *

(Ii) TYPES OF MOLECULES peptide (iii) HYPOTHETICS mo (v) TYPE OF FRAGMENTS internal &lt; (ii) TYPE OF MOLECULES peptide (iii) HYPOTHETICAL mo (v) TYPE OF FRAGMENTS internal &lt;; ix) CHARACTERSTICS (A) NAME / KEYS disulfide bond <B) LOCATION? 3,, 38 &lt; xi &gt; DETERMINATION OF FOLLOW-UP SEGMENTS

Xle Asn Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser He Arg 1 5 10 15

Ile Gin Arg Gly Prc Gly Arg Ala Phe Vai Thr lie Gly Lys He 20 25 30

(I) SEQUENCE CHARACTERISTICS (A) CHARACTERISTICS OF 24 ASRINOACIDS CB) Amino acid TYPES &lt; D &gt; Linear TOPOLOGIES (ii> peptide MOLECULE TYPE &lt; hr) DESCRIPTION OF FOLLOW-UP SEGMENTS # 134

Tyr Asn Lys Arg Lys Arg Ile His Ile Gly Pro Gly Arg Ala Phts 1 5 1® 15

Tyr Thr Thr Lys Asn Ile Ile Sly Thr 2Φ (2) INFORMATION FOR SEQ ID NOs 3s (i) SEQUENCE CHARACTERISTICS (a) LENGTHS 25 amino acids CB) TYPE: amino acid (D) TOPOLOGY: linear (iii) TYPE OF MOLECULES (i.e. peptide) DESCRIPTION OF THE SEQUES: SEQ ID NO: 3;

Asn Asn Thr Thr Arg Ser Ile His 81a Pro Sly Arg Ala Phe 1 5 W 15

Tyr Ala Thr Sly Asp Ile Ile Sly Asp Ile 20 25 (2) INFORMATION FOR SEQ ID NO: 4s &lt; i &gt; SEQUENCE CHARACTERISTICS: CA) COMPLEXES 25 amino acids CB &gt; TIPQs amino acid CD) TOPOLOSIA: linear tii> Peptide MOLECULE TYPE (xi &gt; SEQUENCE DESCRIPTION: SEQ ID NOs 4s

Asn Asn Thr Arg Lys Ser Ile Arg Ile Gin Arg Gly Pro Sly Arg 1 5 10 15

Ala Phe Go to Thr Ile Gly Lys Ile Gly Asn 25 (2) INFORMATION FOR SEQ ID NOs 5s

Ci &gt; SEQUENCE CHARACTERISTICS: CA) LENGTHS 18 amino acids (B) amino acid TYPES (D) TOPOLOGIES linear (ii) TYPE OF MOLECULES peptide (xi) SEQUENCE DESCRIPTION SEQ ID NO: 5s

Arg Ile Gin fírg Gly Pro Oly ftrg Ala Phe Vai Thr IIs Gly Lys 1 5 10 15

Ile Gly Asn (2) INFORMATION FOR SEQ ID NO: (i) SEQUENCE CHARACTERISTICS: (A) LENGTHS ii amino acid <B) amyloid acid TYPES CD) TOPOLOGY: linear Ui) TYPE OF MOLECULES peptide (ix) DESCRIPTION OF SEQUENCES SEQ ID NOs

(Ii) SEQUENCE CHARACTERISTICS: CA) LENGTHS 8 amino acids CB) TYPE aminacid CD &gt;TOPOLOGY; linaar

Cii) TYPE OF MOLECULES peptide

Ckí) SEQUENCE DESCRIPTION; SEQ ID NO; 7; His Ile Sly Pro Sly Arg Ala Phe 15 i2 &gt; INFORMATION FOR SEQ ID NOs 8:

(i) SEQUENCE CHARACTERISTICS &gt; COMPRXHENTDs L · Amino Acids CEO IT PO S am o nd e c e d) D) Linear TOPOLOBIA &lt; ii &gt; TYPE OF MOLECULES peptide &lt; Ki) DESCRIPTION OF THE SEQUENCES SEQ W NOs 8s

