EP1087788A1

EP1087788A1 - Mucosal targeting immunisation

Info

Publication number: EP1087788A1
Application number: EP99926558A
Authority: EP
Inventors: Thérèse JOURDIER; Catherine Moste; Bernard Meignier
Original assignee: Aventis Pasteur SA
Current assignee: Sanofi Pasteur SA
Priority date: 1998-06-26
Filing date: 1999-06-28
Publication date: 2001-04-04
Also published as: WO2000000218A1; US20010021384A1; CA2337823A1; AU4376199A

Abstract

The invention concerns the use of an immunogen specific of a pathogenic agent with a gateway in the buccal mucous membrane region, for producing a vaccine composition to be administered in the floor of the mouth in a human being so as to develop directly a local response in IgA antibodies and in B cells secreting IgA in the buccal mucous membrane, saliva and ganglions draining said mucous membrane. The invention also concerns a vaccine composition capable of being applied in the floor of the mouth in a human being to induce local and systemic immunity in IgA antibodies, substantially consisting of a material adhering or not to the buccal mucous membrane and containing an immunogen specific of the pathogenic agent with a gateway into the buccal mucous membrane.

Description

Mucosal targeting immunization

The present invention relates to an immunization method for inducing a local immune response in the buccal mucous region. It also relates to the use of immunogens for the production of vaccine compositions intended to be administered according to said method.

State of the art

10 The main route of transmission of the AIDS virus is through the mucous membranes, in particular the genital and rectal mucous membranes, or even the oral mucosa. From these mucous membranes, the virus quickly spreads to the draining nodes, before reaching the peripheral blood.

ι - As in the case of other pathogens (virus, bacteria, etc.) has a mucosal entry door, the induction of immunity capable of blocking the virus on its entry into the mucosa or in the first stages of its dissemination in lymph nodes appear large

0 Most of the work generally aims at obtaining a systemic immunity which is detectable by the titer in seπques antibodies, as described by Jian-Ming X et al (vaccine, 993-1000, 1996), Kim JJ et al ( vaccine, 879-883, 1997), Anderson et al. (The Journal of Infectious Diseases, 960-969, 1989), David D et al (vaccine, 1661-1669, 1997), Raskisov et al (US4368191) and Transgene

2 (FR2751879)

The targeting of a local mucosal type immunity can be demonstrated by the presence of specific antibodies contained in the mucous membranes or the secretions (Thibodeau L et al, Aids Research and Human Retrovirus, 1379, 30 1992, Russel MW et al, Reviews of infectious diseases, 5440-5446, 1988)

The work of Lehner et al, (Nature Medicine, 767-775, 1996) carried out in the rhesus macaque have shown that it is possible to induce local immunity against the SIV virus, by performing a sub-injection. "O deep cutaneous in the pelvic region near the iliac nodes This immunization results in the induction of antibodies of the IgA and IgG type in rectal, uπnary fluids and in the serum Such an immunization method is however not applicable to humans

Letchworth GJ et al (US5462734) teach that an intramuscular injection (unspecified of the injection site) of a glycoprotein induces a systemic response only, and the recall carried out at the level of the mucous membranes then makes it possible to obtain local mucosal immunity. trials do not target the oral cavity II has not been reported also obtaining secretory IgA antibodies

A Gaffar et al (US3931398) hypothesize ^that an injection of a

10 cane vaccine in the oral cavity would induce local immunity

No test to measure the IgA antibody response has been reported, however.

