Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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w0 00/0021,7 PCT/FR99/41539
MUCOSAL TARGETING IMMUNISATION
The present invention relates to an immunization method
which. makes it possible to induce a local immune
response, in particular in the rectogenitourinary
mucosal region. .Another subject matter of the invention
is the use of immunogens in the preparation of vaccine
compositions intended to be administered according to
said method.
State of the art
The main route for the transmission of the AIDS virus
is the mucous membranes, in particular genital and
1S rectal mucous membranes, indeed even buccal mucous
membrane. From these mucous membranes, the virus
rapidly spreads to the draining lymph nodes, before
joining the peripheral blood.
As in th.e case of other pathogenic agents (virus,
bacterium, and the like) with a mucosal gateway, the
induction of immunity capable of blocking the virus at
its entry into the mucous membrane or in the first
stages of the spreading t~~ereof into the lymph nodes
appears to be important.
The majority of studies are generally targeted at
obtaining a systemic immunity which is detectable by
the titer of serum antibodies, as described by
Jian-Ming X. et a1. (Vaccine, 993-1000, 1996), Kim J.J.
et a1. (Vaccine, 879-883, 1997), Anderson et a1. (The
Journal of Infectious Diseases, 960969, 19$9),
David D. et a1. (Vaccine, 1661-1669, 1997), Raskisov et
a1. (US 4 368 191) and Transgene (FR 2 751 879).
The targeting of a local immunity of the mucosal type
can. be demonstrated by the presence of specific
antibodies present in the mucous membranes or the
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secretions (Thibodeau L. et a1. Aids Research and Human
Retrovirus, 1379, 1992; Russel M.W. et al., Reviews of
Infectious Diseases, 5440-5446, 1988).
The studies of Lehner et a1. (Nature Medicine, 767-775,
1996), carried out on the rhesus macaque, have shown
that it is possible to induce a local immunity with
respect to the SIV virus by carrying out an extremely
deep subcutaneous injection in the pelvic region in the
vicinity of the iliac lymph nodes. This immunization is
reflected by the induction of antibodies of IgA and IgG
type in the rectal and urinary fluids and in the serum.
However, such an immunization method is not applicable
to man.
Letchworth G.J. et a1. (US 5 462 734) teach that an
intramuscular injection (without specifying the
injection site) of a glycoprotein induces solely a
systemic response and the booster applied in the mucous
membranes then makes it possible to obtain a local
mucosal immunity. It thus seems that the intramuscular
injection does not make possible, by itself alone, the
targeting of a local response.
Gaffar A. et a1. (US 3 931 398) teach that an injection
of a vaccine in the oral cavity would make it possible
to induce a local immunity. No measurement of the
antibodies is reported. It thus seems that the
proximity of the injection site to the site of
targeting of a local immunity is essential, as also
reported by Letchworth G.J. et al. above.
There thus currently exists no mucosal immunization
method targeted at the rectogenitourinary region which
is truly effective and usable in the current practice
of human or veterinary medicine. Furthermore, there was
nothing to allow it to be supposed that an injection at
sites distant from the mucous membranes could bring
about a targeted local response.
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Summary of the invention
In order to meet the need to have available technical
solutions for effectively and easily inducing a local
immunity, the present invention is thus targeted at the
use of an irnmunogen specific for a pathogenic agent
having a gateway in a mucous membrane for the
production of an immunogenic composition intended to be
administered to man by the parenteral route at the
surface of a part of the body distinct and distant from
said mucous membrane, so as to directly develop a local
response in respect of IgA, IgG and/or IgM antibodies
and of B cells which secrete said antibodies in said
mucous membrane and the lymph nodes which drain it.
A particular objective of the invention relates to the
targeting of the rectal, genital and/or urinary mucous
membranes by administration by the parenteral route in
the thigh. In the face of this report, it is now thus
possible to look for multiple sites for injections,
distinct from the mucous membranes, which will make
possible, directly, the induction of a local response
in these mucous membranes, in particular in respect of
antibodies and of 8 cells which secrete antibodies
found in the mucous membranes and the lymph nodes which
drain them.
Another particular objective of the invention is to
provide such an immunization route in developing a
local immunity against the AIDS (HIV) virus.
Another objective of the invention is tv simultaneously
induce a systemic immunity, in particular a humoral
and/or cellular immunity, detectable in the peripheral
blood.