Gly Pro Sly Arg Ala Phe 1 5 (2) INFORMATION FOR SEQ ID NOS 9s U) SEQUENCE CHARACTERISTICS (A) LYMPHOCYCACIDES &lt; B &gt; (Ii) TYPES OF MOLECULES peptide (ix) DESCRIPTION OF THE SEQUENCES SEQ ID NOs 9s

Ile Bln Arg Bly Pro Sly Arg Ala Phe 5 &lt; 2 &gt; INFORMATION FOR SEQ ID NOs 10s &lt; i) SEQUENCE CHARACTERISTICS; CA) LENGTHS 9 amino acids (B) TYPE s amino acid CD) TOPOLOBIAs linear (ii) TYPE OF MOLECULES peptide (i) DESCRIPTION OF SEQUENCES SEQ ID NOs 10s Tyr II Gly Pro Gly Arg Ala Phe i 5

(2) INFORMATION FOR SEQ ID NG: (i) SEQUENCE CHARACTERISTICS CA) LENGTHS 9 amino acids CB) TYPE? CD) TOPOLOSXAs linear

(Iii) TYPE OF MOLECULES peptide (xi5 DESCRIPTION OF SEQUENCES SEQ ID NO: 1 SEQ ID NOs: SEQ ID NOs: 12S

C) SEQUENCE CHARACTERISTICS (SEQ ID NO: 12) SEQUENCE CHARACTERISTICS (SEQ ID NO: 12) SEQUENCE CHARACTERISTICS (SEQ ID NOs: 12)

Go Thr Leu Sly Pro Sly Arg Go Trp i 5 (2) INFORMATION FOR SEQ ID NOs 13?

Ci) SEQUENCE CHARACTERISTICS? CA) LENGTH? 9 amino acids CB) TYPE? linear amino acid CD) TOPOLOSIAs

(Iii) TYPE OF MOLECULES peptide (xi) DESCRIPTION OF SEQUENCES SEQ ID NOs 13si (1) Thr Lys Sly Pro wt Arg Ile Ile 138 (2) INFORMATION FOR SEQ W NOS 145 &lt; I &gt; SEQUENCE CHARACTERISTICS? (A) LENGTHS 9 amino acids CB) TYPE? (D) Linear TOPOLOBIAs (ii) TYPE OF MOLECULE? peptide &lt; ki) SEQUENCE DESCRIPTION? SEQ ID NOs 14? (2) INFORMATION FOR SEQ ID NOs: 15s (i &gt; SEQUENCE CHARACTERISTICS? &lt; A &gt; LENGTH? 9 amino acids (B) TYPES amino acid CD) TOPOLOBIAs linear &lt; ii) TYPE OF MOLECULES peptide (i) SEQUENCE DESCRIPTION? SEQ ID NO? 15?

Thr Pro Ile Biy Leu Bly SIn Ais Leu 1 5 (2) INFORMATION FOR SEQ ID NO? 16?

Ci) SEQUENCE CHARACTERISTICS? (A) LENGTH? 9 amino acids (B) TYPE? (D) Linear TOPOLOBIAs &lt; ii) TYPE OF MOLECULE? peptide

(Mi) DESCRIPTION OF THE SEQUENCES 3EQ ID NOs 16s His Rhe Sly Pro Bly BIn Leu 1 5 <2) INFORMATION FOR SEQ ID NO a 17s <i) SEQUENCE CHARACTERISTICS? CA) LENGTHS 9 amino acids CB) TI PO to amine acid CD &gt; TOPOLOBIAs linear (ii) TYPE OF MOLECULE: peptide (i) DESCRIPTION OF SEQUENCES SEQ ID NOs 172

Ile Arg Ile Sly Pro Sly Lys goes Phs 1 &lt; 2) INFORMATION FOR SEQ ID NOs 18a

Cl) SEQUENCE CHARACTERISTICS CA) LENGTHS 4 amino acids CB) TYPES of amino acids C D) TOPOLOG1A s both <ii) TYPE OF MOLECULES peptid

Cxi) DESCRIPTION OF SEQUENCES SEQ ID NOs 18s Gly Pro Giy Arg (2) INFORMATION FOR SEQ ID NOs 19s