L Thibodeau et al (CR Acad Sci Paris 389-394 1991) report that ι> the repeated topical application of HIV gp160 on the oral mucous membranes of rabbits, followed by a parenteral injection, makes it possible to obtain systemic immunity and local IgA type Topical application to the oral mucosa does not however allow obtaining a systemic response of the IgA type because only the booster by the parenteral route gives this effect Finally, nothing allows to affirm that the 0 results are applicable in humans

J Hinkula et al (Vaccine, 874-878, 1997) show that the injection of HIV DNA under the control of a CMV promoter into the tongue or into the gums of mice makes it possible to obtain a detected systemic IgA response in the peripheral blood 5 It is not reported obtaining an IgA response in the secretions Finally, the injection routes are not really applicable to humans

The present invention has therefore set itself the objective of proposing a simple and effective route o of administration making it possible to directly induce local and systemic immunity, in particular locally in IgA antibodies and B cells secreted by IgA, at the level of the mucous membrane and oral secretions in humans, lymph nodes that drain them 00/00218

Summary of the invention

The invention therefore relates to the use of a specific immunogen of a pathogenic agent having an entry point at the level of the buccal mucous region, for the production of a vaccine composition intended to be administered in the floor of the mouth in humans so as to directly develop a local response to IgA antibodies and to IgA-secreting B cells in the buccal mucosa, saliva and the lymph nodes draining said mucosa.

A particular objective of the invention is to provide such an immunization route for developing local immunity against the AIDS virus (HIV).

Another objective of the invention is to simultaneously induce systemic and local immunity in antibodies by at least one route of administration. By local immunity is meant an IgA, IgG and / or IgM response in the oral mucosa, saliva and ganglia draining said mucosa, while a systemic immunity relates to an IgA, IgG and / or IgM response detected in the peripheral blood.

Yet another objective of the invention is to propose an immunization method which can be very advantageously combined with a conventional immunization to supplement it and in general with any other type of immunization, local or systemic.

Another objective is to propose a route allowing the injection of substantial volumes of a vaccine.

Another object of the present invention consists of a vaccine composition, capable of being applied to the floor of the mouth in humans in order to induce local and systemic immunity in IgA antibodies, consisting essentially of an adhesive material to the buccal mucosa and which contains at least one immunogen specific for a pathogenic agent having a gateway in the buccal mucosa region.

Finally, a last object of the present invention relates to a vaccine composition, capable of being deposited in the floor of the mouth at 00/00218

man in order to induce local and systemic immunity in IgA antibodies, consisting of a material non-adhesive to the oral mucosa which degrades on contact with secretions and which contains at least one specific immunogen of a pathogenic agent having a entrance door at the level of the buccal mucosa, characterized in that this material is provided with invasive elements promoting the penetration of the immunogen through the buccal mucosa

Detailed description of the invention

The present invention thus relates in particular to the development of a local and systemic response at the level of the oral mucosa and of the ganglia which drain it by the administration of an immunogenic composition in the floor of the mouth of man. waiting this route allows I to obtain a local and systemic response directly which had not been demonstrated before

The invention applies both to the field of prophylaxis (vaccines) and to the field of active immunotherapy. The term immunogenic composition therefore covers compositions for prophylactic purposes, in particular vaccines, and compositions for curative purposes in which the immunogenic is antigen-like

It is thus possible to administer the immunogenic composition by parenteral injection, eg by sublingual injection into the floor of the mouth. The production of immunoglobulins IgA, IgG and / or IgM is induced locally at the level of this mucosa, in the secretions (saliva) and regionally. , in the lymph nodes which drain this region and which induce the production of B cells secreted from said antibodies, while inducing systemic immunity This immunity is capable of inducing protection against the entry and dissemination of the pathogen considered from this mucous region buccal

The immunogenic composition can also be administered by deposition in the floor of the mouth even if this technique is not known to give the best results. Preferably therefore, there is a bioadhesive or a capsule releasing the immunogenic composition in the floor of the mouth. bioadhesives specific to the oral mucosa are well known to humans trade, and have been described in particular by the company 3M Pharmaceuticals (Drug Dev & Indus Phar, 2809-2821, 1994), Taylan B et al (J Controlled Release, 1 1 - 20 1996) Nakane S et al (Pharmaceutical Dev & Tec , 251 -259, 1996), Efentakis M et al (STP Pharma sciences, 227-232 1998), and Diarra M et al (Biomateπals, 1523-1527, 1998) The capsules capable of releasing a pharmacological composition according to desired kinetics are also well known to those skilled in the art By way of non-limiting indication, one can thus cite Save T et al, J of Pharmacy & Pharmacology, 192-195 1994, W09618389, WO9908662, W09922767, W09921586, W09917744, EP913149 EP874642 m and WO9908729