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Details of the invention
A subject matter of the present invention is thus the
development of a local response, in particular in the
rectogenitourinary mucous membrane and the lymph nodes
which drain it, by parenteral injection of an
immunogenic composition in a part of the body distinct
from the mucous membrane, such as the thigh. The
parenteral administration in the thigh and in the
higher neighboring regions, in particular the groin,
makes it possible to target the iliac and inguinal
lymph nodes. The intramuscular route in one or both
lower limbs, in particular in the quadriceps,
especially in the right anterior muscle, is preferred_
Such an immunization route proves to be capable of
inducing locally, in this mucous membrane, on the one
hand, the production of immunoglobulins in the
secretions and, on the other hand, regionally, in the
lymph nodes which drain this mucous membrane, the
production of B cells which secrete antibodies, while
inducing a systemic immunity. This immunity is capable
of inducing protection against the entxy and the
spreading of the pathogen under consideration from this
rectogenitourinary mucous membrane region.
The invention applies both to the field of prophylaxis
(e. g. vaccines) and to the field of active
immunotherapy. The term "immunogenic composition" thus
covers compositions with a prophylactic purpose, in
particular vaccines, and compositions with a curative
purpose, in which composi.t~.ons the immunogen is of
antigen type.
A first specific subject matter of the invention is
thus the use of an immunogen specific for a pathogenic
agent having a gateway in the rectogenitourinary mucous
membrane region fox the production of an immunogenic
composition intended to be administered to man by the
parenteral route in the thigh, preferably by the
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intramuscular route, in particular in the quadriceps,
especially in the right anterior muscle (the parenteral
administration in a muscle of the thigh will preferably
take place in the muscle of each right and left lower
S limb) , so as to develop a local response in respect of
IgA antibodies, indeed even also of IgG and/or IgM
antibodies, and of B cells which secrete zgG, IgM
and/or IgA, in respectively, on the one hand, the
rectogenitourinary mucous membranes ar~.d their
secretions and, on the other hand, the lymph nodes
which drain this mucous membrane, in particular
inguinal lymph nodes and external and internal iliac
lymph nodes. This is because the parenteral injection
and in particular the intramuscular injection in the
thigh turns out to make possible the recruitment of B
cells which pxoduce zgG, zgM and/or IgA antibodies in
the lymph nodes which drain the rectogenitourinary
mucous membrane region.
The production of IgA and IgG antibodies of plasma or
secretors origin and the local recruitment of B cells
which secrete IgA and IgG are thus achieved, as was
observed in the tests reported in points ZIZ and V of
the examples.
Mention will very particularly be made, among the
pathogenic agents to which the invention can be
applied, of: the HIV virus, Herpes viruses, e.g. the
Herpes simplex virus, in particular of type 2, Candida
species, Chlamydia species, the human papillomavirus,
genital mycoplasmas, Treponema pallidum, papovaviruses,
e.g. Condyloma accuminatum, and gonococcal infections.
It should be noted that this method of administration
is not limited to inducing a local response and can
also make it possible to induce, at the same time, a
systemic response, the two actions combining together
and complementing one another, indeed even reinforcing
one another, in a particularly advantageous way.
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Consequently, the use in accordance with the invention
is also targeted at developing, in addition to a local
response according to the invention, a systemic
response of IgG and/or IgM and optionally IgA type
(antibodies and secretory B cells).
without it being necessary to specify it each time, it
is obvious. that, when speaking of a response in respect
of IgG, IgM or IgA, as antibodies and secretory B
cells, it is a response specific to the immunogen used.
Fvr other immunogens, the response may be in addition
or instead a local cellular immune response (cytotoxic
T lymphocytes, THl and TH2 response, optionally
suppressor response).
Another subject matter of the invention is the method
of immunization against pathogenic agents, such as
those described above, which consists in administering,
by any means known per se, the appropriate immunogenic
composition by the parenteral route, in particular the
intramuscular route, in the thigh of one or both lower
limbs, preferably quadriceps, in particular right
anterior muscle. Without it being necessary to restate
it each time, the method of immunization can have each
of the characteristics, alone or ~.n combination, set
out here in the context of the use.
In the context of the use and of the method of
immunization, it may be further specified that the
invention applies to all known types of immunogenic
composition and in particular known vaccines, whether
they are of conventional type or of recombinant type.
