CA2169453A1 - Inhibition of hiv mucosal infection - Google Patents
Inhibition of hiv mucosal infectionInfo
- Publication number
- CA2169453A1 CA2169453A1 CA002169453A CA2169453A CA2169453A1 CA 2169453 A1 CA2169453 A1 CA 2169453A1 CA 002169453 A CA002169453 A CA 002169453A CA 2169453 A CA2169453 A CA 2169453A CA 2169453 A1 CA2169453 A1 CA 2169453A1
- Authority
- CA
- Canada
- Prior art keywords
- peptide
- mucosal
- hiv
- cells
- antibodies
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
- A61K39/21—Retroviridae, e.g. equine infectious anemia virus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
- A61K2039/541—Mucosal route
- A61K2039/542—Mucosal route oral/gastrointestinal
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Abstract
Disclosed is a method of inhibiting the entry of HIV into the vaginal and rectal mucosal epithelium by administering a peptide vaccine. Antibodies generated against peptides corresponding to epitopes of gp120 involved in entry into mucosal cells are generated in vivo by introduction of peptides or peptide-cholera toxin conjugates into epithelial cells. The resulting neutralizing antibodies are able to block subsequent infection of these tissues by HIV.
Description
~ WO95/11701 21 694~3 PCT~S91/12152 INHIBITION OF HIV MUCOSAL INFECTION
FIELD OF THE INVENTION
This invention relates to the inhibition of binding of HIV to the genital and rectal mucosal epithelium.
Dispersed aggregates of non-encapsulated lymphoid tissue are often localized to the submucosal areas of the gastrointestinal, respiratory and urogenital tracts. These tracts are a main means of entry into the body by foreign 10microorganisms. Secretory immunoglobulin A (IgA) is an antibody capable of crossing mucosal membranes and protecting them against invasion by pathogens. Mucosal lymphoid tissue thus plays an important role in the local immune response which occurs at mucosal surfaces.
15It is well established that mucosal epithelial cells, regardless of whether they express the cell surface CD4 receptor used by the HIV to enter T-cells, macrophages and Langerhans cells, can be latently infected by HIV (Fantini et al., (1992) J. Virol., 66: 5805; Fantini et al., ~1991) 20Virology, 185: 904). Although the receptor~s) for HIV entry into mucosal intestinal epithelial cells appear to be glycolipids (Yahi et al., (1992) J. Virol., 66: 4848), there is no information regarding the HIV epitope(s) which mediates attachment to these cells. Such knowledge would be of 25paramount importance since this epitope(s) would be an ideal target against which the local mucosal immune system could act to prevent the mucosal entry of HIV.
The induction of a mucosal immune response to prevent entry of human immunodeficiency virus (HIV-1) through the 30rectal and genital (vaginal) mucosa has not been significantly explored as an approach in preventing AIDS. Conventionally administered vaccines derived from the viral glycoprotein gpl20 provide little immunity to HIV. Systemic immunization strategies have protected against intravenous challenge with 35simian immunodeficiency virus (SIV), the monkey counterpart of HIV, in monkeys, but have failed to prevent infection by SIV
introduced via the vaginal mucosa (Miller et al., (1990) J.
WO95/11701 ~ ~ G q 4 5 ~- PCT~S9~/121~2 Immunol., 144: 122).
In general, mucosal delivery of antigens does not evoke a strong immune response. A notable exception, however, is cholera toxin (CT), produced by the bacterium Vibrio cholera, S which i5 among the strongest mucosal immunogens known. CT
binds strongly to a glycosphingolipid called ganglioside GMl on mucosal cell surfaces using its B subunit. Mucosal administration of minute amounts of antigens covalently linked to the B subunit (CTB) has been shown to elicit vigorous mucosal as well as extramucosal immune responses in experimental animals including nonhuman primates (Czerkinsky et al., (1989) Infect. Immun., 57: 1072-1077; Liang et al., (1988) J. Immunol., 141, 3781-3787; Lehner et al., (1992) Science, 258: 1365-1369; Holmgren et al., (1993) Vaccine, 11:
1179-1184). The possibility of disseminating a specific B-cell response from the gut to other mucosal tissues in orally immunized hl7m~n.s has also been documented (Czerkinsky et al., (1991) Infect. Immun., 59: 996-1001). In addition, it has been demonstrated that mucosal immune responsiveness in HIV-l infected individuals remains relatively stable compared to a dramatic hyporesponsiveness to parenterally administered vaccines (Eriksson et al., (1993) AIDS, 7: 1087-1091). This study not only underscores the relative independence of mucosal and systemic immunity, but also raises the possibility of inducing HIV-specific mucosal immunity in an already infected individual, thus interfering with subsequent mucosal transmission. Immunization strategies effective in inducing an immune response in the genital and rectal mucosa have been evaluated in nonhuman primates (Lehner et al., (1992) Science, 258: 1365-1369).
In view of the incidence of sexually transmitted HIV
infection (over 75~ of all cases), the alarming increase in the number of new AIDS cases and the inability of systemic immunization strategies to induce a significant mucosal immune response, a vaccine able to produce an immune response at the mucosal surfaces through which HIV gains access to the circulation would have significant value as part of an overall WO 95/11701 ! ,, i _j PCT~S9~/12152
FIELD OF THE INVENTION
This invention relates to the inhibition of binding of HIV to the genital and rectal mucosal epithelium.
Dispersed aggregates of non-encapsulated lymphoid tissue are often localized to the submucosal areas of the gastrointestinal, respiratory and urogenital tracts. These tracts are a main means of entry into the body by foreign 10microorganisms. Secretory immunoglobulin A (IgA) is an antibody capable of crossing mucosal membranes and protecting them against invasion by pathogens. Mucosal lymphoid tissue thus plays an important role in the local immune response which occurs at mucosal surfaces.
15It is well established that mucosal epithelial cells, regardless of whether they express the cell surface CD4 receptor used by the HIV to enter T-cells, macrophages and Langerhans cells, can be latently infected by HIV (Fantini et al., (1992) J. Virol., 66: 5805; Fantini et al., ~1991) 20Virology, 185: 904). Although the receptor~s) for HIV entry into mucosal intestinal epithelial cells appear to be glycolipids (Yahi et al., (1992) J. Virol., 66: 4848), there is no information regarding the HIV epitope(s) which mediates attachment to these cells. Such knowledge would be of 25paramount importance since this epitope(s) would be an ideal target against which the local mucosal immune system could act to prevent the mucosal entry of HIV.
The induction of a mucosal immune response to prevent entry of human immunodeficiency virus (HIV-1) through the 30rectal and genital (vaginal) mucosa has not been significantly explored as an approach in preventing AIDS. Conventionally administered vaccines derived from the viral glycoprotein gpl20 provide little immunity to HIV. Systemic immunization strategies have protected against intravenous challenge with 35simian immunodeficiency virus (SIV), the monkey counterpart of HIV, in monkeys, but have failed to prevent infection by SIV
introduced via the vaginal mucosa (Miller et al., (1990) J.
WO95/11701 ~ ~ G q 4 5 ~- PCT~S9~/121~2 Immunol., 144: 122).
In general, mucosal delivery of antigens does not evoke a strong immune response. A notable exception, however, is cholera toxin (CT), produced by the bacterium Vibrio cholera, S which i5 among the strongest mucosal immunogens known. CT
binds strongly to a glycosphingolipid called ganglioside GMl on mucosal cell surfaces using its B subunit. Mucosal administration of minute amounts of antigens covalently linked to the B subunit (CTB) has been shown to elicit vigorous mucosal as well as extramucosal immune responses in experimental animals including nonhuman primates (Czerkinsky et al., (1989) Infect. Immun., 57: 1072-1077; Liang et al., (1988) J. Immunol., 141, 3781-3787; Lehner et al., (1992) Science, 258: 1365-1369; Holmgren et al., (1993) Vaccine, 11:
1179-1184). The possibility of disseminating a specific B-cell response from the gut to other mucosal tissues in orally immunized hl7m~n.s has also been documented (Czerkinsky et al., (1991) Infect. Immun., 59: 996-1001). In addition, it has been demonstrated that mucosal immune responsiveness in HIV-l infected individuals remains relatively stable compared to a dramatic hyporesponsiveness to parenterally administered vaccines (Eriksson et al., (1993) AIDS, 7: 1087-1091). This study not only underscores the relative independence of mucosal and systemic immunity, but also raises the possibility of inducing HIV-specific mucosal immunity in an already infected individual, thus interfering with subsequent mucosal transmission. Immunization strategies effective in inducing an immune response in the genital and rectal mucosa have been evaluated in nonhuman primates (Lehner et al., (1992) Science, 258: 1365-1369).
In view of the incidence of sexually transmitted HIV
infection (over 75~ of all cases), the alarming increase in the number of new AIDS cases and the inability of systemic immunization strategies to induce a significant mucosal immune response, a vaccine able to produce an immune response at the mucosal surfaces through which HIV gains access to the circulation would have significant value as part of an overall WO 95/11701 ! ,, i _j PCT~S9~/12152
2 ~ 5 ~
approach to reducing HIV-l infection.