Preferably, the bioadhesives or capsules used are provided or coupled to a system promoting the penetration of the immunogenic composition through the floor of the mouth, for example by means of abrasive particles, on the surface or not of the bioadhesives or capsules, or at the means of a particular formulation of the immunogenic composition such as liposomes, for example As an indication, mention may be made of the article by Gandhi et al (Advanced Drug Delivery Reviews, 43-74, 1994)

o The invention therefore therefore also relates to any vaccine composition capable of being applied to the floor of the mouth in humans in order to induce local and systemic immunity in IgA antibodies, consisting essentially of material adhesive to the oral mucosa, such as that one of those described in the above literature, and which contains at least one immunogen specific for a pathogenic agent having an entry gate at the level of the buccal mucous region. This adhesive material can be provided or paired with a system promoting the penetration of the immunogen through the oral mucosa such as abrasive particles consisting for example of hydroxyapatite or other salt crystals, or even as a particular formulation of the immunogen

^ o

The invention also relates to a vaccine composition, capable of being deposited in the floor of the mouth in humans in order to induce local and systemic immunity in IgA antibodies, consisting of a material non-adhesive to the oral mucosa which degrades on contact with secretions and which contains at least one immunogen specific for a pathogenic agent having an entry point in the oral mucosa region, characterized in that this material is provided with invasive elements promoting penetration of the immunogen through of the oral mucosa. This composition can therefore take the form of a capsule based on starch, maltodextπnes or other polysaccharides containing invasive elements, such as crystals of a salt, or hydroxyapatite, for example.

Against all expectations, the route of immunization according to ^the invention allows in particular to obtain secretory IgA in the form of dimers of antibody attached to the secretory component, having the property of being particularly resistant and therefore likely to '' be more suited to a neutralization activity. The 10 definitions of secretory IgA taken up by Thibodeau L (supra) and Russel MW (supra) are incorporated by reference in the description of the present invention

A first object of the invention is therefore the use of a specific immunogen ι ^ of a pathogenic agent having an entry point at the level of the buccal mucous region, for the production of an immunogenic composition intended to be administered from so as to develop a local response in IgA, IgG and / or IgM antibodies and in B cells secreting IgA IgG and / or IgM in the buccal mucosa, saliva and lymph nodes draining this mucosa, in particular lymph nodes under -maxillary Sublingual injection into the floor of the mouth is the preferred means in particular to allow the best recruitment of B cells in the lymph nodes draining the oral mucosa region. In addition, the amounts of immunogenic composition injected can vary from 0.1 to 3 ml, in particular 0.5 to 2 ml, for example

Among the pathogens to which the invention can be applied, particular mention will be made of: the HIV virus, herpes viruses, in particular herpes simplex, candida, hepatitis viruses, especially hepatitis A, picornaviridae, in particular enteroviruses, such as poliomyelitis reoviruses, in particular rotaviruses, adenoviruses, human papillomavirus, periodontoses, cytomegalovirus, Epstein-Barr virus, all aerosol-borne pathogens such as M. tuberculosis, N. meningitidis, Streptococcus type B, S pneumoniae, B pertussis , for example.