As is known per se, the compositions, e.g. vaccines, of
conventional type include attenuated or inactivated
live whole compositions, e.g. vaccines, or subunits
(proteins or peptides). They can be adjuvanted or non-
adjuvanted and be presented in the combined form
grouping together different valences and/or different
immunogenic forms with the same valency. The
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recombinant compositions, e.g. vaccines, include the
living vectors expressing one or more immunogens of the
pathogen under consideration and the polynucleotide
plasmid vectors composed of a DNA, which can, for
example, be naked or included in a liposome (see, e.g.,
WO-A-90 11092, WO-A-93 19813, WO-A-94 21797,
WO-A-95 20660), which express one or more immunogens.
As regards recombinant living vectors, mention may in
particular be made, as vectors, of pox viruses, such as
the vaccinia virus and especially avian pox viruses
(canarypox, fowlpox, pigeonpox, and the like), such as
those described in Tartaglia et al., Virol., 1992, 188,
217, and adenoviruses. As regards a novel
administration route, it is very obvious that the
invention cannot be limited to a specific type of
composition, e.g. vaccines, but can be applied to all
types of immunogenic compositions, e.g. vaccines, and
to all available compositions, e.g. vaccines, which can
be used by the parenteral route, in particular the
intramuscular route.
Likewise, the immunization protocol will depend on the
type of composition or on the composition used. It will
include the number of administrations generally used
for a given composition, which will generally
correspond to more than orie administration, in
particular from 2 to 4. A person skilled in the art is
in any case fully able, by routine tests, to determine
the optimum number of administrations (e. g. primary
vaccination and booster). However, it should be noted
that the injection in a site distinct from the mucous
membranes can make possible, by itself alone, the
targeting of a local response, without requiring a
booster to be applied in the mucous membranes.
This targeted immunization can also be combined with a
conventional systemic immunization by the same
composition or another composition combating the same
pathogen.
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It is also possible to combine with it, in the same
host, an immunization protocol targeted at the buccal
mucous membrane comprising the administration of an
identical or different inununvgenic composition, in
particular of the same composition, which combats the
same pathogen, targeted at inducing a local response in
respect of IgG, IgM and/or IgA and of B cells which
secrete IgG, IgM and/or IgA respectively in the saliva
and the lymph nodes which drain the buccal mucous
membrane, in particular the submaxillary lymph nodes
(it can also be accompanied by a systemic reaction),
preferably by sublingual injection in the floor of the
mouth. The test reported in points III and IV shows
that a sublingual injection is an appropriate means,
other administration routes in the oral cavity
remaining possible. It is, of course, possible to use
other means capable of suitably activating the lymph
nodes which drain the buccal mucous membrane, in
particular the submaxillary lymph nodes.
Such a Combination is particularly useful for
preventing or treating an infection by a pathogen
having both buccal and rectogenitourinary gateways.
Mention may in particular be made of the HIV virus, the
Herpes virus, and the like.
Consequently, according to an advantageous development
of the present invention, the invention also relates to
the use of an immunogenic composition intended to be
administered to a host by the parenteral route in the
thigh, in particular by the intramuscular route in the
thigh, preferably in the quadriceps, in particular in
the right anterior muscle, and, moreover, to the use of
another immunogenic composition, identical to or
different from the preceding one, intended to be
administered to the same host by sublingual injection
in the floor of the mouth.
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The use in accordance with the invention and the method
of immunization in accordance with the invention have a
preferred application in the context of vaccination
against the HIV virus.
A specific example is the use of a vaccine combining
together a vector expressing HIV gp120/gp160 and of
[sic] the gp120/gp160 glycoprotein subunit of this same
virus. A specific example is described later.
In the context of the present invention, the use is
thus anticipated of this anti-HIV vaccine for its
administration by the intramuscular route in the thigh,
optionally in combination with a sublingual
administration and/or a systemic administration of
conventional type, e.g. by intramuscular injection in
the deltoid.