SUMMARY OF THE INVENTION
'- One embodiment of the present lnvention is a method for inhibiting the infection of mucosal cells by HIV-l by .- 5 administering a vaccine to the mucosa, thereby delivering to the mucosa a peptide of HIV-l gpl20 having from about l0 to about 50 amino acids, whereby antibodies against the peptide are generated in the mucosa, the peptide being selected such that the antibodies inhibit infection of HIV-l in mucosal epithelial cells.
In another aspect of this preferred embodiment, the peptide includes an epitope effective to generate mucosal production of antibodies that inhibit infection of mucosal cells by HIV-l, the peptide consisting essentially of SEQ ID
NOS: 9, l0, ll, 12, or 13. Advantageously, the vaccine further includes an agent for enhancing delivery of the peptide to the mucosa. Preferably, the agent is a mucosal binding protein; most preferably, it is either the binding subunit of cholera toxin or that of E. coli heat labile enterotoxin. The invention also provides that the peptide and the mucosal binding protein are bound together to form a chimeric protein which may advantageously be the expression product of recombinant DNA. In another embodiment of the invention, the agent is a lipid. Preferably, the lipid is in the form of a lipid vesicle. Another aspect of this preferred embodiment provides that the administering step comprises administering to the mucosa a polynucleotide operably encoding the peptide, whereby the peptide is produced by cells of the mucosa.
A further embodiment of the invention provides a vaccine for inhibiting the infection of mucosal cells by HIV-l, comprising a l0 to 50 amino acid peptide of HIV-l gpl20 having an epitope selected such that antibodies against this epitope inhibit the infection of mucosal epithelial cells by HIV-l, and a compound or structure associated with the peptide for facilitating delivery of the peptide to the mucosa.
Preferably, this peptide consists essentially of SEQ ID NO 9, WO95/11701 ~1 6 9 4 5 ~ PCT~S9~/121~2 10, 11, 12 or 13 and the compound or structure is a lipid vesicle. Most preferably, the compound or structure is a mucosal binding protein. In a particularly preferred embodiment, the binding protein is a cholera toxin protein which may advantageously be the binding subunit. In another aspect of this preferred embodiment, the binding protein is the binding subunit of E. coli heat labile enterotoxin.
DETAILED DESCRIPTION QF THE INVENTION
The present invention discloses the identiication of synthetic peptides derived from the sequence of the envelope glycoprotein gpl20 of HIV-l. These peptides were used to generate neutralizing antibodies which inhibited infection of transformed human vaginal and colorectal cell lines in vi tro.
These peptides will induce the production of a localized mucosal immune response, generating antibodies able to neutralize infection of human colorectal and vaginal epithelial cells by HIV-1. The peptides are set forth herein as SEQ ID NOS: 9-13. In one aspect of the invention, one or more of the peptides of SEQ ID NO:9, 10, 11, 12, and 13 is used to generate antibodies. These antibodies can be generated in any conventional manner, including by intramuscular, intraperitoneal, subcutaneous, or mucosal administration to an animal. Generation of both monoclonal and polyclonal antibodies are contemplated. These antibodies are then used to prevent infection of cells of the mucosal epithelium by providing the antibodies in association with the mucosal cells and then challenging the cells with HIV-1. The antibodies inhibit or prevent binding of the virus to the cells, and thereby inhibit or prevent infection of the cells by the virus.
The antibodies can be exogenous or endogenous antibodies, and the cells can be in vi tro or in vivo . When the cells are in vitro, the antibodies are typically generated in laboratory or domestic animals or are monoclonal antibodies. More importantly, it provides a valuable tool for analyzing the mechanism and structure involved in that binding.
When the cells are in vivo, the antibodies are preferably PCT~S9~/12152 2 ~ ~?453 endogenous mucosal antibodies that have been generated by administering one or more of the peptides of SEQ ID NOs 9-13 ^ to the anlmal in which the cells are located. Mucosal vaccination, as described below, is particularly preferred.
.- 5 However, exogenous antibodies may also be administered to the animal to inhibit HIV-1 infection of mucosal cells. In all of the treatments described herein, the mucosal cells are preferably of human origin.
The peptides of the present invention can be utilized alone or in combination and can also be uncoupled or coupled to other epithelial cell binding proteins including CT, CTB
and the binding subunit of E. coli heat labile enterotoxin.
The peptides may be coupled by either chemical or recombinant means. DNA encoding the peptldes can be joined to DNA
encoding cholera toxin, or its B subunit, by well known methods, inserted into a eukaryotic expression vector and delivered to epithelial cells using lipid vesicles or lamellar structures. The production of these peptide-CT, CTB or enterotoxin conjugates in vivo will then elicit a localized mucosal immune response and will protect against subsequent infection by HIV. The inclusion of muco~al eplthelial cell binding proteins, such as cholera toxin, will advantageously increase the efficiency of entry of peptides into these cells.
Since the B subunit of the cholera toxin A-B dimer is responsible for binding to cell surface receptors, a peptide-CTB conjugate will also bind efficiently to epithelial cells.
The literature also reports methods for forming compositions of immunogenic peptides and other gut binding proteins (Wenneras et al., (1990) FEMS Microbiol. Lett., 66: 107-112).
Techniques for forming peptide-CTB conjugates are well known (Liang et al., (1988) J. Immunol., 141: 3781-3787; Sanchez et al., (1990) Res. Microbiol., 141: 971-979). Liposome formation and delivery of peptides encapsulated in liposomes is also well known as described by Lowell rNew Generation Vaccines, Woodrow, G.C. and Levine, M.M., eds., Marcel Dekker, Inc., New York, pp. 141-160). These peptides are also useful in the production of monoclonal and polyclonal antibodies.
WO95111701 2 1 6 9 4 5 3 PCT~S9~/12152 These antibodies have a distinct neutralizing effect on HIV-l.
These peptides, either alone or after coupling to CT or other molecules, may be administered orally, rectally, vaginally, or in a combination of these routes in an amount sufficient to generate a mucosal antibody response sufficient to inhibit HIV-l entry into the mucosal epithelial cells. The amounts of peptides used will depend on their pharmaceutical formulation and the site and route of delivery; however, for an adult human, a suitable immunogenic amount of peptide is generally between about 50 ~g and about l mg, administered one to four times over a period of two weeks to one year or longer.
The peptides, peptide-binding protein conjugates, and other compositions of the present invention can be administered orally to generate a localized gastrointestinal mucosal immune response or intravaginally or intrarectally to produce a localized mucosal immune response in these areas prone to viral entry by sexual contact. These peptides can be administered in unit dosage in an amount necessary to produce localized mucosal immunity against HIV-l infection.
Pharmaceutical compositions envisioned for oral administration include tablets, capsules, liquids, and the like and those contemplated for intravaginal or intrarectal administration include injectable carriers, suppositories, ointments, gels, creams, foams, sprays, dispersions, suspensions, pastes and the like in an amount from about lO ~g to about lO mg or more.
These preparations can be in any suitable form, and generally comprise the active ingredient in combination with any of the well known pharmaceutically acceptable carriers. The preparations may further advantageously include preservatives, antibacterials, antifungals, antioxidants, osmotic agents, and similar materials in composition and quantity as is conventional. For assistance in formulating the compositions of the present invention, one may refer to Remington's Pharmaceutical Sciences, 15th Ed., Mack Publishing Co., Easton, PA (1975).
The ~ontemplated modes of administration assume that the peptides or conjugates are able to be directly taken up by the WO95/11701 PCT~S9~tl2152 ~ 3 epithelial cells lining these areas. These peptides and conjugates may advantageously be enclosed in liposomes to ~~ facilitate delivery of these agents to cells. Direct injection of the peptides or peptide-binding protein - 5 conjugates, either alone or in combination with lipid vesicles or other lamellar structures, into the mucosal endothelium in a similar dose range is also envisioned as a method of eliciting an anti-HIV response in these tissues.
Example 1 Susceptibility of colorectal and vaqinal epithelial cells to infection bY HIV-1 HIV-1 infectious virus stocks of HTLV-IIIB-infected H9 T
cell lymphoma cells (ATCC HTB-176) (Popovic et al., (1984) Science, 224: 497-500) were used in the following experiments.
The cells were maintained in RPMI-1640 medium containing 20 fetal calf serum (FCS), 100 units/ml penicillin and 100 ~g/ml streptomycin. Virus stocks were prepared using well known procedures and frozen at -90C. One stock of HTLV-IIIB with endpoint titer of 1 x 104 tissue culture infectious doses (TCID50) was used for all experiments.