35 It should be noted that this mode of administration is not limited to inducing a local response, and can also make it possible to induce at the same time „„ ,, „

WO 00/00218

systemic response, the two actions combining and complementing, even reinforcing, in a particularly advantageous way

Consequently, the use in accordance with the invention also aims to develop, in addition to a local response in IgA, IgG and / or IgM antibodies and in secret B cells of said antibodies, a systemic response of the IgA, IgG and / or IgM (antibodies and secreted B cells) Preferably the local and systemic response is of the IgA and IgG type at least

Without it being necessary to specify it each time, it goes without saying that when we speak of a response in IgG, IgM or IgA, antibodies and secret B cells it is a specific response to the immunogen used

Another object of the invention is the method of immunization against i - pathogenic agents as described above, consisting in administering by any means known per se, the appropriate immunogenic composition so as to induce a local response as described above Without needing to be reminded each time, the immunization method can use each of the characteristics, alone or in combination, stated HERE in the context of 0 use. It will be recalled that the sublingual injection in the floor of the mouth is the preferred medium

In the context of the use and of the immunization method, it can also be specified that the invention applies to all types of compositions

2 _^ immunogenic and in particular known vaccines, whether of the conventional type or of the recombinant type As is known per se, the compositions, eg vaccines of the conventional type group together the compositions, eg whole live attenuated attenuated or inactivated vaccines, the subunits (proteins or peptides), these compositions, eg conventional vaccines which may be adjuvanted or non-adjuvanted and which may be presented

^ o in combined form grouping together different valences and / or different immunogenic forms of the same valence The compositions, eg recombinant vaccines group together the live vectors expressing one or more immunogens of the pathogen thus consider that the polynucleotide plasmid vectors constituted by DNA which can for example be naked or included in a liposome (see

3- eg WO-A-90 11092, WO-A-93 19813, WO-A-94 21797, WO-A-95 20660) and which express one or more immunogens With regard to recombinant living vectors, mention may be made in particular as vectors poxviruses, such as 00/00218

vaccinia virus and especially avian poxviruses (canarypox fowlpox pigeonpox, etc.) such as those described in Tartaglia et al, Virol 1992, 188 217, as well as adenoviruses As this is a new route of administration, it is obvious that I invention can not be limited to a particular type of composition, eg vaccine, but intended to apply to all types of immunogenic compositions, eg vaccines and all compositions, eg vaccines available for use by this route

Similarly, the immunization protocol will depend on the type of composition or vaccine or the composition or vaccine used II will include the number of administrations usually used for a given vaccine which, in general, will correspond to more than 1 administration, in particular from 2 to 4 Anyone skilled in the art is in any case perfectly able by routine tests to determine the optimum number of administrations (eg primary vaccination and booster)

This targeted immunization could also be associated with a conventional systemic immunization by the same composition or another composition against the same pathogen.

It can also be associated with the same host an immunization protocol targeting the recto-genito-uπnal mucosa comprising the administration of an immunogenic composition against the same identical or different pathogen, in particular of the same composition, preferably by parenteral injection. in the thigh (one or both lower limbs, right and left), preferably intramuscularly, in particular in the quadπceps, in particular in the anterior right muscle This immunization of the recto-genito-uπnal mucosa aims to induce a response local IgA, IgG and / or IgM antibodies, and in secret B cells of said antibodies at the level of this mucosa and of the lymph nodes draining this mucosa, in particular of the external and internal iliac nodes and of the inguinal nodes (it may also be accompanied by systemic reaction)

Such a combination is particularly useful for preventing or treating an infection by a pathogen having both the oral and rectogenitalunion entry gates. The HIV virus and the Herpes virus may in particular be mentioned. Consequently, according to an advantageous development of the present invention, the use of a specific pathogen-specific immunogen is aimed at the production of a portion of an immunogenic composition intended for administration to humans in the floor of the mouth so to induce an IgA, IgG and / or IgM antibody response and secretπces B cells of said antibodies in the buccal mucosa, saliva and lymph nodes draining this mucosa, in particular submaxillary lymph nodes, and preferably by sublingual injection in the floor of the mouth, and on the other hand an immunogenic composition, identical or different from the previous one, intended to be administered i to the same host parenterally in the thigh, preferably intramuscularly especially in the quadriceps in particular in the anterior rectus muscle, so as to induce a local response in IgA IgG and / or IgM antibodies and in secret B cells of said anti body at the level of the recto-genitourinary membranes

