EP1089758A1 - Mucosal targeting immunisation - Google Patents

Abstract

The invention concerns the use of an immunogen specific of a pathogenic agent having a gateway in a mucous membrane for producing an immunogenic composition to be administered to a human by parenteral route at the surface of part of the body distinct from the mucous membrane so as to directly develop a local response in IgA, IgG and/or IgM antibody in said mucous membrane.

Description

Mucosal targeting immunization

The present invention relates to an immunization method for inducing a local immune response, in particular in the rectogenitourinary 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

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

As in the case of other pathogens (viruses, bacteria, etc.) with 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 the lymph nodes appear large.

Most of the work generally aims at obtaining a systemic immunity which is detectable by the titer in serum antibodies, as described by Jian-Ming X. et al. (vaccine, 993-1000, 1996), Kim J.J. 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 (FR2751879).

The targeting of local mucosal-type immunity can be demonstrated by the presence of specific antibodies contained in the mucous membranes or secretions (Thibodeau L. et al., Aids Research and Human Retrovirus, 1379, 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 an injection under - extremely deep skin in the pelvic region near the iliac nodes. This immunization results in the induction of antibodies of the IgA and IgG type in rectal and urinary fluids and in the serum. However, such an immunization method is not applicable to humans.

Letchworth G.J. 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. It therefore seems that intramuscular injection alone does not allow targeting of a local response.

Gaffar A. et al. (US3931398) teach that an injection of a vaccine into the oral cavity would induce local immunity. No antibody measurements are reported. It therefore seems that the proximity of the injection site to the place of targeting of local immunity is essential, as also reported by Letchworth G.J. et al. above.

There is therefore currently no mucosal immunization method targeting the recto-genitourinary region, which is really effective and usable in the current practice of human or veterinary medicine. Furthermore, there is nothing to suggest that an injection into sites far from the mucous membranes can establish a targeted local response.

Summary of the invention

In order to overcome the need for technical solutions to effectively and easily induce local immunity, the present invention thus aims at the use of specific immunogen of a pathogenic agent having an entry point at the level of a mucosa for the production of an immunogenic composition intended for administration to humans parenterally on the surface of a separate part of the body remote from said mucosa, so as to directly develop a local response in IgA, IgG and / or IgM antibodies and in B cells secreting said antibodies at the level of said mucosa and of the ganglia which drains it.

A particular objective of the invention relates to the targeting of the rectal, genital and / or urinary mucosa by parenteral administration in the thigh. Faced with this observation, we can now search for multiple sites injections distinct from the mucous membranes which will directly allow the induction of a local response in these mucous membranes, in particular in antibodies and in B cells secreting antibodies found in the mucous membranes and the ganglia which drain them.

Another 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 immunity, in particular humoral and / or cellular, detectable in the peripheral blood.

Detail of the invention

The present invention thus relates to the development of a local response, in particular at the level of the recto-genito-urinary mucosa and of the ganglia which drain it by parenteral injection of an immunogenic composition into a part of the body distinct from the mucosa, such as the thigh. Parenteral administration in the thigh and in the surrounding upper regions, in particular the groin, makes it possible to target the iliac and inguinal nodes. The intramuscular route is preferred in one or both of the lower limbs, particularly in the quadriceps, particularly in the anterior rectus muscle. Such an immunization pathway proves capable of locally inducing at the level of this mucosa on the one hand the production of immunoglobulins in the secretions and, on the other hand regionally, at the level of the ganglia draining this mucosa, the production of antibody secreting B cells, while inducing systemic immunity. This immunity is capable of inducing protection against the entry and spread of the pathogen considered from this recto-genito-urinary mucous region.

The invention applies both to the field of prophylaxis (e.g. 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 immunogen is of antigen type.

A first particular object of the invention is therefore the use of an immunogen specific for a pathogenic agent having an entry point at the level of the recto-genito-urinary mucous region, for the production of an immunogenic composition. intended to be administered to man parenterally in the thigh, preferably intramuscularly, especially in the quadriceps, in particular in the anterior rectus muscle (parenteral administration in a thigh muscle will preferably be in the muscle of each right and left lower limb), so as to develop a local response in IgA antibody, or even also in IgG and / or IgM antibody, and in B cells secreting IgG, IgM and / or IgA at the level of respectively on the one hand the recto-genitourinary mucous membranes and their secretions, and on the other hand the lymph nodes which drain this mucous membrane, in particular external and internal iliac nodes and inguinal nodes. Parenteral and in particular intramuscular injection into the thigh indeed appears to allow the recruitment of B cells producing IgG, IgM and / or IgA antibodies in the lymph nodes draining the recto-genito-urinary mucous region.