Endpoint titration of the HTLV-IIIB isolate of HIV-1 in two clones of transformed vaginal epithelial cells (Hs 760 T
and Hs 769.Vg cells; ATCC CRL-7491 and 7499, respectively) and 12 subclones of colon adenocarcinoma HT-29 cells (ATCC HTB-38) were performed by inoculation of respective cell lines with serial 10-fold dilutions of virus with 100 ~l/well (ranging from 1 TCID50/cell to 0.00001 TCID50/cell) in 24-well plates (Costar) for 2 hours at 37C. After adsorption, cells were washed five times with Modified Eagle's Medium (MEM) and supplemented with 1.5 ml growth medium (DMEM for vaginal cells and MEM for colon cells, both containing 10~ fetal calf serum (FCS), 1~ L-glutamine, and antibiotics). Seven days after infection, epithelial cells were washed five times with MEM
and treated with 0.1~ trypsin in phosphate buffered saline (PBS) for 5 min at 37C. HTLV-IIIB-infected H9 cells (106 cells) in H9 maintenance medium were added to each well and cocultured with epithelial cells for 24 hours. The H9 W095/11701 2 1.6 9 4 5 3 PCT~S9~/12152 cultures were microscopically followed for 7 days for the presence of HIV-induced syncytium formation and p24 antigen production using an ELISA able to detect as little as lOO pg p24/ml). The vaginal cell lines Hs 760.T and Hs 769.Vg were permissive. Viral infection of Hs 760.T was detected by coculture at a high multiplicity (l TCIDsO/cell) and 6 of the HT-29 colon cell clones were permissive at multiplicities ranging from O.l-O.Ol TCID50/cell. Of these clones, cloOne L20 was chosen for further study.
WO95/11701 PCT~S9~/12152 2-1 6q453 Table 1 Susceptibility of colorectal and vaginal epithelial cells to infection by HIV-l (HTLV-IIIB).
. 5 multi~licity of infection (TCID50/cell) cell Line subclone method~ 1 lO-I 10~ X103 HT-29 EOcoculture E5coculture 0 " E8coculture - - - -L2coculture " L4coculture Ll6coculture " Ll8Bcoculture +
~ Ll8Acoculture + +
" Ll2coculture + +
" LlOcoculture + + +
" Ll4coculture + + +
~ L20coculture + + +
" L20 PCR 2.5-- 2 0.5 0.125 HS 769 Vg coculture " PCR 2.5 0.6 0.5 0.125 HS 760 T coculture +
~ PCR 10 * coculture with H9 cells and subsequent p24 antigen detection or detection of proviral DNA by PCR.
** copy number, x10-2 per cell WO95/11701 ~ t 6 q 4 5 ~ PCT~S9~/12152 ~
HIV-1 RNA and DNA was detected both in epithelial cells and in the culture supernatants as described in the following examples. .
Example 2 Detection of ~roviral DNA bY PCR -~
Epithelial cells were harvested seven days post-infection and DNA was extracted (Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory Press, 2: 9.16-9.19). Primers specific for the HIV-1 env gene (5'-GTAACGCACA~'l"l"l"l'AATTGTGGAGGGGAA-3'; SEQ ID NO: 1) and (5'-CCTCATATTTCCTCCTCCAGGTCT-3'; SEQ ID NO: 2) were used for detection of proviral DNA. DNA (20Q ng) was amplified on a DNA thermal cycler (Perkin-Elmer, Norwalk, CT) using ~-32P-dCTP
to label the fragments. The reaction mixture consisted of 10 ~l of 10x PCR buffer (Promega, Madison, WI), 1.5 mM MgCl2, 20 pmol primers, 0.125 mM dNTPs, 5 ~Ci ~-32P-dCTP and 0.5 units Taq DNA polymerase (Promega). The amplification was for 35 cycles and included denaturation at 94C for 1 min, annealing at 55C for 1 min and extension at 72C for 1 min. One-tenth of the final reaction mixture was analyzed by electrophoresis on 5~ polyacrylamide gels. The gels were dried and exposed to X-ray film (X-OMAT; Eastman Kodak, Rochester, NY) for 13-16 hours using an intensifying screen.
To measure HIV copy number (the number of HIV genomes), two-fold serial dilutions of DNA isolated from ACH-2 cells, which contain one proviral copy per cell (Clouse et al., (1989) ~. Immunol., 142: 431-438; Seshamma et al., (1992) ~.
Virol. Methods, 40: 331-346; Graziosi et al., (1993) Proc.
Natl . Acad. Sci . U. S.A., 90: 6405-6409). The total amount of DNA in each dilution was normalized to 200 ng using DNA
extracted from H9 cells and PCR was performed as above. HIV
copy number was estimated by comparison of the intensities of the amplified bands. PCR analysis using a pair of human ~-actin primers was performed in parallel as an internal standard.
WO95/11701 PCT~S9~/12152 Example 3 Detection of HIV RNA exPression by RT-PCR
Epithelial cells were harvested 7 days post-infection and total RNA was extracted by the RNAzol method (Biotex ,~ 5 Laboratories, Houston, TX). For each sample, 500 ng of total RNA was incubated with lO units RNase-free DNase I (Boehringer Mannheim, Mannheim, Germany) at 37C for l hour. Samples were then heated to 80C for lO min to degrade the DNase. cDNA was synthesized in a reverse transcriptase (RT) reaction with lO
pmol downstream PCR prlmer (described below), 0.625 mM dNTPs, 5 x reaction buffer (Promega) and 200 units Moloney murine leukemia virus RT (Promega) to a final volume of 20 ~l. The mixture was incubated at 37C for l hour. The cDNA was amplified for 30 cycles by PCR as described in Example 3. The primers used to detect HIV-l regulatory RNA were as follows:
5'-GAAGAAGCGGAGACAGCGACG-3' (SEQ ID NO: 3) 5'-GGCCTGTCGGGTCCCCTCG-3' (SEQ ID NO: 4) Primers specific for the HIV-l major splice donor (MSD) site used to detect HIV-l structural RNA were as follows:
5'-CTCTCGACGCAGGACTCGGC-3' (SEQ ID NO: 5) 5'-CTTTCCCCCTGGCCTTAACCG-3' (SEQ ID NO: 6) 32P-dCTP was incorporated into the amplified fragments and one-tenth of the final reaction mixture was analyzed by electrophoresis on 8~ polyacrylamide gels. In each sample, RNA without reverse transcriptase was also amplified by PCR to demonstrate that the amplified fragments were from HIV cDNA, not from contamination of HIV DNA.
Example 4 Detection of HIV RNA in culture su~ernatants by RT-nested PCR
RNA was extracted from 500 ~l of culture supernatants by the RNAzol method. After DNase I treatment, cDNA was synthesized by a RT reaction with SEQ ID NO:8 as an RT primer.
The synthesized cDNA was amplified by the nested PCR method.
The primers for the first PCR were as follows:
5'-GAAGAAGAGATAGTAATTAGATCT-3' (SEQ ID NO: 7) 5'-GGTGGGTGCTACTCCTAATTGTTCAATTC-3' (SEQ ID NO: 8) The primers used for the second (nested) PCR were SEQ ID
WO95/11701 2 1 6 q 4 5 ~ PCT~S94112152 NO: 7 and SEQ ID NO: 2 . One-tenth of the first PCR product was added to the second PCR reaction. The PCR conditions were as described in Example 3, except that 40 cycles of amplification were performed. One-tenth of the final reaction mixture was analyzed by electrophoresis on 2~ agarose gels and stained with ethidium bromide. RNA samples without RT were also amplified by the nested primers as a test for DNA
contamination. DNA content and RNA expression in HIV-l infected epithelial cells is shown in Table 2.
Approximately l~ of HT29 L20 cells are infected with HIV-l, if HIV-infected cells contain l copy of proviral DNA per cell. As can be seen in Table 2, expression of regulatory RNA
in HIV-l infected HT29 L20 cells is lower than that in HIV-l infected H9 cells and ACH-2 cells. Expression of structural RNA is barely detectable.
Table 2 HIV-l (HTLV-IIIB) RNA expression in colorectal and vaginal epithelial cells cell line HIV-l DNA HIV-1 RNA Virus ln cell medium (copy/cell) expression Regulatory Structural HIV P24 RNA RNA RNAantigen HT-29 0.025 + + - -clone L20 Hs 769.Vg 0.025 + +
Hs 760.T 0.1 + + - -~ ACH-2 1 + + + +
H-9 20 + + + +
Exam~le 6 Neutralization of HTLV-IIIB infectivity in L20 and Hs769 cells by antipe~tide antisera Hyperimmune sera was isolated from monkeys immunized with the five peptides derived from the gpl20 sequence listed below (Table 3).
~ WO95/11701 2 1 6 9 4 5 ~ PCT~S9~112152 Table 3 peptide no. sequence SEQ ID N0.
LTSCNTSVITQACPKVSFEPIPIHYC lO
~- 5 16 PKVSFEPIPIHYCAPAGFAILKCNN
l9 THGIRP WSTQLLLNGSLAEEE 12 Solid phase peptide synthesis was performed using an Applied Biosystems (Foster City, CA) 430A peptide synthesizer.
An amino-terminal cysteine residue was added to each peptide to facilitate coupling to a carrier protein. Peptides were covalently coupled to ovalbumin, grade V (Sigma, St. Louis, MO) at an approximate lO:l (peptide:ovalbumin) molar ratio using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP;
Pharmacia, Uppsala, Sweden). Three to five year old male and female monkeys (Macaca fasciculari6) were immunized by three consecutive intramuscular injections of lO0 ~g ovalbumin-conjugated peptides emulsified in Freund's complete (first injection) or incomplete (second and third injections) given three weeks apart. Blood was collected from the ~emoral vein before immunization and one or two weeks after the final immunization. Pre- and post-immune sera were prepared and stored at -20C.