The corresponding immunization method therefore provides for this double administration. This immunization targeting the recto-genito-uπnary mucosa is also accompanied by a systemic immunization which advantageously combines with that resulting from the immunization targeting the buccal mucosa. It is also possible to combine these two local immunizations to a conventional systemic immunization, using the same composition or different compositions directed against the same pathogen. The use and the method of immunization in accordance with the invention find a preferred application in the context of vaccination against the virus. HIV A particular example is

2- the use of a vaccine grouping together a vector expressing gp120 / gp160 of HIV and of the glycoprotein subunit gp120 / gp160 of this same virus A particular example is described below

In the context of the present invention, it is thus planned to implement

30 this anti-HIV vaccine for its administration targeting the oral mucosa as described above, optionally combined with a conventional intramuscular administration in the thigh and / or systemic, eg by intramuscular injection in the deltoid

" ^• Finally, the subject of the invention is also a vaccine composition comprising a vaccine against a pathogen having an entry point at the level of the buccal mucous region and a pharmaceutically carrier or excipient acceptable, this vehicle or excipient, or this composition, leading, in conjunction with the vaccine, to a local response in IgA IgG and / or IgM antibodies and in B cells secreting said antibodies in this mucous region when the composition is in particular administered in the floor of the mouth In the context of this vaccine composition, the characteristics stated HERE with regard to the other objects of the invention can be taken up alone or in combination

The invention will now be described in more detail using embodiments taken by way of nonlimiting examples. It must be understood that the invention defined by the appended claims is not limited to the embodiments. particulars indicated in the description above but encompasses variants thereof which do not depart from the scope or the spirit of the present invention

Example I - Vaccine ι

1-1. vCP205 ALVAC-HIV recombinant live vaccine

vCP205 is an ALVAC canarypox virus whose construction is described in Example 14 of WO-A-95 27507 to which a person skilled in the art may refer II

20 is capable of expressing the env, gag and pro genes of the HIV-1 virus These genes are inserted into the C3 locus and are regulated by the H6 and I3L promoters of the vaccinia virus

A plasmid pHIV32 containing the expression cassettes for the env glycoprotein gene gp120 MN (plus the transmembrane part of gp41 LAI) and the genes of the LAI strain coding for gag and for protease pro, was used as donor plasmid in an in vivo recombination procedure to produce vCP205. These cassettes were inserted into the C3 locus, between the flanking sequences of ALVAC, in a 5'-5 'configuration, and linked to the H6 and I3L promoters.

VCP205 was produced on fibroblasts from chick embryos in the middle

DMEM-Ham F12 without serum, added lactoglutamate and clarified by centrifugation The mean titer was 10 ^{8 0} DICC ₅₀ / ml on QT35 On cells

3 prepares the vaccinating solutions by dilution in PBS ("phosphate buffer saline") with Ca ⁺⁺ and Mg ^{* +} 1-2 gp160MN / LAI-2 subunit vaccine

The subunit produced is a hybrid gp160 subunit obtained from a pox vector

A vaccine vector VVTG9150 is used to produce gp160. This vector codes for a soluble hybrid gp160 in which the gp120 part is derived from the HIV-1 MN strain and the gp41 part comes from the LAI isolate. The corresponding DNA sequences were fused with ^an artificial restriction site IO Smal not modifying neither amino acid sequences of gp120 and gp41. The construction is briefly described below.

The sequence coding for gp120 MN was amplified from cells

SupTI infected with HIV-MN, by PCR technique with oligonucleotides

15 introducing a SphI restriction site and a SmaI site respectively immediately downstream of the sequence coding for the leader peptide and upstream of the cleavage sites situated between gp120 and gp41.