Finally, a further subject matter of the invention is
an immunogenic composition comprising at least one
immunogen specific for a pathogen having a gateway in
the rectogenitourinary mucous membrane region and a
pharmaceutically acceptable vehicle or excipient, this
vehicle or excipient or this composition resulting, in
combination with the immunogen, in a local response in
respect of IgG, IgM and/or IgA antibodies and of 8
cells which secrete IgG, IgM and/or IgA in this mucous
membrane region when the composition is administered by
the parenteral route in the thigh, in particular by the
intramuscular route. In the context of this immunogenic
composition, the characteristics set out here with
respect to the other subject matters of the invention
can be taken up again, alone or in combination.
The invention will now be described in more detail with
the help of embodiments taken as nonlimiting examples.
It must be clearly understood that the invention
defined by the appended claims is not limited to the
spec~_fic embodiments indicated in the above description
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but encompasses the alternative forms thereof which do
not depart either from the scope ox from the spirit of
the present invention.
Example 1 - Targeting of the lymph nodes
The targeting of the lymph nodes involved in the
rectogenitourinary responses by the intramuscular route
in the thigh is illustrated by experiments carried out
20 on the cynomolgus macaque: in these experiments, a 2~
solution of Evans blue dye in saline solution was
inj ected ( 0 . 5 to 1 ml ) in the right anterior muscle at
a site situated at a distance of one third from the
groin and of two thirds from the knee.
Dissection of the euthanized animals 4 hours after
injection of the dye allowed the lymph nodes draining
the inoculation region to be located by virtue of their
blue coloring: inguinal and iliac lymph nodes were thus
identified.
Example II - Vaccine
II-1 Recombinant live vaccine vCP205, RLVAC-HIV:
vCP205 is an ALVAC canarypox virus, the construction of
which is disclosed in Example 14 of w0-A-95 27507, to
which a person skilled in the art may refer. It is
capable of expressing the env, gag and p.ro genes of the
HIV-1 virus. These genes are inserted in the C3 locus
and axe regulated by the H6 and I3L promoters of the
vaccinia virus.
A pHIV32 plasmid comprising the expression cassettes
for the gene of the env gp7.20 I~'B~1 glycoprotein (plus the
transmembrane part of gp41 LRI) and the genes of the
LAI strain coding for gag and for the pro protease was
used as donor plasmid in an in vivo recombination
procedure for producing vCP205. These cassettes were
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inserted in the C3 locus, between the ALVAC flanking
sequences, in a 5'-5' configuration, and linked to the
H6 and I3L promoters.
The vCP205 was produced on chicken embryo fibroblasts
in a serum-free DMEM-Ham F12 medium with lactoglutarnate
added and clarified by centrifuging. The mean titer was
108'° CCzDS°/ml with regard to QT35 cells. The
vaccinating solutions are prepared by diluting in PBS
tphosphate buffer saline) with Ca++ and Mg+'.
II-2 gp160I~T/LAI-2 subunit vaccine
The subunit produced is a hybrid gp160 subunit obtained
from a pox vector.
VVTG9150 vaccine vector is used to produce gp160.
This vector codes for a hybrid soluble gp160 in which
the gp120 part is derived from the HIV-J. MN strain and
the gp41 part originates from the LAI isolate. The
corresponding DNA sequences were fused using an
artificial Smal restriction site modifying neither of
the two amino acid sequences of gp120 and gp4l. The
construction is briefly described below.
The sequence coding for gp120 1~1 was amplified from
SupTl cells infected with HIV-MN by the PCR technique
with oligonucleotides introducing an Sphl restriction
site and an Smaz site respectively immediately
downstream of the sequence coding for the leader
peptide and upstream of the cleavage sites situated
between gp120 and gp4l.
The sequence coding for gp41 was thus produced: the
complete coding sequence of env HIV-1 LAI was placed
under the contz~ol of the PHSR promoter of the vaccinia
virus. Several modifications were introduced. An SphI
restriction site was created immediately downstream of
the sequence coding for the leader peptide, without
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detrimentally affecting the amino acid sequence. An
Smal restriction site was also created inunediately
upstream of the sequence coding for the cleavage sites
between gp120 and gp4l, without detrimentally affecting
the amino acid sequence. The two cleavage sites at
position 507-516 (numbering of the amino acids
according to Myers et al. in: Human retroviruses and
AIDS (1994), Los Alamos National Lab. (USA)) were
mutated (original sequence: KRR...REKR mutated to
QNFi...QEHN). The sequence coding for the transmembrar~e
hydrophobic peptide IFIMIVGGLVGLRIVFAVLSIV (amino acids
689-710 according to Myers et al. above) was deleted. A
stop codon was introduced instead of the second codon E
of the sequence coding for PEGIEE (amino acids 735-740
according to Myers et al.), that is to say the 29th
amino acid of the intracytoplasmic domain.