These peptides have been shown to elicit neutralizing antibodies to HIV in monkeys (Vahlne et al., (l99l) Proc.
Natl . Acad. Sci . U.S.A., 88 : 10744-10748). These antibodies, a guinea pig hyperimmune serum with high neutralizing HTLV-IIIB capacity and a monoclonal antibody against gpl20 (the latter two kindly provided by L. Akerblom, Uppsala, Sweden) were assayed for their ability to neutralize HTLV-IIIB
infectivity by primary inhibition of HIV-l infectivity in HT-29, clone L20, colon cells and in Hs 760.T vaginal cells and subsequently assayed by cocultivation with highly permissive H9 lymphoid cells.
Stock virus was diluted to 104 TCIDso for neutralization in colon cells and used undiluted (106 TCIDso) for WO95/11701 2 1 ~ 9 4 5 3 PCT~S9-l/12152 neutralization in vaginal cells and mixed with serial four fold dilutions of heat-inactivated monkey sera starting at 1:5. The monkey sera were used at a final dilution of 1:10 or 1:20. The guinea pig hyperimmune serum served as a positive control. After incubation for 2 hours at 37C, the serum virus mixture was incubated with the epithelial cells for 2 hours at 37C. The cells were washed twice with medium and supplemented with 1.5 ml of respective maintenance medium/well. Seven days after infection the cells were washed five times and treated with 0.1~ trypsin at 37C for 5 minutes. Hg cells (lo6) were added to each well and cocultures were monitored for 7 days for syncytia formation and presence of p24 antigen. Results for HS 760.T cells, Hs769 cells and HT-29 L20 cells are indicated in Tables 4/5, 6, and 7, respectively, and are expressed as mean neutralization titers, defined as the reciprocal of the serum dilution that reduced the p24 antigen by at least go~. The HIV-1 copy number is also shown for HIV-1 infected HS 760.T
cells (Tables 4 and 5).
Table 4 Neutralization of HIV-1 ~HTLV-IIIB) infectivity in Hs 760.T cells by monkey hyperimmune sera against gpl20 peptides.
HIV-1 DNA neutralization assayed (copy/lO'cells) by cocultivation sera to peptides pre-immune post-immune pre-immune post-immune gpl20-12 125<12.5 ND +
gpl20-15 ND c12.5 ND +
gpl20-16 100 25 ND +
gpl20-19 125<12.5 ND +
gpl20-24 100c12.5 ND +
mixture gpl20- 125~12.5 - +
(12+15+16+19+24) Guinea pig anti-gpl20 125 c12.5 - +
ND, not done ~ WO95/11701 2 1 6 ~ ~ 5 3 PCT~S9~/12152 Table 5 Neutralization of HIV-l ~HTLV-IIIB) infectivity in Hs 760.T cells by guinea pig anti-gpl20 serum and monkey hyperimmune sera against gpl20 peptides.
HIV-l DNA neutralization assayed (copv/3xlO'cells) bv cocultivation Serumi Pre-immune Post-immune Pre-immune Post-immune Guinea pig anti-gpl20 375<37.5 _ +
gpl20-12 375~37.5 ND +
gpl20-15 ND~37.5 ND +
0 gpl20-16 30075 ND +
gpl20-19 375~37.5 ND +
gpl20-24 300~37.5 ND +
mixture gpl20- 375 ~37 5 _ +
~12+15+16+19+24) ~ Guinea pig anti-gpl20 serum and monkey hyperimmune sera against gpl20 peptides were tested at a final dilution of 1/40 and 1/10, respectively.
Mixture of gpl20 peptides antisera was tested at a final dilution of 1/20.
ND, not done.
Table 6 Neutralization of HIV-l (HTLV-IIIB) infectivity in HS 769.Vg cells by guinea pig and monkey hyperimmune sera against gpl20.
Guinea piq anti qpl20 dilutionHIV-l copy number (copy/lQ~ cells) pre-immune post-immune x40 500 c12.5 x160 250 lO0 x640 250 lO0 Monkey anti-peptide 24 dilution HIV-l copy number (copy/104 cells) pre-immune post-immune xlO 500 12.5 x40 250 25 WO9S/11701 2 1 69~53 PCT~S9~/12152 ~
Table 7 Neutralization of HIV-l (HTLV-IIIB) in~ectivity in HT-29 L20 cells by guinea pig anti-gp 120 serum and monkey hyperimmune sera against -~5 gpl20 peptides.
Neutralization assaYed bY cocultivation by PCR
Serum Pre-immune Post-immune Post-immune Guinea pig anti-gpl20 - + +
gpl20-1 to gpl20-11 - - ND
(aa 1-164) gpl20-12 - + +
(aa 152-176) gpl20-13 to gpl20-14 - - ND
(aa 165-205) gpl20-15 - + +
(aa 193-218) gpl20-16 - + +
(aa 206-230) gpl20-17 to gpl20-18 - - ND
(aa 219-257) gpl20-19 ND ND ND
(aa 248-269) gpl20-20 to gpl20-23 - - ND
(aa 258-320) gpl20-24 - + +
(aa 307-330) gpl20-25 to gpl20-40 - - ND
(aa 321-511) mixture of gpl20- - + ND
(12+15+16+19+24) ND, not done.
WO95/11701 PCT~S9~/12152 ` 21 6q453 The results indicated that the level of proviral DNA was markedly decreased by incubation of HT29 L20 cells with anti-gpl20 guinea pig serum. A decrease in viral load was also detected in cells incubated with the antisera to peptides ' 5 corresponding to SEQ ID NOS: 9, 10, 11 and 13 (Table 3). HIV-l copy number was also markedly decreased in HS769 vaginal epithelial cells by an antiserum to the peptide of SEQ ID NO:
13.
Example 7 Protection from HIV-l mucosal infection i~ vivo with a vaccine aqainst apl20 epitopes DNA corresponding to peptides having the sequence of SEQ
ID NO: 9-13 is linked to DNA encoding the B subunit of cholera toxin by standard methods of molecular biology. The resulting chimeric construct is placed in a commercially available eukaryotic expression vector such as pGEX (Pharmacia, Piscataway, NJ) containing the appropriate translation initiation and termination signals. This construct is then incorporated into a lipid vesicle by methods well known in the art. The lipid vesicle is then formulated into a foam or suppository composition by well known pharmacolological preparation methods and administered vaginally and/or rectally to humans at high risk for HIV infection. The dose range administered i5 in the range of from about lO ~g to lO mg.
The administration is repeated at two week intervals for a total of three administrations. The presence of anti-HIV
antibodies in the vaginal and rectal mucosa is assayed by isolating protein from vaginal secretions and feces (which contains cells shed from the vaginal and rectal epithelium, respectively) and performing a p24 ELISA to determine whether any antibodies are present. These antibodies can then be used in HIV-l virus neutralization assays (Vahlne et al., (l99l) Proc. Natl. Acad. Sci. U.S.A., 88: 10744-10748).
WO 9S/11701 PCT/US9~1/12152 ~1~945~
SEQUENCE LISTING
(1) GENERAL INFORMATION: .
(i) APPLICANT: SYNTELLO, Inc.
(ii) TITLE OF INVENTION: Inhibition of HIV Mucosal Infection (iii) NUMBER OF SEQUENCES: 13 (iv) CORRESPONDENCE ADDRESS:
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(B) REGISTRATION NUMBER: 29.655 (C) REFERENCE/DOCKET NUMBER: METRICS.036QPC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (619) 235-8550 (B) TELEFAX: (619) 235-0176 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
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GAAGAAGCGG AGACAGCGAC G 2l (2) INFORMATION FOR SEQ ID NO:4:
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WO 95/11701 PCT/US9~/12152 2t~q;453 (iv) ANTI-SENSE: NO -' (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:4:
GGC~l~lCGG GTCCCCTCG 19 (2) INFORMATION FOR SEQ ID NO:5:
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G~l~G~lGCT ACTCCTAATT GTTCAATTC 29 (2) INFORMATION FOR SEQ ID NO:9:
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(A) LENGTH: 25 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
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(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Gly Glu Ile Lys Asn Cys Ser Phe Asn Ile Ser Thr Ser Ile Arg Gly l 5 l0 15 PCT/IJS9~/12152 WO 95/11701 ~ t ~ ~ 4 ~
Lys Val Gln Lys Glu Tyr Ala Phe Phe (2) INFORMATION FOR SEQ ID NO:10: ,, (i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
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Leu Thr Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Val 5 lo 15 Ser Phe Glu Pro Ile Pro Ile His Tyr Cys ( 2 ) INFORMATION FOR SEQ ID NO:11:
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Pro Lys Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala lo 15 Gly Phe Ala Ile Leu Lys Cys Asn Asn WO 95/11701 PCT~US9~/12152 ~- (2) INFORMATION FOR SEQ ID NO:12:
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Thr His Gly Ile Arg Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly l 5 l0 15 Ser Leu Ala Glu Glu Glu (2) INFORMATION FOR SEQ ID NO:13:
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(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Ile Arg Ile Gln Arg Gly Arg Gly Arg Ala Phe Val Thr Ile Gly Lys l 5 l0 15 Ile Gly Asn Met Arg Gln Ala His
approach to reducing HIV-l infection.