The coding sequence for gp41 was thus produced the complete coding sequence for env HIV-1 LAI was placed under the control of the vaccinia virus pH5R promoter Several modifications were made A Sphl restriction site was created immediately downstream of the sequence coding for the leader peptide, without altering the amino acid sequence. A SmaI restriction site was also created immediately upstream of the sequence coding for the 25 cleavage sites between gp120 and gp41, without altering the amino acid sequence. The two cleavage sites in position 507-516 (amino acid numbering according to Myers et al. In: Human retroviruses and AIDS (1994) Los Alamos National Lab. (USA)) were mutated (original sequence 'KRR ... REKR mutated in QNH ... QEHN). The sequence encoding the transmembrane hydrophobic peptide IFIMIVGGLVGLRIVFAVLSIV (amino acids 689-710 from Myers et al. Supra) has been deleted. A stop codon was introduced in place of the second E codon of the sequence coding for PEGIEE (amino acids 735-740 according to Myers et al.), That is to say the 29 ^th ~ ^e amino acid of the domain intracytoplasmic.

35

The plasmid in which the LAI sequence was inserted between the homologous regions of the TK gene of the vaccinia virus, was cut by Sphl and Smal, then linked to the gp120 MN sequence. VVTG9150 was then constructed by conventional homologous recombination and propagated to ensure the expression of gp160 according to the method usually used for vCP205 on BHK21 cells. Gp160 was then purified by immunoaffinity chromatography.

Example II - Test 1

Two female rhesus macaques (P9224 and P9225) already immunized intramuscularly (in the left or right thighs, alternately) 2 times o with 10 ^{S 5} DICC ₅₀ of ALVAC-HIV (vCP205) clarified, then 3 times with 100 μg of gp160 MN / LAI-2 adjuvanted with OspA ("outer surface protein A" of Borrelia burgdorferi) and aluminum hydroxide, were inoculated twice at 1 month intervals, in the floor of the mouth (sublingual), with a mixture containing 10 ⁶ DICC ₅₀ of clarified vCP205 and 100 μg of gp160 MN / LAI-2. Saliva, urine, vaginal and rectal secretions, and serum were analyzed by ELISA to detect the presence of anti-gp160 and anti-CPpp IgA and IgG (against the canarypox virus itself).

One of the two monkeys (P9225) received an additional injection of the same o mixture (vCP205 + gp160 MN / LAI-2) in the floor of the mouth and the upper right thigh, three months after the last injection. Lymphocytes from peripheral blood and certain lymph nodes (submaxillary, axillary, inguinal and iliac) were analyzed in ELISPOT for the detection of B cells producing IgA and IgG antibodies specific for gp160 and 5 CPpp.

It has been possible to show the appearance of anti-gp160 and anti-CPpp IgA of local origin in the mouthwash of macaque P9224 after the first and second sublingual injections, and anti-gp160 IgA of plasma origin in the o vaginal lavage of this same monkey. The specific anti-gp160 IgG responses appeared from the first injection in most of the secretions tested and were maintained throughout the study.

Furthermore, the sera of the two macaques showed a significant increase in the specific IgA and IgG of gp160 and CPpp. , „„ „, _O 00/00218

Finally, it was demonstrated by ELISPOT, in the P9225 monkey, a preferential induction of B cells secreting lgA ⁺ and lgG ⁺ anti-gp160 and anti-CPpp antibodies in the lymph nodes targeted by the immunizations, namely submaxillary nodes and right iliac and inguinal nodes. These cells were also present in the peripheral blood, but at a lower frequency.

In conclusion, this test demonstrated the ability ^to induce local antibody response and anti-HIV-1 systemic rhesus monkey after immunization nearby nodes draining the oral mucosa and recto-genito urinary.

Example III - Tests 2

The vaccine is a mixture containing 10 ^{6 3} DICC50 of vCP205 and 100 μg of gp160 subunit.

An ALVAC vector comprising no HIV sequence was also used as a control.