The production of antibodies IgA and IgG of plasma or secretory origin is thus reported, and the local recruitment of B cells secreting IgA and IgG as was observed in the tests reported in points III and V of the examples.

Among the pathogens to which the invention can be applied, there will be mentioned very particularly: the HIV virus, Herpes viruses, eg Herpes simplex, in particular type 2, Candida, Chlamydia, Human papillomavirus, Genital Mycoplasmas, Treponema pallidum, Papovaviruses, eg Condyloma accuminatum and gonococcal infections.

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 a systemic response, the two actions combining, and complementing, or even reinforcing, so particularly advantageous. Consequently, the use in accordance with the invention also aims to develop, in addition to a local response according to the invention, a systemic response of the IgG and / or IgM type, and optionally IgA (antibodies and secretory B cells).

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 secretory B cells, it is a specific response to the immunogen used. For other immunogens, the response may be in addition to or instead of a local cellular immune response

(cytotoxic T lymphocytes, TH1 and TH2 response, possibly suppressor). Another object of the invention is the method of immunization against pathogenic agents as described above, consisting in administering by any means known per se, the appropriate immunogenic composition by parenteral route, in particular intramuscular, in the thigh, one or both lower limbs, preferably quadriceps, particularly the anterior rectus muscle. 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 use.

In the context of the use and of the immunization method, it can also be specified that the invention applies to all types of immunogenic composition and in particular known vaccines, whether they are of the conventional type or of the recombinant type. As is known per se, the compositions, e.g. vaccines of the conventional type group together the compositions, e.g. whole live attenuated or inactivated vaccines, the subunits (proteins or peptides). They can be adjuvanted or non-adjuvanted and be presented in combined form bringing 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 considered as well as the polynucleotide plasmid vectors made up of DNA which can for example be naked or included in a liposome (see eg WO-A-90 11092, WO-A-93 19813, WO-A-94 21797, WO-A-95 20660) and which express one or more immunogens. As regards live recombinant vectors, there may be mentioned in particular as vectors the poxviruses, such as the vaccinia virus and especially the 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 the invention cannot be limited to a particular type of composition, eg vaccines, but is intended to apply to all types of immunogenic compositions, eg vaccines, and to all the compositions, eg vaccines, available which can be used parenterally, in particular intramuscularly.

Likewise, the immunization protocol will depend on the type of composition or the composition used. It will include the number of administrations usually used for a given composition 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 capable of routine to determine the optimum number of administrations (eg primary vaccination and booster). It should be noted, however, that injection into a site separate from the mucous membranes can alone target a local response, without the need for a reminder in the mucous membranes.

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

It is also possible to associate it with the same host an immunization protocol targeting the oral mucosa comprising the administration of an immunogenic composition against the same pathogen, identical or different, in particular of the same composition, aimed at inducing a local IgG response. , IgM and / or IgA and in B cells secreting IgG, IgM and / or IgA respectively in the saliva and the lymph nodes draining the oral mucosa, in particular the submaxillary lymph nodes (it can also be accompanied by systemic reaction), preferably by sublingual injection into the floor of the mouth. The test reported in points

III and IV show that a sublingual injection is an appropriate means, other routes of administration in the oral cavity remaining possible. One can of course use other means capable of properly activating the lymph nodes draining the oral mucosa, in particular the submaxillary lymph nodes.

Such a combination is particularly useful for preventing or treating infection by a pathogen having both the oral and recto-genitourinary entry points. Mention may in particular be made of the HIV virus, Herpesvirus, etc.

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 parenterally in the thigh, in particular intramuscularly in the thigh, preferably in the quadriceps, in particular in the anterior rectus muscle, and on the other hand the use of another immunogenic composition, identical or different from the previous one, intended to be administered to the same host by sublingual injection in the floor of the mouth.

The use and the immunization method in accordance with the invention find a preferred application in the context of vaccination against the HIV virus.

A particular example is the use of a vaccine combining a vector expressing gp120 / gp160 of HIV and of the gp120 / gp160 glycoprotein subunit of this same virus. A particular example is described below.

In the context of the present invention, it is thus planned to implement this anti-HIV vaccine for its administration by intramuscular route in the thigh, possibly combined with conventional sublingual and / or systemic administration, e.g. by intramuscular injection in the deltoid.