SUMMARY OF THE INVENTION
'- One embodiment of the present lnvention is a method for inhibiting the infection of mucosal cells by HIV-l by .- 5 administering a vaccine to the mucosa, thereby delivering to the mucosa a peptide of HIV-l gpl20 having from about l0 to about 50 amino acids, whereby antibodies against the peptide are generated in the mucosa, the peptide being selected such that the antibodies inhibit infection of HIV-l in mucosal epithelial cells.
In another aspect of this preferred embodiment, the peptide includes an epitope effective to generate mucosal production of antibodies that inhibit infection of mucosal cells by HIV-l, the peptide consisting essentially of SEQ ID
NOS: 9, l0, ll, 12, or 13. Advantageously, the vaccine further includes an agent for enhancing delivery of the peptide to the mucosa. Preferably, the agent is a mucosal binding protein; most preferably, it is either the binding subunit of cholera toxin or that of E. coli heat labile enterotoxin. The invention also provides that the peptide and the mucosal binding protein are bound together to form a chimeric protein which may advantageously be the expression product of recombinant DNA. In another embodiment of the invention, the agent is a lipid. Preferably, the lipid is in the form of a lipid vesicle. Another aspect of this preferred embodiment provides that the administering step comprises administering to the mucosa a polynucleotide operably encoding the peptide, whereby the peptide is produced by cells of the mucosa.
A further embodiment of the invention provides a vaccine for inhibiting the infection of mucosal cells by HIV-l, comprising a l0 to 50 amino acid peptide of HIV-l gpl20 having an epitope selected such that antibodies against this epitope inhibit the infection of mucosal epithelial cells by HIV-l, and a compound or structure associated with the peptide for facilitating delivery of the peptide to the mucosa.
Preferably, this peptide consists essentially of SEQ ID NO 9, WO95/11701 ~1 6 9 4 5 ~ PCT~S9~/121~2 10, 11, 12 or 13 and the compound or structure is a lipid vesicle. Most preferably, the compound or structure is a mucosal binding protein. In a particularly preferred embodiment, the binding protein is a cholera toxin protein which may advantageously be the binding subunit. In another aspect of this preferred embodiment, the binding protein is the binding subunit of E. coli heat labile enterotoxin.
DETAILED DESCRIPTION QF THE INVENTION
The present invention discloses the identiication of synthetic peptides derived from the sequence of the envelope glycoprotein gpl20 of HIV-l. These peptides were used to generate neutralizing antibodies which inhibited infection of transformed human vaginal and colorectal cell lines in vi tro.
These peptides will induce the production of a localized mucosal immune response, generating antibodies able to neutralize infection of human colorectal and vaginal epithelial cells by HIV-1. The peptides are set forth herein as SEQ ID NOS: 9-13. In one aspect of the invention, one or more of the peptides of SEQ ID NO:9, 10, 11, 12, and 13 is used to generate antibodies. These antibodies can be generated in any conventional manner, including by intramuscular, intraperitoneal, subcutaneous, or mucosal administration to an animal. Generation of both monoclonal and polyclonal antibodies are contemplated. These antibodies are then used to prevent infection of cells of the mucosal epithelium by providing the antibodies in association with the mucosal cells and then challenging the cells with HIV-1. The antibodies inhibit or prevent binding of the virus to the cells, and thereby inhibit or prevent infection of the cells by the virus.
The antibodies can be exogenous or endogenous antibodies, and the cells can be in vi tro or in vivo . When the cells are in vitro, the antibodies are typically generated in laboratory or domestic animals or are monoclonal antibodies. More importantly, it provides a valuable tool for analyzing the mechanism and structure involved in that binding.
When the cells are in vivo, the antibodies are preferably PCT~S9~/12152 2 ~ ~?453 endogenous mucosal antibodies that have been generated by administering one or more of the peptides of SEQ ID NOs 9-13 ^ to the anlmal in which the cells are located. Mucosal vaccination, as described below, is particularly preferred.
.- 5 However, exogenous antibodies may also be administered to the animal to inhibit HIV-1 infection of mucosal cells. In all of the treatments described herein, the mucosal cells are preferably of human origin.
The peptides of the present invention can be utilized alone or in combination and can also be uncoupled or coupled to other epithelial cell binding proteins including CT, CTB
and the binding subunit of E. coli heat labile enterotoxin.
The peptides may be coupled by either chemical or recombinant means. DNA encoding the peptldes can be joined to DNA
encoding cholera toxin, or its B subunit, by well known methods, inserted into a eukaryotic expression vector and delivered to epithelial cells using lipid vesicles or lamellar structures. The production of these peptide-CT, CTB or enterotoxin conjugates in vivo will then elicit a localized mucosal immune response and will protect against subsequent infection by HIV. The inclusion of muco~al eplthelial cell binding proteins, such as cholera toxin, will advantageously increase the efficiency of entry of peptides into these cells.
Since the B subunit of the cholera toxin A-B dimer is responsible for binding to cell surface receptors, a peptide-CTB conjugate will also bind efficiently to epithelial cells.
The literature also reports methods for forming compositions of immunogenic peptides and other gut binding proteins (Wenneras et al., (1990) FEMS Microbiol. Lett., 66: 107-112).
Techniques for forming peptide-CTB conjugates are well known (Liang et al., (1988) J. Immunol., 141: 3781-3787; Sanchez et al., (1990) Res. Microbiol., 141: 971-979). Liposome formation and delivery of peptides encapsulated in liposomes is also well known as described by Lowell rNew Generation Vaccines, Woodrow, G.C. and Levine, M.M., eds., Marcel Dekker, Inc., New York, pp. 141-160). These peptides are also useful in the production of monoclonal and polyclonal antibodies.
WO95111701 2 1 6 9 4 5 3 PCT~S9~/12152 These antibodies have a distinct neutralizing effect on HIV-l.
These peptides, either alone or after coupling to CT or other molecules, may be administered orally, rectally, vaginally, or in a combination of these routes in an amount sufficient to generate a mucosal antibody response sufficient to inhibit HIV-l entry into the mucosal epithelial cells. The amounts of peptides used will depend on their pharmaceutical formulation and the site and route of delivery; however, for an adult human, a suitable immunogenic amount of peptide is generally between about 50 ~g and about l mg, administered one to four times over a period of two weeks to one year or longer.
The peptides, peptide-binding protein conjugates, and other compositions of the present invention can be administered orally to generate a localized gastrointestinal mucosal immune response or intravaginally or intrarectally to produce a localized mucosal immune response in these areas prone to viral entry by sexual contact. These peptides can be administered in unit dosage in an amount necessary to produce localized mucosal immunity against HIV-l infection.
Pharmaceutical compositions envisioned for oral administration include tablets, capsules, liquids, and the like and those contemplated for intravaginal or intrarectal administration include injectable carriers, suppositories, ointments, gels, creams, foams, sprays, dispersions, suspensions, pastes and the like in an amount from about lO ~g to about lO mg or more.
These preparations can be in any suitable form, and generally comprise the active ingredient in combination with any of the well known pharmaceutically acceptable carriers. The preparations may further advantageously include preservatives, antibacterials, antifungals, antioxidants, osmotic agents, and similar materials in composition and quantity as is conventional. For assistance in formulating the compositions of the present invention, one may refer to Remington's Pharmaceutical Sciences, 15th Ed., Mack Publishing Co., Easton, PA (1975).
The ~ontemplated modes of administration assume that the peptides or conjugates are able to be directly taken up by the WO95/11701 PCT~S9~tl2152 ~ 3 epithelial cells lining these areas. These peptides and conjugates may advantageously be enclosed in liposomes to ~~ facilitate delivery of these agents to cells. Direct injection of the peptides or peptide-binding protein - 5 conjugates, either alone or in combination with lipid vesicles or other lamellar structures, into the mucosal endothelium in a similar dose range is also envisioned as a method of eliciting an anti-HIV response in these tissues.
Example 1 Susceptibility of colorectal and vaqinal epithelial cells to infection bY HIV-1 HIV-1 infectious virus stocks of HTLV-IIIB-infected H9 T
cell lymphoma cells (ATCC HTB-176) (Popovic et al., (1984) Science, 224: 497-500) were used in the following experiments.
The cells were maintained in RPMI-1640 medium containing 20 fetal calf serum (FCS), 100 units/ml penicillin and 100 ~g/ml streptomycin. Virus stocks were prepared using well known procedures and frozen at -90C. One stock of HTLV-IIIB with endpoint titer of 1 x 104 tissue culture infectious doses (TCID50) was used for all experiments.