The plasmid in which the LAI sequence was inserted
between the homologous regions of the TK gene of the
vaccinia virus was cleaved by Sphl and SrnaI and then
linked to the gp120 MN sequence. The VVTG9150 was
subsequently constructed by conventional homologous
recombination and propagated in order to ensure the
expression of the gp160 according to the method
generally used for vCP205 on HHK21 cells. The gp160 was
subsequently purified by immunoaffinity chromatography.
Example III - Test
Two female rhesus macaques (P9224 and P9225), already
immunized by the intramuscular route (in the left or
right thighs, alternately) twice with 106'5 CCIDso of
clarified ALVAC-HIV (vCP205) and then three times with
100 ~cg of gp160 MN/LAI-2 adjuvanted with OspA (outer
surface prote~.n A of Borrelia burgdorferi) and aluminum
hydroxide, were inoculated twice at intervals of 1
month in the floor of the mouth (sublingual) with a
mixture comprising 10~ CCIDSO of clarified vCP205 and
100 ~,g of gp160 MN/LAI-2. The saliva, the urine, the
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vaginal and rectal secretions, and the serum were
analyzed by ELISA in order to detect the presence of
anti-gp160 and anti-CPpp (which combat the canarypox
virus itself) IgA and IgG.
One of the two monkeys (P9225) received an additional
injection of the same mixture (vCP205 + gp160 N~1/LAT-2)
in the floor of the mouth and the top of the right
thigh, three months after the final injection. The
lymphocytes of the peripheral blood and of some lymph
nodes (submaxillary, axillary, inguinal and iliac)
lymph nodes were analyzed by ELISPOT for the detection
of 8 cells which produce IgA and IgG antibodies
specific for gp160 and CPpp.
The appearance of anti-gp160 and anti-CPpp IgA in the
mouthwash from the macaque P9224 and of anti-gp160 IgA
in the vaginal wash from the macaque P9224 could be
shown. The specific anti-gp160 IgG responses appeared
from the first injection in the majority of the
secretions tested and were maintained throughout the
study.
Furthermore, the sera of the two macaques showed a
significant increase in the IgAs and IgGs specific for
gp160 and CPpp.
Finally, a preferential induction of B cells which
secrete anti-gp160 and anti-CPpp IgA+ and IgG
antibodies in the lymph nodes targeted by the
immunizations, namely the submaxillary lymph nodes and
the right inguinal and iliac lymph nodes, was
demonstrated by ELISPOT in the monkey Pg225. These
cells were also present in the peripheral blood but at
a lower frequency.
To conclude, this test showed the possibility of
inducing a local and systemic anti-HIV-1 antibody
response in the rhesus monkey after immunization close
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to lymph nodes which drain the buccal and rectogenito-
ur~.nary mucous membranes.
Example IV - Test 2
The vaccine is a mixture comprising 106'3 CCZD50 of
vCP205 and 100 ug of gp160 subunit. An ALVAC vector not
comprising any IiIV sequence was also used, as control.
Group 1: 4 monkeys (rhesus macaques) received, on
-
4 occasions at an interval of 1 month, an
injection of the vaccinal mixture by the
sublingual route in the floor of the mouth; vol ume
for volume mixture of 106'9 CCZDsp/ml vCP205 and of
400 ~.glm1 gp160; 0.25 ml on the right and 0.25 ml
on the left.
- Group 2: 4 monkeys (rhesus macaques) received, on
4 occasions at an interval of 1 month, an
injection of the vaccinal mixture by the
intramuscular route in the thigh (perpendicular to
and in the right anterior muscle); volume for
volume mixture of 106' CCIDSO/ml vCP205 and of
200 ~.g/ml gp160; 0.5 ml on the right and 0.5 ml on
the left.
Group 3 (contxols): 3 monkeys (rhesus macaqu es)
-
received, on 4 occasions at an interval of
1 month, an injection of the ALVAC vector by the
intramuscular route in the thigh; 106'3 CCIDgp /ml
ALVAC (CPpp); 0.5 ml on the right and 0.5 ml on
the left.