- Group 1: 4 monkeys (rhesus macaques) received 4 times, 1 month apart, an injection of the vaccine mixture sublingually into the floor of the mouth; mixing, at equal volume, vCP205 at 10 ^{5 9} DICC ₅₀ / ml and gp160 at 400 μm / ml; 0.25 ml on the right and 0.25 ml on the left. - Group 2: 4 monkeys (rhesus macaques) received 4 times, 1 month apart, an injection of the vaccine mixture intramuscularly into the thigh (perpendicular to, and into the anterior rectus muscle); mixture of vCP205 at 10 ^{6 6} DICC ₅₀ / ml and gp160 at 200 μg / ml at equal volume; 0.5 ml on the right and 0.5 ml on the left. - Group 3 (controls): 3 monkeys (rhesus macaques) received 4 times, 1 month apart, an injection of the ALVAC vector intramuscularly into the thigh; ALVAC (CPpp) at 10 ^{6 3} DICC _5C / ml; 0.5 ml on the right and 0.5 ml on the left.

We measured the number of B cells secreting IgG ⁺ Total specific for gp160 (resulting from the two types of vaccines) and specific control ALVAC vector per 10 ⁶ mononuclear cells, sampling and each group of macaques. The mean and ^the calculated standard deviation have been rounded to the nearest unit.

The right and left nodes of each category (submaxillary, axillary, inguinal, internal iliac, external iliac) were removed after sacrifice of the animal, crushed (the right and left submandibular lymph nodes were pooled), then subjected to the analysis of antibody-producing cells by the ELISPOT technique (adapted from Eriksson K. et al., Journal of Immunological methods, 153: 107-113, 1992).

A systemic response in IgA and IgG antibodies has also been observed.

The results of counting the B lymphocytes secreting IgG are collated in the table which follows. Similar results have been obtained with IgA.

Example IV - Test 3

ELISA was assayed by IgA antibodies and IgG anti-gp160 serum and mucosal secretions (urine, mouth washes, vaginal and rectal) of the four monkeys injected sublingually of ^Example III. The samples were taken before immunization (S0), then after IO immunization just before the sacrifice of the animal (S14). The results are expressed for each secretion, as a ratio of ^the specific activity IgA or IgG anti-gp160 measured at sacrifice with respect to that measured prior to immunization. Secretion was considered to be significantly positive for specific IgA or IgG if this ratio was greater than 3.

I 5

Note: the specific activity of a secretion corresponds to the specific IgA or IgG titer (here gp160) divided by the total IgA or IgG titer.

Furthermore, the local or plasma origin of specific IgA or IgG in a secretion was evaluated according to two different techniques described below.

1. Measurement of the relative coefficient of excretion lg (A or G) with respect to ^the albumin (protein essentially plasma-derived), according to the technique described by Bélec L. et al., AIDS Research and Human Retroviruses, 1_2 : 157-5167, 1996.

2. measurement of the local production coefficient of lg (A or G), comparing the specific activity measured in the secretion with that in the serum taken at the same time, according to the technique described by Van Cott T. et al., Journal of Immunology, 160: 2000-2012, 1998.

30

The results are presented in the tables below and show the preferential production of IgA and IgG in the oral secretions, the origin of which is local (secretaries). Results

Example V - Bioadhesive

A quantity of bioadhesive material containing 10 ³ DICC50 of vCP205 and 400 μg of gp160 subunit (see the examples above) is prepared according to the method of Kriwet B. et al. (J Controlled Release, 1-3, 1998). For this, microparticles based on polyacrylate with a diameter of about 1 mm are synthesized. The system is stabilized by an emulsifier SpanTM 80 and TweenTM 80 dispersed in an oily layer. The initial polymerization medium therefore contains drops of emulsions and inverted micelles. The polymerization is initiated by radicals and allows the encapsulation of vCP205 and gp160 previously formulated also in liposomes. The final bioadhesive is disposed in the floor of a man's mouth in order to ^induce local immunity in IgA, IgG and / or IgM anti-gp160, and B cells secreting said antibodies in the biuccales mucous, secretions mouth and the lymph nodes that drain them, as well as systemic immunity for said antibodies and B cells. Example VI - Bioadhesive

