Note: Descriptions are shown in the official language in which they were submitted.
~ 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.
- 5BACKGROUND OF THE INVENTION
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
WO95111701
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 ~
--10--
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
2169453
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
-12-
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.
- 12 GEIKNCSFNISTSIRGKVQKEYAFF 9
LTSCNTSVITQACPKVSFEPIPIHYC lO
~- 5 16 PKVSFEPIPIHYCAPAGFAILKCNN
l9 THGIRP WSTQLLLNGSLAEEE 12
24 IRIQRGPGRAFVTIGKIGNMRQAH 13
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
-14-
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
-15-
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 ~
-16-
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
-17-
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~
-18-
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:
GTAACGCACA GTTTTAATTG TGGAGGGGAA 30
(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:
CCTCATATTT CCTCCTCCAG GTCT 24
(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
-20-
(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:
CTCTCGACGC AGGACTCGGC 20
(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:
CTTTCCCCCT GGCCTTAACC G 21
(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:
GAAGAAGAGA TAGTAATTAG ATCT 24
(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 ~
--22--
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 1 69453
~- (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