The number of B lymphocytes which secrete total zgG's
specific for the gp160 (resulting from the two types of
vaccine) and specific for the control ALVAC vector per
106 mononucleated cells was measured by sampling from
each group of macaques. The calculated mean and the
calculated standard deviation were rounded off to the
nearest unit.
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The right and left lymph nodes of each category (sub-
maxillary, axillary, inguinal, internal iliac and
external iliac) were removed after sacrificing the
animal, milled (the right and left submaxillary lymph
nodes were pooled) and then subjected to analysis of
the antibody-producing cells by the ELISPOT technique
(adapted from Erikson K. et al., Journal of
Immunological Methods, 153, 107-113, 1992).
A systemic response in respect of IgG antibodies was
also observed.
The results of counting the IgG-secreting B lymphocytes
are combined in the following table:
CA 02335506 2000-12-20
ro
°'
M O
O y-I ~ N v-I M rl O r-i r-i t0 v-W -I W rl O d~ N +1
-H ~ -1-I +1 -H tl ~1 -H -H +I tl tl tl ti +1 -H +1 O
V V ~ ,~ N N M N N N N d~ l0 ~-1 M
O ~ N v-1 ~ N
N N
O
C
O
O
ra
~O
t~ O N ~, ap
O r1 ~D O r-1 O ~~i e-1 ~-1 O e-1 O ~ ,~ pp p O O O O O
ri H +I tl +I +I tl tl tl tl fl tl ti i-1 -H +1 +1 tl +1 tl
~-I O~ O N rl rl .-1 N N G1 I'~ N O O O O O O
N h~ X17
N
U
r1 tl1 OO C~ ~ tf1 OD M 0 l0 ~ CO O~ Q1 ~ OC N N
r1 'cft N ri er O O Q, ll~ 00 f~- 01 ~ M
,u c ~ ono '~ '~ o ~i '~ ~ +1 ~ .+~ ~ + + ~ . ~ +~
' O lD 10 O N t0 N y11 ~ C'~ N L~ n O C~ ~ O M
E ri ~~-i ("7 ~O CT1 ~"~ '",~ t~ N U1 lf1 e~ ~ ~ N ~ p
pl "i d~ ~o w u, ~ ~,p ~ oo uW o r
ro N ~ ~ ~ v a
ow°a~~ ~oa~~c~v ov
" ~ ~ x .~ o ~ o .>~ .~ o ~ o ~ x o
GlW, a ~ 1~ i). G
w ~ ~ b a ,-~ ..-W , .n Q, .c
0 0
a an ~ ~, ;1 ~ ~-, s.'' '''~ '''
C ~ rt
'--W-, ~i .rl ..1 ~-1 .~-, r-, ~.-/ ~r1 .1 r-, r-~ ~rl
°i~ ~~~ao~ '~~~a~, ~~~c~
N N H ,;q N N H ~ G1 N H
Cn W
H W ~ H W N H
i
N i
1J O N ~~ N
O U N i .~ i
-O 1.-~
H
+ ~ ~ N ~ 0 5 a
w ..-~ w
o ~ x ~ ~ ~ ~ ~ N
H
~r ~
tla ~ H H
w
O
O tn ~ -~.
x o a
N M
O
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Example V - Test 3
The vaccine is composed of two products injected
separately, first of all 106'5 CCIDSO of vCP205 and then
50 ~,g of gp160 I~1/LAZ subunit adjuvanted with a
conventional adjuvant.
2 female rhesus macaques (#P952 and #P9315) received:
- on 2 occasions at weeks 0 and 4, an injection of
vCP205 by the intramuscular route in the thigh
(perpendicular to and in the right anterior
muscle), on the right and then on the left
alternately, at a dose of 106' S CCIDSO in a volume
of 1 ml,
- on 5 occasions at weeks 8, 12, 25, 99 (for P952)
or 100 ( fox- P9315 ) and 103 ( for P952 ) or 104 ( for
P9315), an injection of adjuvanted gp160 MN/LAZ by
the intramuscular route in the thigh
(perpendicular to and in the right anterior
muscle), on the right and on the Left, at a dose
of 50 ug (+ 500 ~,g of adjuvant) in a volume of
1 ml (0.5 ml in each thigh).