A quantity of bioadhesive material containing 10 ³ DICC50 of vCP205 and 400 μg of gp160 subunit (see the examples above) is prepared according to the method of Li C. (Drug Dev Ind. Pharm, 919-926, 1998). For this, the immunogenic composition consisting solely of gp160 and of vCP205 is encapsulated in a polymer consisting of silicone and Carbopol 974P. The adhesive particles are also coated with hydroxyapatite crystals in order to facilitate penetration of the composition through the mucosa. The final bioadhesive is placed in the floor of a man's mouth in order to induce local immunity in anti-gp160 IgA, IgG and / or IgM antibodies, and in B cells secreting said antibodies at the level of the oral mucous membranes, secretions mouth and the lymph nodes that drain them, as well as systemic immunity for said antibodies and B cells.

Example VII - Capsules

Capsules are prepared based on starches and particles of hydroxyapatites containing lyophilized antigens of cytomegalovirus, hepatitis A, or pathogens conventionally transmitted by aerosol such as M. tuberculosis, N. meningitidis, Streptococcus type BS pneummaniae, B. pertussis, for example. These capsules are designed to dissolve slowly in the mouth. The hydroxyapatite crystals facilitate the penetration of the composition through the mucosa. A capsule is placed in the floor of a man's mouth in order to induce local immunity in anti-gp160 IgA, IgG and / or IgM antibodies, and in B cells secreting said antibodies at the level of the oral mucous membranes, of the oral secretions. and the lymph nodes that drain them, as well as systemic immunity for said antibodies and B cells.

Claims

claims

1. Use of specific immunogen from a pathogen having a gate ^input at the oral mucosa region, for the manufacture of a vaccine composition to be administered in the floor of the mouth in ^humans so to directly develop a local response in IgA antibodies and in IgA-secreting B cells in the buccal mucosa, saliva and ganglia draining said mucosa.

2. Use according to claim 1, characterized in that the immunogenic composition is intended to be administered by sublingual injection, by deposition, or by means of a bioadhesive. or a capsule releasing the immunogenic composition into the floor of the mouth.

3. Use according to claim 2, characterized in that the bioadhesive or the capsule is provided or coupled to a system promoting the penetration of the immunogenic composition.

4. Use according to claim 1, characterized in that the immunogenic composition is intended to also develop a systemic response in IgA, IgG and / or IgM antibodies.

5. Use according to claim 1, characterized in that the immunogenic composition is intended to develop a response in secretory IgA antibodies.

6. Use according to any one of claims 1 to 5, characterized in that the immunogenic composition is directed against a pathogen chosen from the group consisting of: the HIV virus, herpes viruses in particular herpes simplex, Candida, viruses of hepatitis in particular hepatitis A, Picornaviridae, in particular enteroviruses such as poliomyelitis virus, Reoviruses in particular Rotavirus, Adenoviruses, human Papillomavirus, periodontoses, cytomegalovirus, Epstein-Barr virus, all aerosol-transmitted pathogens such as M. tuberculosis, N. meningitidis, Streptococcus type B, S. pneumoniae and B. pertussis.

7. Vaccine composition, capable of being applied in the floor of the mouth in humans in order to induce local and systemic immunity in IgA antibodies, consisting substantially of an adhesive material to the oral mucosa and which contains at least one specific immunogen of a pathogen having a gateway to the oral mucosa region.

8. Composition according to claim 7, characterized in that the adhesive material is provided or coupled to a system promoting the penetration of the immunogen through the oral mucosa.

9. Vaccine composition, capable of being deposited in the floor of the mouth in humans in order to induce local and systemic immunity in IgA antibodies, consisting of a material non-adhesive to the oral mucosa which degrades on contact secretions and which contains at least one specific immunogen from a pathogen having a gate ^input at the oral mucosa region, characterized in that this material is provided with invasive elements promoting the penetration of the immunogen through the oral mucosa.