Endpoint titration of the HTLV-IIIB isolate of HIV-1 in two clones of transformed vaginal epithelial cells (Hs 760 T
and Hs 769.Vg cells; ATCC CRL-7491 and 7499, respectively) and 12 subclones of colon adenocarcinoma HT-29 cells (ATCC HTB-38) were performed by inoculation of respective cell lines with serial 10-fold dilutions of virus with 100 ~l/well (ranging from 1 TCID50/cell to 0.00001 TCID50/cell) in 24-well plates (Costar) for 2 hours at 37C. After adsorption, cells were washed five times with Modified Eagle's Medium (MEM) and supplemented with 1.5 ml growth medium (DMEM for vaginal cells and MEM for colon cells, both containing 10~ fetal calf serum (FCS), 1~ L-glutamine, and antibiotics). Seven days after infection, epithelial cells were washed five times with MEM
and treated with 0.1~ trypsin in phosphate buffered saline (PBS) for 5 min at 37C. HTLV-IIIB-infected H9 cells (106 cells) in H9 maintenance medium were added to each well and cocultured with epithelial cells for 24 hours. The H9 W095/11701 2 1.6 9 4 5 3 PCT~S9~/12152 cultures were microscopically followed for 7 days for the presence of HIV-induced syncytium formation and p24 antigen production using an ELISA able to detect as little as lOO pg p24/ml). The vaginal cell lines Hs 760.T and Hs 769.Vg were permissive. Viral infection of Hs 760.T was detected by coculture at a high multiplicity (l TCIDsO/cell) and 6 of the HT-29 colon cell clones were permissive at multiplicities ranging from O.l-O.Ol TCID50/cell. Of these clones, cloOne L20 was chosen for further study.
WO95/11701 PCT~S9~/12152 2-1 6q453 Table 1 Susceptibility of colorectal and vaginal epithelial cells to infection by HIV-l (HTLV-IIIB).
. 5 multi~licity of infection (TCID50/cell) cell Line subclone method~ 1 lO-I 10~ X103 HT-29 EOcoculture E5coculture 0 " E8coculture - - - -L2coculture " L4coculture Ll6coculture " Ll8Bcoculture +
~ Ll8Acoculture + +
" Ll2coculture + +
" LlOcoculture + + +
" Ll4coculture + + +
~ L20coculture + + +
" L20 PCR 2.5-- 2 0.5 0.125 HS 769 Vg coculture " PCR 2.5 0.6 0.5 0.125 HS 760 T coculture +
~ PCR 10 * coculture with H9 cells and subsequent p24 antigen detection or detection of proviral DNA by PCR.
** copy number, x10-2 per cell WO95/11701 ~ t 6 q 4 5 ~ PCT~S9~/12152 ~
HIV-1 RNA and DNA was detected both in epithelial cells and in the culture supernatants as described in the following examples. .
Example 2 Detection of ~roviral DNA bY PCR -~
Epithelial cells were harvested seven days post-infection and DNA was extracted (Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory Press, 2: 9.16-9.19). Primers specific for the HIV-1 env gene (5'-GTAACGCACA~'l"l"l"l'AATTGTGGAGGGGAA-3'; SEQ ID NO: 1) and (5'-CCTCATATTTCCTCCTCCAGGTCT-3'; SEQ ID NO: 2) were used for detection of proviral DNA. DNA (20Q ng) was amplified on a DNA thermal cycler (Perkin-Elmer, Norwalk, CT) using ~-32P-dCTP
to label the fragments. The reaction mixture consisted of 10 ~l of 10x PCR buffer (Promega, Madison, WI), 1.5 mM MgCl2, 20 pmol primers, 0.125 mM dNTPs, 5 ~Ci ~-32P-dCTP and 0.5 units Taq DNA polymerase (Promega). The amplification was for 35 cycles and included denaturation at 94C for 1 min, annealing at 55C for 1 min and extension at 72C for 1 min. One-tenth of the final reaction mixture was analyzed by electrophoresis on 5~ polyacrylamide gels. The gels were dried and exposed to X-ray film (X-OMAT; Eastman Kodak, Rochester, NY) for 13-16 hours using an intensifying screen.
To measure HIV copy number (the number of HIV genomes), two-fold serial dilutions of DNA isolated from ACH-2 cells, which contain one proviral copy per cell (Clouse et al., (1989) ~. Immunol., 142: 431-438; Seshamma et al., (1992) ~.
Virol. Methods, 40: 331-346; Graziosi et al., (1993) Proc.
Natl . Acad. Sci . U. S.A., 90: 6405-6409). The total amount of DNA in each dilution was normalized to 200 ng using DNA
extracted from H9 cells and PCR was performed as above. HIV
copy number was estimated by comparison of the intensities of the amplified bands. PCR analysis using a pair of human ~-actin primers was performed in parallel as an internal standard.
WO95/11701 PCT~S9~/12152 Example 3 Detection of HIV RNA exPression by RT-PCR
Epithelial cells were harvested 7 days post-infection and total RNA was extracted by the RNAzol method (Biotex ,~ 5 Laboratories, Houston, TX). For each sample, 500 ng of total RNA was incubated with lO units RNase-free DNase I (Boehringer Mannheim, Mannheim, Germany) at 37C for l hour. Samples were then heated to 80C for lO min to degrade the DNase. cDNA was synthesized in a reverse transcriptase (RT) reaction with lO
pmol downstream PCR prlmer (described below), 0.625 mM dNTPs, 5 x reaction buffer (Promega) and 200 units Moloney murine leukemia virus RT (Promega) to a final volume of 20 ~l. The mixture was incubated at 37C for l hour. The cDNA was amplified for 30 cycles by PCR as described in Example 3. The primers used to detect HIV-l regulatory RNA were as follows:
5'-GAAGAAGCGGAGACAGCGACG-3' (SEQ ID NO: 3) 5'-GGCCTGTCGGGTCCCCTCG-3' (SEQ ID NO: 4) Primers specific for the HIV-l major splice donor (MSD) site used to detect HIV-l structural RNA were as follows:
5'-CTCTCGACGCAGGACTCGGC-3' (SEQ ID NO: 5) 5'-CTTTCCCCCTGGCCTTAACCG-3' (SEQ ID NO: 6) 32P-dCTP was incorporated into the amplified fragments and one-tenth of the final reaction mixture was analyzed by electrophoresis on 8~ polyacrylamide gels. In each sample, RNA without reverse transcriptase was also amplified by PCR to demonstrate that the amplified fragments were from HIV cDNA, not from contamination of HIV DNA.
Example 4 Detection of HIV RNA in culture su~ernatants by RT-nested PCR
RNA was extracted from 500 ~l of culture supernatants by the RNAzol method. After DNase I treatment, cDNA was synthesized by a RT reaction with SEQ ID NO:8 as an RT primer.
The synthesized cDNA was amplified by the nested PCR method.
The primers for the first PCR were as follows:
5'-GAAGAAGAGATAGTAATTAGATCT-3' (SEQ ID NO: 7) 5'-GGTGGGTGCTACTCCTAATTGTTCAATTC-3' (SEQ ID NO: 8) The primers used for the second (nested) PCR were SEQ ID
WO95/11701 2 1 6 q 4 5 ~ PCT~S94112152 NO: 7 and SEQ ID NO: 2 . One-tenth of the first PCR product was added to the second PCR reaction. The PCR conditions were as described in Example 3, except that 40 cycles of amplification were performed. One-tenth of the final reaction mixture was analyzed by electrophoresis on 2~ agarose gels and stained with ethidium bromide. RNA samples without RT were also amplified by the nested primers as a test for DNA
contamination. DNA content and RNA expression in HIV-l infected epithelial cells is shown in Table 2.
Approximately l~ of HT29 L20 cells are infected with HIV-l, if HIV-infected cells contain l copy of proviral DNA per cell. As can be seen in Table 2, expression of regulatory RNA
in HIV-l infected HT29 L20 cells is lower than that in HIV-l infected H9 cells and ACH-2 cells. Expression of structural RNA is barely detectable.
Table 2 HIV-l (HTLV-IIIB) RNA expression in colorectal and vaginal epithelial cells cell line HIV-l DNA HIV-1 RNA Virus ln cell medium (copy/cell) expression Regulatory Structural HIV P24 RNA RNA RNAantigen HT-29 0.025 + + - -clone L20 Hs 769.Vg 0.025 + +
Hs 760.T 0.1 + + - -~ ACH-2 1 + + + +
H-9 20 + + + +
Exam~le 6 Neutralization of HTLV-IIIB infectivity in L20 and Hs769 cells by antipe~tide antisera Hyperimmune sera was isolated from monkeys immunized with the five peptides derived from the gpl20 sequence listed below (Table 3).
~ WO95/11701 2 1 6 9 4 5 ~ PCT~S9~112152 Table 3 peptide no. sequence SEQ ID N0.
LTSCNTSVITQACPKVSFEPIPIHYC lO
~- 5 16 PKVSFEPIPIHYCAPAGFAILKCNN
l9 THGIRP WSTQLLLNGSLAEEE 12 Solid phase peptide synthesis was performed using an Applied Biosystems (Foster City, CA) 430A peptide synthesizer.