The number of B lymphocytes which secrete IgG and IgA
specific for the gp160 per 106 mvnvnucleated cells was
measured in each sample (lymph nodes and blood) from
the two macaques. The mean and the standard deviation
of the counts carried out in triplicate for each sample
are :represented. The right and left lymph nodes of each
category (submaxillary, axillary, inguinal, internal
iliac and external iliac) were removed after sacrifice
of the animal and milled, and the right and left lymph
nodes of each category were pooled and then subjected
to analysis of the antibody-producing cells by the
ELISPOT technique (adapted from Eriksson K. et al.,
Journal of Immunological Methods, 153, 107-113, 1992).
The anti-gp160 IgA and IgG antibodies in the serum and
the mucous secretions (urine, vaginal washes and rectal
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washes) from the two macaques were also assayed by the
EL=SA technique. The samples were taken before the two
late boosters (W99) and then after immunisation,
immediately before the sacrifice of the animal (W104 or
W105). The results are expressed, for each secretion,
in the form of the ratio of the specific anti-gp160 IgA
or IgG activity measured at the time of the sacrifice
to that evaluated before the two late boosters (W9g).
It was considered that a secretion was significantly
positive in respect of specific TgAs or IgGs if this
ratio was greater than 3 (the specific activity of a
secretion corresponds to the titer in respect of
specific IgAs or IgGs (in this instance for gp160)
divided by the titer in respect of total IgAs ox IgGs).
Furthermore, the local or plasma origin of the specific
TgAs or IgGs in a secretion was evaluated according to
two different techniques:
1. Measurement of the relative excretion coefficient
of the zg(A or G)s with respect to albumin
(protein of essentially plasma origin), according
to the technique described by Belec L. et al.,
AIDS Research and Human Retroviruses, 12, 157-167,
1996.
2. Measurement of the local production coefficient of
the Ig(A or G)s, which compares the specific
activity measured in the secretion with that in
the serum withdrawn at the same time, according to
the technique described by Van Cott T. et al.,
Journal of Immunology, 160, 2000-2012, 1998.
CA 02335506 2000-12-20
N
b
~ U
N p, r0 ~"W l1 01 M
O N y O O O N M N p O O M M d~
to N ro +I +1 +1 +t +I +1 +1 +1 +1 +1 +1 +1
U '-I o 0 0 ,.~i ~ ~ ~"~ o e-i ~ a~ N
N M ri
O
G
D
N
O
ri ,_.
lv
O U
N (1 b 01 u'1 T'~ ~
O Ul ~ ~ O N M ~r-i ~ ri O r-I M N
+I +1 +I +1 +I ~ tl ~I tl ~ .1.1 +1
U r1 N v-i f~ CO N ~ M N N ~ N
O ~ N
C
O
ri
Q
r1
VJ UI U1 N
tw u~ ro Gl N 'b 'd
1 O
O ~ F; fU O 0l C ~ O!
li ~ ~ .0,..~ ~ R K1~ f~ G." .C R fr1 41, f-,''
()
o ~~~~~,~ ~a,~'~'a
0
"' '"' ~' ~ ~ ,~ ''a '-1
b
r~ -'' ''' c
-rl ,-, ,~ r-, -.-1 _rl ° .-r r-1 .r~~i
ro
.Ll N Ol N ~ N N 1-i
,L~ ~ ~ H W ~ H W
~,
1~ 1~
C'r ~l1
N
1J O ~ ~ ''a H
v ~' ~ x u, ,J a
b, ~ o c c
.a ~ > U
~ > ~r-, o
3
x N
~° w w
CA 02335506 2000-12-20
- 20 -
Results with respect to the immunoglobulins present in
the secretions
Ratio of the specific
aati-X160 =gG aativita.es
at W104
or w105 versus W99
Secretions Monkey# Ratio
Urine P952 e.9
P9315 51.3
Rectal washes P952 3.5
P9315 57.5
Vaginal washes P952 6.3
P9315 57.9
IgAs were also detected in some urinary, vaginal and
rectal secretions.
Example VI
Vaccinal preparations against Herpes simplex, a
Candida, a Chlamydia, a human papillomavirus, a genital
mycoplasma or Treponema pallidum weze injected in the
muscles of the thigh in order to stimulate and target a
local response in respect of IgA antibodies at least in
the rectal, genital and/or uxinary mucous membranes.
