EP4313141A1

EP4313141A1 - Novel use of an immunogenic or vaccinal composition against covid-19

Info

Publication number: EP4313141A1
Application number: EP22717147.7A
Authority: EP
Inventors: Norbert Schrage; Frank Holzer; Joël Blomet; Daniel Desmecht
Original assignee: Eye Vacc
Current assignee: Eye Vacc
Priority date: 2021-03-22
Filing date: 2022-03-22
Publication date: 2024-02-07
Also published as: US20240165219A1; BR112023019270A2; WO2022200384A1; AU2022245195A1; MX2023011173A

Abstract

The present invention relates to an anti-SARS-CoV-2 immunogenic or vaccinal composition for use in the treatment of COVID-19, the composition being characterised in that it is administered to the ocular mucosa and/or the urogenital mucosa.

Description

NEW APPLICATION OF AN IMMUNOGENIC OR VACCINE COMPOSITION AGAINST

COVID-19

In addition to the systemic immune system, our body contains a mucosa-associated immune system (also called MALT, Mucosa Associated Lymphoid Tissue). The mucous membranes locally contain their own immune system, for example in the digestive tract (GALT, Gut Associated Lymphoid Tissue), in the nose (NALT, Nasal Associated Lymphoid Tissue), or even in the eye (CALT, Conjunctiva Associated Lymphoid Tissue).

Indeed, the sites of entry of pathogens into the body are generally located in the mucous membranes of the eye, nose, mouth or gastrointestinal tract. Our body thus contains a large number of cellular and biochemical defense mechanisms, directly at the level of these mucous membranes, which are activated on contact with pathogens. Typically the mucosal immune system includes epithelial cells, innate immune cells and dendritic cells which are at the interface between innate immunity and specific (acquired) immunity. The mucous membranes can thus contain cells of innate immunity, immunocompetent cells, memory cells and antibody-producing cells.

After surviving a primary infection with a pathogen, the body develops systemic (serum) immunity against that agent. On the other hand, if the pathogen has been in contact with the mucous membranes of the body, mucosal immunity is also developed, in addition to systemic immunity.

Vaccines to develop mucosal immunity are already known. These are mainly administered nasally or orally. They offer the singular advantage of inducing a protective immune response, both at the mucosal level and at the systemic level. They also aim to prevent the crossing of the mucous membranes, the entry point for most bacterial and viral pathogens. This is for example the case of the flu vaccine FluMist® which is administered by nasal spray, or the vaccines against poliomyelitis which are administered orally. Vaccination against an influenza virus (influenza A virus), by the ocular route, in ferrets was also studied in the article Eyedrop Vaccination Induced Systemic and Mucosal Immunity against Influenza Virus in Ferrets by Sangchul Yoon, Eun-Do Kim, Min-Suk Song, Soo Jung Han, Tae Kwann Park, Kyoung Sub Choi, Young-Ki Choi, and Kyoung Yul Seo; PLoS One. 2016; 11 (6): e0157634. Ocular vaccination was also studied in mice by the Kyoung Yul Seo, Soo Jung Han, Hye-Ran Cha, Sang-Uk Seo, Joo-Hye Song, So-Hyang Chung, and Mi-Na Kweon team ( Eye Mucosa: An Efficient Vaccine Delivery Route for Inducing Protective Immunity; J Immunol 2010; 185:3610-3619), although mice are not natural hosts of the virus.

In the case of highly contagious mucosal-colonizing pathogens, the pathogen multiplies so rapidly after colonizing the mucous membranes that the infection of the mucous membranes and deeper layers progresses faster than the organism cannot establish a effective immunity to eliminate the pathogen. This is typically the case with the current coronavirus infection. 2 of severe acute respiratory syndrome (SARS-CoV-2 or Severe Acute Respiratory Syndrome CoronaVirus 2). Indeed, Covid-19 (or coronavirus disease 2019) mainly presents with infection and inflammation of the mucous membranes with secondary serious general illness (as is also the case with influenza, herpes or poliomyelitis).