Finally, the subject of the invention is also an immunogenic composition comprising at least one immunogen specific for a pathogen having an entry point at the level of the recto-genitourinary mucous region and a pharmaceutically acceptable vehicle or excipient, this vehicle or excipient, or this composition, leading, in conjunction with the immunogen, to a local response in IgG, IgM and / or IgA antibodies and in B cells secreting IgG, IgM and / or IgA in this mucous region, when the composition is administered parenterally to the thigh, in particular intramuscularly. In the context of this immunogenic composition, the characteristics set out here with regard to the other objects of the invention can be used alone or in combination.

The invention will now be described in more detail using embodiments taken by way of nonlimiting examples. It should be understood that the invention defined by the appended claims is not limited to the particular embodiments 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 - Targeting of lymph nodes

The targeting of the nodes involved in recto-genito-urinary responses by the intramuscular route in the thigh is illustrated by experiments carried out on the cynomolgus macaque: in these experiments, a solution of Evans Blue dye at 2% in saline a was injected (0.5 to 1 ml) into the anterior rectus muscle at a site located one third of the groin and two thirds of the knee.

The dissection of the euthanized animals 4 hours after injection of the dye, allowed the identification of the lymph nodes draining the inoculation zone thanks to their blue coloration: inguinal and iliac nodes were thus identified. Example II - Vaccine

11-1 Live recombinant vaccine vCP205, ALVAC-HIV:

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. It 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 gene for the glycoprotein env gp120 MN (plus the transmembrane part of gp41 LAI) and the genes of the LAI strain coding for gag and for the 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 chicken embryos in DMEM-Ham F12 medium without serum, added with lactoglutamate and clarified by centrifugation. The mean titer was 10 ^8.0 TCID ₅ o ml on QT35 cells. The vaccine solutions are prepared by dilution in PBS ("phosphate buffer saline") with Ca ⁺⁺ and Mg ⁺⁺ .

II-2 gp160MN / LAI-2 subunit vaccine

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

A WTG9150 vaccine vector 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 using an SmaI artificial restriction site not modifying either of the two amino acid sequences of gp120 and gp41. The construction is briefly described below.

The sequence coding for gp120 MN was amplified from SupT1 cells infected with HIV-MN, by PCR technique with oligonucleotides 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 located between gp120 and gp41.

The sequence coding for gp41 was thus produced: the complete coding sequence for env HIV-1 LAI was placed under the control of the vaccinia virus promoter PH5R. Several changes have been made. A SphI 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 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 coding for the transmembrane hydrophobic peptide IFIMIVGGLVGLRIVFAVLSIV (amino acids 689-710 according to 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 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 cut with SphI and Smal, then linked to the sequence gp120 MN. WTG9150 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 III - Test 1

Two female rhesus macaques (P9224 and P9225) already immunized intramuscularly (in the left or right thighs, alternately) 2 times with 10 ⁶⁵ DICCso of ALVAC-HIV (vCP205) clarified, then 3 times with 100 μg of gp160 MN / LAI- 2 adjuvanted with OspA ("outer surface protein A" from Borrelia burgdorferi) and aluminum hydroxide, were inoculated 2 times 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 secretions and rectals as well as the serum were analyzed by ELISA to detect the presence of IgA and IgG anti-gp160 and anti-CPpp (against the canarypox virus itself).

One of the two monkeys (P9225) received an additional injection of the same mixture (vCP205 + gp160 MN / LAI-2) in the floor of the mouth as well as 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 CPpp.

It has been possible to show the appearance of anti-gp160 and anti-CPpp IgA in the mouthwash of the P9224 macaque, and of anti-gp160 IgA in the vaginal washing of the P9224 macaque. The specific anti-gp160 IgG responses appeared from the first injection in most of the secretions tested and were maintained throughout the study.

In addition, the sera of the two macaques showed a significant increase in the specific IgA and IgG of gp160 and CPpp.

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 inguinal and iliac nodes. 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 near lymph nodes draining the oral and recto-genitourinary mucous membranes.

Example IV - Test 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, at 1 month in the interval, an injection of the vaccine mixture sublingually into the floor of the mouth; mixture at equal volume of vCP205 at 10 ⁶ ' ⁹ DICC ₅₀ / ml and gp160 at 400 μg / 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 at equal volume from vCP205 to 10 ⁶ ' ⁶ DICC ₅₀ / ml and from gp160 to 200 μg / ml; 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 ₅₀ / 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, out and in each group of monkeys. The calculated mean and standard deviation have been rounded to the nearest unit.