An amino-terminal cysteine residue was added to each peptide to facilitate coupling to a carrier protein. Peptides were covalently coupled to ovalbumin, grade V (Sigma, St. Louis, MO) at an approximate lO:l (peptide:ovalbumin) molar ratio using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP;
Pharmacia, Uppsala, Sweden). Three to five year old male and female monkeys (Macaca fasciculari6) were immunized by three consecutive intramuscular injections of lO0 ~g ovalbumin-conjugated peptides emulsified in Freund's complete (first injection) or incomplete (second and third injections) given three weeks apart. Blood was collected from the ~emoral vein before immunization and one or two weeks after the final immunization. Pre- and post-immune sera were prepared and stored at -20C.
These peptides have been shown to elicit neutralizing antibodies to HIV in monkeys (Vahlne et al., (l99l) Proc.
Natl . Acad. Sci . U.S.A., 88 : 10744-10748). These antibodies, a guinea pig hyperimmune serum with high neutralizing HTLV-IIIB capacity and a monoclonal antibody against gpl20 (the latter two kindly provided by L. Akerblom, Uppsala, Sweden) were assayed for their ability to neutralize HTLV-IIIB
infectivity by primary inhibition of HIV-l infectivity in HT-29, clone L20, colon cells and in Hs 760.T vaginal cells and subsequently assayed by cocultivation with highly permissive H9 lymphoid cells.
Stock virus was diluted to 104 TCIDso for neutralization in colon cells and used undiluted (106 TCIDso) for WO95/11701 2 1 ~ 9 4 5 3 PCT~S9-l/12152 neutralization in vaginal cells and mixed with serial four fold dilutions of heat-inactivated monkey sera starting at 1:5. The monkey sera were used at a final dilution of 1:10 or 1:20. The guinea pig hyperimmune serum served as a positive control. After incubation for 2 hours at 37C, the serum virus mixture was incubated with the epithelial cells for 2 hours at 37C. The cells were washed twice with medium and supplemented with 1.5 ml of respective maintenance medium/well. Seven days after infection the cells were washed five times and treated with 0.1~ trypsin at 37C for 5 minutes. Hg cells (lo6) were added to each well and cocultures were monitored for 7 days for syncytia formation and presence of p24 antigen. Results for HS 760.T cells, Hs769 cells and HT-29 L20 cells are indicated in Tables 4/5, 6, and 7, respectively, and are expressed as mean neutralization titers, defined as the reciprocal of the serum dilution that reduced the p24 antigen by at least go~. The HIV-1 copy number is also shown for HIV-1 infected HS 760.T
cells (Tables 4 and 5).
Table 4 Neutralization of HIV-1 ~HTLV-IIIB) infectivity in Hs 760.T cells by monkey hyperimmune sera against gpl20 peptides.
HIV-1 DNA neutralization assayed (copy/lO'cells) by cocultivation sera to peptides pre-immune post-immune pre-immune post-immune gpl20-12 125<12.5 ND +
gpl20-15 ND c12.5 ND +
gpl20-16 100 25 ND +
gpl20-19 125<12.5 ND +
gpl20-24 100c12.5 ND +
mixture gpl20- 125~12.5 - +
(12+15+16+19+24) Guinea pig anti-gpl20 125 c12.5 - +
ND, not done ~ WO95/11701 2 1 6 ~ ~ 5 3 PCT~S9~/12152 Table 5 Neutralization of HIV-l ~HTLV-IIIB) infectivity in Hs 760.T cells by guinea pig anti-gpl20 serum and monkey hyperimmune sera against gpl20 peptides.
HIV-l DNA neutralization assayed (copv/3xlO'cells) bv cocultivation Serumi Pre-immune Post-immune Pre-immune Post-immune Guinea pig anti-gpl20 375<37.5 _ +
gpl20-12 375~37.5 ND +
gpl20-15 ND~37.5 ND +
0 gpl20-16 30075 ND +
gpl20-19 375~37.5 ND +
gpl20-24 300~37.5 ND +
mixture gpl20- 375 ~37 5 _ +
~12+15+16+19+24) ~ Guinea pig anti-gpl20 serum and monkey hyperimmune sera against gpl20 peptides were tested at a final dilution of 1/40 and 1/10, respectively.
Mixture of gpl20 peptides antisera was tested at a final dilution of 1/20.
ND, not done.
Table 6 Neutralization of HIV-l (HTLV-IIIB) infectivity in HS 769.Vg cells by guinea pig and monkey hyperimmune sera against gpl20.
Guinea piq anti qpl20 dilutionHIV-l copy number (copy/lQ~ cells) pre-immune post-immune x40 500 c12.5 x160 250 lO0 x640 250 lO0 Monkey anti-peptide 24 dilution HIV-l copy number (copy/104 cells) pre-immune post-immune xlO 500 12.5 x40 250 25 WO9S/11701 2 1 69~53 PCT~S9~/12152 ~
Table 7 Neutralization of HIV-l (HTLV-IIIB) in~ectivity in HT-29 L20 cells by guinea pig anti-gp 120 serum and monkey hyperimmune sera against -~5 gpl20 peptides.
Neutralization assaYed bY cocultivation by PCR
Serum Pre-immune Post-immune Post-immune Guinea pig anti-gpl20 - + +
gpl20-1 to gpl20-11 - - ND
(aa 1-164) gpl20-12 - + +
(aa 152-176) gpl20-13 to gpl20-14 - - ND
(aa 165-205) gpl20-15 - + +
(aa 193-218) gpl20-16 - + +
(aa 206-230) gpl20-17 to gpl20-18 - - ND
(aa 219-257) gpl20-19 ND ND ND
(aa 248-269) gpl20-20 to gpl20-23 - - ND
(aa 258-320) gpl20-24 - + +
(aa 307-330) gpl20-25 to gpl20-40 - - ND
(aa 321-511) mixture of gpl20- - + ND
(12+15+16+19+24) ND, not done.
WO95/11701 PCT~S9~/12152 ` 21 6q453 The results indicated that the level of proviral DNA was markedly decreased by incubation of HT29 L20 cells with anti-gpl20 guinea pig serum. A decrease in viral load was also detected in cells incubated with the antisera to peptides ' 5 corresponding to SEQ ID NOS: 9, 10, 11 and 13 (Table 3). HIV-l copy number was also markedly decreased in HS769 vaginal epithelial cells by an antiserum to the peptide of SEQ ID NO:
13.
Example 7 Protection from HIV-l mucosal infection i~ vivo with a vaccine aqainst apl20 epitopes DNA corresponding to peptides having the sequence of SEQ
ID NO: 9-13 is linked to DNA encoding the B subunit of cholera toxin by standard methods of molecular biology. The resulting chimeric construct is placed in a commercially available eukaryotic expression vector such as pGEX (Pharmacia, Piscataway, NJ) containing the appropriate translation initiation and termination signals. This construct is then incorporated into a lipid vesicle by methods well known in the art. The lipid vesicle is then formulated into a foam or suppository composition by well known pharmacolological preparation methods and administered vaginally and/or rectally to humans at high risk for HIV infection. The dose range administered i5 in the range of from about lO ~g to lO mg.
The administration is repeated at two week intervals for a total of three administrations. The presence of anti-HIV
antibodies in the vaginal and rectal mucosa is assayed by isolating protein from vaginal secretions and feces (which contains cells shed from the vaginal and rectal epithelium, respectively) and performing a p24 ELISA to determine whether any antibodies are present. These antibodies can then be used in HIV-l virus neutralization assays (Vahlne et al., (l99l) Proc. Natl. Acad. Sci. U.S.A., 88: 10744-10748).
WO 9S/11701 PCT/US9~1/12152 ~1~945~
SEQUENCE LISTING
(1) GENERAL INFORMATION: .
(i) APPLICANT: SYNTELLO, Inc.
(ii) TITLE OF INVENTION: Inhibition of HIV Mucosal Infection (iii) NUMBER OF SEQUENCES: 13 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Knobbe, Martens, Olson & Bear (B) STREET: 620 Newport Center Drive, Sixteenth Floor (C) CITY: Newport Beach (D) STATE: CA
(E) ~UN'l'~Y: U.S.A.
(F) ZIP: 92660 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC co~patible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version ~1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Israelsen, Ned A.