The current Covid-19 pandemic has given rise to unprecedented research, in particular in order to develop vaccines, and to date, several anti-Covid-19 vaccines have already been authorized to vaccinate the world population. Such vaccines are for example (1) the Pfizer/BioNTech mRNA vaccine, generally referred to as Bnt162b2 or Comirnaty®; (2) Moderna's mRNA vaccine, commonly referred to as Moderna mRNA-1273, Spikevax, or COVID-19 Vaccine Moderna; (3) AstraZeneca's non-replicating viral vector vaccine, commonly referred to as Vaxzevria®, ChAdOx1-S or COVID-19 Vaccine AstraZeneca; (4) the non-replicating viral vector vaccine from Janssen/Johnson & Johnson, generally referred to as Ad26COV2.S, JMJ Vaccine or J&J COVID-19; (5) Gamaleya's non-replicating viral vector vaccine, generally referred to as Sputnik V or Gam-COVID-Vac; (6) the whole inactivated Covid-19 vaccine with adjuvant from Sinovac R&D, generally referred to as CoronaVac; or (7) the adjuvanted recombinant nanoparticle subunit vaccine (Matrix M) from Novavax, generally referred to as Nuvaxovid or NVX-CoV2373.

All currently available Covid-19 vaccines are administered intramuscularly. They thus generate a good systemic immunity, making it possible to limit the severe evolutions of the disease, but they do not make it possible to develop the immunity of the mucous membranes. However, this mucosal immunity is essential for the prevention of new infections, otherwise the risk of infection with SARS-CoV-2 will remain high.

Moreover, the quantities of vaccines currently available are still too low, and the possible secondary risks of these vaccines (such as the rare cases of thrombosis) are also a source of concern for some people, who are therefore reluctant to be vaccinated.

There is therefore still a need to improve the vaccination of the world population against Covid-19, thus allowing the exit from this pandemic crisis.

The present invention meets this need thanks to a new method of vaccination against Covid-19 which allows an improvement in immunity (the double immunity -systemic and of the mucous membranes- in fact provides better protection against the pathogen, and in so doing contributes to the development of herd immunity), while reducing the risk of serious side effects linked to vaccination. The present invention thus makes it possible to develop immunoglobulins of the IgM type then of the IgG type, but especially immunoglobulins of the secretory IgA type (slgA on the mucous membranes). The present invention also aims to provide a new mode of vaccination against Covid-19 which is as effective as possible.

The present invention also aims to provide a new method of vaccination against Covid-19, which is simpler to implement and faster, in order to increase the vaccination rates. The present invention makes it possible to develop mucosal immunity against SARS-CoV-2, by targeting the ocular mucosa and/or the uro-genital mucosa. The present invention also aims to develop a so-called sterilizing immunity. Indeed, one of the objectives of the present invention is to target the mucous membranes, in order to mainly develop immunoglobulins of the IgA type (monomeric IgAs, but also dimeric IgAs and secretory IgAs on the mucous membranes), in particular with the aim of blocking the replication virus in the mucous membranes of the respiratory tract (which is possible using immunoglobulins of the IgA type and not of the IgG type).

The present invention thus relates to an immunogenic or vaccinal composition against SARS-CoV-2, for its use in the prevention and/or treatment of Covid-19, characterized in that it is administered to the ocular mucosa and/or the urogenital mucosa.

The advantage of the administration of such a composition is to establish both mucosal immunity and serum immunity against SARS-CoV-2. This double immunity makes it possible to better protect the body against the virus and limits the risk of infections, without suffering the serious systemic side effects which can be observed with certain vaccines currently injected intramuscularly (in particular the rare cases of thromboses). Indeed, in the case of the present invention, given that serum immunity is acquired indirectly via immunization of the mucous membranes, a thrombotic accident is therefore not expected.

The expression “immunogenic composition” and/or “vaccine composition” according to the invention means a composition which induces an immune response against SARS-CoV-2 after administration to the subject. A vaccine composition makes it possible in particular to generate immunity, more particularly a protective and adaptive immune response against SARS-CoV-2. This immune response can be humoral and/or cellular. This immunity also aims to be immunizing.

The term “SARS-CoV-2” according to the invention means the virus belonging to the Coronaviridae family, to the genus Betacoronavirus, and to the subgenus Sarbecovirus. It is an enveloped virus with a helical capsid whose genome consists of single-stranded RNA of approximately 30,000 nucleotides. The structure of the SARS-CoV-2 virion includes in particular a helical capsid formed of protein N, a matrix formed of protein M and a lipid envelope in which at least two types of protein are embedded: (glyco)protein S (spike) and small envelope protein (E) (and optionally hemagglutinin esterase (HE)). Protein S and variants is the main target of the immune response by production of neutralizing antibodies. This surface protein binds to the ACE2 receptor (which is expressed in many tissues), which allows the virus to enter the cells of the infected body. Protein S contains two subunits, S1 and S2. S1 includes the Receptor Binding Domain (RBD) which contains the Receptor Binding Motif (RBM). The S2 subunit contains a fusion peptide which allows the fusion between the cell membrane of the host cell (of the contaminated organism) and the viral envelope.