The right and left lymph nodes of each category (submandibular, 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 Erikson K. et al., Journal of Immunological methods, 153: 107-113, 1992).

A systemic IgG antibody response has also been observed.

The results of counting the B lymphocytes secreting IgG are collated in the following table:

Example V - Test 3

The vaccine consists of two products injected separately, first of all vCP20) 55 to 1100 ⁶⁶⁵⁵ DDIICCC50, then of the gp160 MN / LAI subunit at 50 μg adjuvanted by a conventional adjuvant

2 female rhesus macaques (# P952 and # P9315) received:

- twice, at weeks 0 and 4, an injection of vCP205 intramuscularly into the thigh (perpendicular to, and into the anterior right muscle), to the right and then to the left alternately, at a dose of 10 ⁶⁵ DICC50 in one volume 1 ml.

- 5 times, in weeks 8, 12, 25, 99 (for P952) or 100 (for P9315) and 103 (for P952) or 104 (for P9315), an injection of gp160 MN / LAI adjuvanted intramuscularly in the thigh (perpendicular to, and in the anterior right muscle), right and left, at a dose of 50 μg (+ 500 μg of adjuvant) in a volume of 1 ml (0.5 ml in each thigh).

The number of B lymphocytes secreting IgG and IgA specific for gp160 was measured for 10 ⁶ mononuclear cells, in each sample (lymph nodes and blood) of the two macaques. The mean and standard deviation of triplicate counts for each sample are shown. 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 lymph nodes of each category 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).

The anti-gp160 antibodies IgA and IgG antibodies were also assayed by the ELISA technique in the serum and mucous secretions (urine, vaginal and rectal washings) of the two macaques. The samples were taken before the two late boosters (S99), then after immunization just before the animal was sacrificed (S104 or S105). The results are expressed, for each secretion, as the ratio of the specific anti-gp160 IgA or IgG activity measured at the time of sacrifice. compared to that evaluated before the two late recalls (S99). A secretion was considered to be significantly positive in specific IgA or IgG if this ratio was greater than 3 (the specific activity of a secretion corresponds to the specific IgA or IgG titer (here of gp160) divided by the titer in IgA or total IgG).

Furthermore, the local or plasma origin of specific IgA or IgG in a secretion was evaluated using two different techniques:

1. measurement of the relative excretion coefficient of lg (A or G) compared to albumin (protein of essentially plasma origin), according to the technique described by Bélec L. et al., AIDS Research And Human Retroviruses, 12 : 157-167, 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.

Results on antibody-secreting cells:

Results on immunoqlobulins present in secretions

IgA has also been detected in some urinary, vaginal and rectal secretions.

Example VI

Vaccines against Herpes simplex, Candida, Chiamydia, Human Papillomavirus, Genital Mycoplasma or Treponema pallidum have been injected into the thigh muscles in order to stimulate and target a local IgA antibody response at least in rectal, genital and / or urinary mucosa.

Claims

claims 1. Use of an immunogen specific for a pathogenic agent having an entry point at the level of a mucosa for the production of an immunogenic composition intended to be administered to humans parenterally on the surface of a part from the body distinct and distant from said mucosa, so as to directly develop a local response in antibodies IgA, IgG and / or IgM and in B cells secreting said antibodies at the level of said mucosa and of the ganglia which drains it. 2. Use according to claim 1 characterized in that the mucosa consists of the rectal, genital and / or urinary mucosa, and that the administration by parenteral route is carried out in the thigh. 3. Use according to claim 1, characterized in that secretory IgA is developed. 4. Use according to claim 1, characterized in that one develops a response in B cells secreting IgG, IgM and / or IgA at the level of the rectal, genital and / or urinary mucous membranes respectively and of the lymph nodes which drain them. 5. Use according to claim 2, characterized in that the immunogenic composition is intended to be administered intramuscularly into the thigh. 6. Use according to claim 5, characterized in that the immunogenic composition is intended to be administered in the / quadriceps, in particular the / anterior right muscles. 7. Use according to one of claims 1 to 5, characterized in that the immunogenic composition is intended to also develop a systemic response. 8. 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, Chiamydia, Human Papillomavirus, Genital Mycoplasma, Treponema pallidum, and gonococcal infections. 9. Use according to one of claims 1 to 8, in combination with the use of another immunogenic composition, identical or different from the previous one, intended to be administered in the oral cavity to the same subject so as to induce furthermore, an IgG and / or IgA antibody response and also IgG and / or IgA secreting B cells in the oral mucosa and the lymph nodes which drain it.