Ci) SEQUENCE CHARACTERISTICS CA) LENGTHS 4 amino acids &lt; 8) TI PO s am i n o a e .1. cl or CD) TOPOLOSIAs both (iii) TYPE OF MOLECULES peptide

&lt; k &gt; SEQUENCE DESCRIPTION; SEQ ID NOs 19;

Gly Pro Gly Lys 1 &lt; 2 &gt; INFORMATION FOR SEQ ID MO; 2Θ; (ii) SEQUENCE CHARACTERISTICS; (A) LENGTHS 4 amino acids B &gt;TYPE; smincácidc (D) TOPOLOGY; both i i.3.) TYPE OF MOLECULE; peptide (Ki) SEQUENCE IDENTIFICATION SEQ ID NOs: 2S0 and Pro Gly Gin i2) INFORMATION FOR BEQ ID NO; 21s (i) SEQUENCE CHARACTERISTICS; (A) COMPRINATIONS 4 amino acids (B) TYPE; CD) TOPOLOGY; both iii) TYPE OF MOLECULE; peptide (i) SEQUENCE DESCRIPTION; SEQ ID NOs 21s

Gly Leu Gly Gin i2) INFORMATION FOR SEQ ID NO = 22; (ii) SEQUENCE CHARACTERISTICS; CA &gt;LENGTH; 26 amino acids

CB)! (I) TYPE OF MOLECULE: peptide <ik) CHARACTER: t ST I CA% (A) NAME / CHAVES modified site <B &gt; LOCATIONS 1 (D) OTHER INFORMATION / classification ^ Nls / note = "norleucins" ix CHARACTERISTICS 1A) NAME / CHAVES disulfide bond CB) LOCATIONS 2 »= 26 (i) DESCRIPTION OF SEQUENCES BEQ ID NOs 22ϊ

Leu Cys Tyr Asn Lys Arg Lys Arg lis His Ile Sly for Gly Arg 5 5 5 5

Ala Phe Tyr Thr Thr Lys Asn Ile Ile Gly Cys 20 25 (2) INFORMATION FOR SEQ ID NOs 23s (li) SEQUENCE CHARACTERISTICS2 (A) LENGTH? 11 amino acids (B) amino acid TYPES (D) TOPOLOGIES both &lt; ii &gt; TYPE OF MOLECULES peptide &lt; i.k) CHARACTERISTICS (A &gt; NAME / CHAVEs modified site 1B5 LOCATIONS 1 (D) OTHER INFORMATIONdh / classification = NIe / RDta = unDrlsLicine <kí> SEQUENCE DESCRIPTION SEQ ID NOs 23s

(Ii) INFORMATION FOR SEQ ID NOs 24π (i) SEQUENCE CHARACTERISTICS (i) COMPOSITIONS 4 amino acids (b) TYPE aminDACID (D) TOPOLOGIES both (ii &gt; Peptide MOLECULE TYPE κI &gt; SEQUENCE DESCRIPTION SEQ ID NOs 24s

Gly Pro Sly Go 1

Claims (3)

A method for the preparation of a vaccine for use in mammals comprising the Class II outer membrane Class II protein of serogroup 8 serogroup 8 coupled to an antigen in the range of from 0.1% to 50% % of a class II protein in an antigen conjugate, a vaccine which in mammals will induce or enhance the formation of antibodies specific for said antigen. 2. A process according to claim 1, characterized in that the antigens are derived from bacteria, viruses, mammalian cells, fungi, fungi, allergens, viruses or poisons, synthetic peptides, fragments of polypeptides " A method according to claim 1, characterized in that the outer membrane Class II protein of Heissaria meninqitidis, serogroup B, is a recombinant protein produced in a recombinant host cell. A process for the preparation of a composition comprising mixing an effective amount of at least 1æg for the immunization of the mammalian species of the polysaccharide / protein conjugate, which comprises the polysaccharide of the HaSBBBF1, and the B polysaccharide, Meisseria meninqitidis Class II outer membrane protein. serogroup B, is a pharmaceutically acceptable carrier. Lisbon, July 18, 19vl J. PEREIRA DA CRUZ Official Agent for Industrial Property RUA VICTOR CORDON, 10-A 3. * 1200 LISBOA