(B) REGISTRATION NUMBER: 29.655 (C) REFERENCE/DOCKET NUMBER: METRICS.036QPC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (619) 235-8550 (B) TELEFAX: (619) 235-0176 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
W 0 95/11701 PCTrUS9~/12152 21 6~453 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GAAGAAGCGG AGACAGCGAC G 2l (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: l9 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
WO 95/11701 PCT/US9~/12152 2t~q;453 (iv) ANTI-SENSE: NO -' (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:4:
GGC~l~lCGG GTCCCCTCG 19 (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: cDNA
(iii~ HYPOTHETICAL: NO
(iv~ ANTI-SENSE: NO
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:5:
(2~ INFORMATION FOR SEQ ID NO:6:
(i~ SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 21 base pairs (B) TYPE: nuclPir acid (C~ STRANDEDNESS: single (D~ TOPOLOGY: linear (ii~ MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear W O 95/11701 PCT~US9l/12152 2 1 694~:3 (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
G~l~G~lGCT ACTCCTAATT GTTCAATTC 29 (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Gly Glu Ile Lys Asn Cys Ser Phe Asn Ile Ser Thr Ser Ile Arg Gly l 5 l0 15 PCT/IJS9~/12152 WO 95/11701 ~ t ~ ~ 4 ~
Lys Val Gln Lys Glu Tyr Ala Phe Phe (2) INFORMATION FOR SEQ ID NO:10: ,, (i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Leu Thr Ser Cys Asn Thr Ser Val Ile Thr Gln Ala Cys Pro Lys Val 5 lo 15 Ser Phe Glu Pro Ile Pro Ile His Tyr Cys ( 2 ) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Pro Lys Val Ser Phe Glu Pro Ile Pro Ile His Tyr Cys Ala Pro Ala lo 15 Gly Phe Ala Ile Leu Lys Cys Asn Asn WO 95/11701 PCT~US9~/12152 ~- (2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Thr His Gly Ile Arg Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly l 5 l0 15 Ser Leu Ala Glu Glu Glu (2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Ile Arg Ile Gln Arg Gly Arg Gly Arg Ala Phe Val Thr Ile Gly Lys l 5 l0 15 Ile Gly Asn Met Arg Gln Ala His
Claims (33)
1. A method for inhibiting infection of mucosal epithelium cells by HIV-1, comprising the step of administering a vaccine to the epithelium, said vaccine comprising a peptide of HIV-1 gp120 having from about 10 to about 50 amino acids, whereby antibodies against said peptide are generated in said mucosa, said peptide being selected such that said antibodies inhibit infection of said cells by HIV-1.
2. The method of Claim 1, wherein said peptide includes an epitope effective to generate mucosal production of antibodies that inhibit binding between said mucosal cells and HIV-1, said epitope being found in SEQ ID NO: 9, 10, 11, 12 or 13.
3. The method of Claim 2, wherein said vaccine further comprises an agent for enhancing delivery of said peptide to the mucosa.
4. The method of Claim 3, wherein said agent comprises a mucosal binding protein.
5. The method of Claim 4, wherein said mucosal binding protein is the binding subunit of cholera toxin.
6. The method of Claim 4, wherein said mucosal binding protein is the binding subunit of E. coli heat labile enterotoxin.
7. The method of Claim 4, wherein said peptide and said mucosal binding protein are bound together to form a chimeric protein.
8. The method of Claim 7, wherein said chimeric protein is the expression product of recombinant DNA.
9. The method of Claim 3, wherein said agent comprises a lipid.
10. The method of Claim 9, wherein said lipid is in the form of a lipid vesicle.
11. The method of Claim 1, wherein said administering step comprises administering to the mucosa a polynucleotide operably encoding said peptide, whereby said peptide is produced by cells of the mucosa.
12. A composition for inhibiting the infection Or mucosal cells by HIV-1, comprising:
a lo to 50 amino acid peptide of HIV-1 gp120 having an epitope selected such that antibodies against such epitope inhibit the binding of HIV-1 to mucosal epithelial cells; and a compound or structure associated with said peptide for facilitating delivery of said peptide to the mucosa.
a lo to 50 amino acid peptide of HIV-1 gp120 having an epitope selected such that antibodies against such epitope inhibit the binding of HIV-1 to mucosal epithelial cells; and a compound or structure associated with said peptide for facilitating delivery of said peptide to the mucosa.
13. The composition of Claim 12, wherein said epitope is found in SEQ ID NO: 9, 10, 11, 12 or 13.
14. The composition of Claim 12, wherein said compound or structure is a lipid vesicle.
15. The composition of Claim 12, wherein said compound or structure is a mucosal binding protein.
16. The composition of Claim 15, wherein said binding protein is a cholera toxin protein.
17. The composition of Claim 15, wherein said binding protein is the binding subunit of cholera toxin.
18. The composition of Claim 15, wherein said binding protein is the binding subunit of E. coli heat labile enterotoxin.
9. A peptide of HIV-1 gp120 having from about 10 to about 50 amino acids for use in inhibiting the injection of mucosal epithelial cells by HIV-1, wherein said peptide is administered to the mucosa, said peptide being selected such that antibodies against said peptide are generated in said mucosa, said antibodies inhibiting infection of said mucosal epithelial cells by said HIV-1.
20. The peptide of Claim 19, wherein said peptide includes an epitope effective to generate mucosal production of antibodies that inhibit infection of said mucosal cells by HIV-1, said epitope being found in SEQ ID NO: 9, 10, 11, 12 or 13.
21. The peptide of Claim 20, wherein said peptide further comprises an agent for enhancing delivery of said peptide to the mucosa.
22. The peptide of Claim 21, wherein said agent comprises a mucosal binding protein.
23. The peptide of Claim 22, wherein said mucosal binding protein is the binding subunit of cholera toxin.
24. The peptide of Claim 22, wherein said mucosal binding protein is the binding subunit of E. coli heat labile enterotoxin.
25. The peptide of Claim 22, wherein said peptide and said mucosal binding protein are bound together to form a chimeric protein.
26. The peptide of Claim 25, wherein said chimeric protein is the expression product of recombinant DNA.
27. The peptide of Claim 21, wherein said agent comprises a lipid.
28. The peptide of Claim 27, wherein said lipid is in the form of a lipid vesicle.
29. The peptide of Claim 19, wherein said administering step comprises administering to the mucosa a polynucleotide operably encoding said peptide, whereby said peptide is produced by cells of the mucosa.
30. A method for inhibiting infection of mucosal cells by HIV-1, comprising the steps of:
generating mucosal antibodies against one or more of the peptides of SEQ ID NO: 9, 10, 11, 12 or 13;
providing said antibodies in contact with mucosal epithelial cells; and contacting said cells with HIV-1, whereby said antibodies inhibit the infection of said cells by HIV-1.
generating mucosal antibodies against one or more of the peptides of SEQ ID NO: 9, 10, 11, 12 or 13;
providing said antibodies in contact with mucosal epithelial cells; and contacting said cells with HIV-1, whereby said antibodies inhibit the infection of said cells by HIV-1.
31. The method of Claim 30, wherein said antibodies are generated by administering an immunogen to mucosal tissue in vivo.
32. The method of Claim 31, wherein said immunogen is coupled to a mucosal binding protein.
33. The method of Claim 32, wherein said mucosal binding protein is the B subunit of cholera toxin.
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US14357793A | 1993-10-26 | 1993-10-26 | |
US08/143,577 | 1993-10-26 |
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JP (1) | JPH09504296A (en) |
AU (1) | AU8087994A (en) |
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WO (1) | WO1995011701A1 (en) |
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US6348449B1 (en) | 1993-09-21 | 2002-02-19 | The Trustees Of The University Of Pennsylvania | Methods of inducing mucosal immunity |
US7135191B2 (en) * | 1997-09-04 | 2006-11-14 | Zsolt Istvan Hertelendy | Urogenital or anorectal transmucosal vaccine delivery system |
CA2358385C (en) | 1998-12-31 | 2013-08-06 | Chiron Corporation | Polynucleotides encoding antigenic hiv type c polypeptides, polypeptides and uses thereof |
JP4701532B2 (en) | 2001-04-26 | 2011-06-15 | 東ソー株式会社 | Amplification and detection of HIV-1 RNA |
JP5033303B2 (en) | 2001-07-05 | 2012-09-26 | ノバルティス バクシンズ アンド ダイアグノスティックス,インコーポレーテッド | Polynucleotides encoding polypeptides with antigenic type C HIV, polypeptides and uses thereof |
ITMI20021781A1 (en) * | 2002-08-06 | 2004-02-07 | San Raffaele Centro Fond | GP41 EPITOPE AND ITS USES IN THE TREATMENT OF HIV INFECTIONS. |
KR101012507B1 (en) * | 2008-01-08 | 2011-02-08 | (주)디딤바이오텍 | A composition for sanitary supplies comprising mucosal immunoadjuvant and a preparation method thereof |
AU2012308212A1 (en) * | 2011-09-17 | 2014-03-27 | Y-Clone Medical Sciences Co., Ltd | Antibody recognizing arbitrarily designed epitope of three or more amino acid residues in a peptide and method of generating thereof |
US10660951B2 (en) | 2012-09-17 | 2020-05-26 | Zhiwei Allen Wu | Antibody recognizing arbitrarily designed epitope of three or more amino acid residues in a peptide and method of generating thereof |
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US4808700A (en) * | 1984-07-09 | 1989-02-28 | Praxis Biologics, Inc. | Immunogenic conjugates of non-toxic E. coli LT-B enterotoxin subunit and capsular polymers |
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- 1994-10-25 JP JP7512750A patent/JPH09504296A/en active Pending
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- 1994-10-25 CA CA002169453A patent/CA2169453A1/en not_active Abandoned
- 1994-10-25 AU AU80879/94A patent/AU8087994A/en not_active Abandoned
- 1994-10-25 WO PCT/US1994/012152 patent/WO1995011701A1/en not_active Application Discontinuation
Also Published As
Publication number | Publication date |
---|---|
JPH09504296A (en) | 1997-04-28 |
EP0726776A1 (en) | 1996-08-21 |
WO1995011701A1 (en) | 1995-05-04 |
AU8087994A (en) | 1995-05-22 |
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