More particularly, the term “SARS-CoV-2” according to the invention means the virus whose sequence was initially described in GenBank, under the accession code MN908947, as well as all the variants of this virus, in particular the so-called English variant, so-called South African variant, so-called Indian variant or the so-called Brazilian/Japanese variant. In general, this includes the so-called Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1. 529). This also includes variants B.1.640, B.1.427, B.1.429, B.1.616, B.1.525, P.3, B.1.617.2, B.1.620, B.1.617.3,

B.1.214.2, A.23.1, A.27, A.28, C.16, B.1.351, B.1.526, B.1.526.1, B.1.526.2, P.2, B.1.1.519, AV.1, AT.1,

C.36, B.1.621, C.37, AY.4.2, B.1.1.318, B.1.617.2, C.1.2 or BA.2. Information regarding SARS-CoV-2 sequences, mutations and variant sequences can be found, for example, at the following links: https://www.ncbi.nlm.nih.gov/sars-cov-2/ ; https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/variant-surveillance/variant-info.html; https://www.ecdc.europa.eu/en/covid-19/variants-concern; or https://www.gisaid.org/.

The expression “the prevention of Covid-19” according to the invention means prophylaxis. The administration of the immunogenic or vaccine composition according to the invention makes it possible in particular to prevent or reduce the risk of developing Covid-19, and/or reduces the risk of developing so-called severe forms of the disease (i.e. i.e. with serious symptoms such as difficulty breathing, and/or requiring hospitalization, often in the intensive care unit). The administration of the immunogenic or vaccinal composition according to the invention could also prevent or reduce the risk of transmission of the virus, typically from one person to another. According to a particular embodiment, the immunogenic or vaccine composition for the prevention of Covid-19 according to the invention is administered to a subject not being contaminated, preferably not being infected, by SARS-CoV-2 .

The expression “the treatment of Covid-19” according to the invention means therapy. The administration of the immunogenic or vaccinal composition according to the invention can indeed be envisaged, in order to stimulate the natural defenses of the body, even when the person is at least already contaminated by the virus. According to a particular embodiment, the immunogenic or vaccine composition for the treatment of Covid-19 according to the invention is administered to a subject being contaminated, and/or being infected, by SARS-CoV-2.

Preferably, the present invention relates to an immunogenic or vaccinal composition against SARS-CoV-2, for its use in the prevention of Covid-19, characterized in that it is administered to the ocular mucosa and/or the uro mucosa -genital.

The expression “the ocular mucosa” according to the invention means the conjunctiva and the cornea. The conjunctiva is the transparent mucous membrane that lines the inner surface of the upper and lower eyelids and covers the anterior surface of the eyeball. More specifically, the ocular mucosa therefore includes both the tarsal conjunctiva and the bulbar conjunctiva, as well as the conjunctival culs-de-sac.

The expression "the uro-genital mucosa" according to the invention means the mucous membranes of the urinary and/or genital tract, both the male and the female tract. More precisely, the urogenital mucosa therefore means the urogenital tract.

Targeting the ocular mucosa and/or the urogenital mucosa makes it possible to target mucous membranes which (i) only participate in themselves to a small extent in the spread of the pathogenic agent, but (ii) which present in at the same time a very high immunocompetence, which makes it possible to immunize the organism very quickly, in particular before an infection of the respiratory tract. One of the hypotheses, non-limiting, of the inventors is indeed that an infection of the eye by SARS-CoV-2 leads to the rapid formation of immunity, allowing the body to gain time, so as to that when contamination and/or infection progresses to the nose, the body is already immune. This progression of contamination and/or infection from the eye to the nose could be explained by the presence of the nasolacrimal duct which connects the nose to the eyes. An infection through the nasolacrimal duct has already been described in the case of keratitis caused by APC viruses (Adeno-Pharyngo Conjunctivales, Adenovirus type keratitis epidemica). The typical course of this disease begins with infection of the conjunctiva with droplets and manifests as severe conjunctivitis followed by pharyngitis. This could also explain why subjects, including medical personnel, developed systemic immunity to SARS-CoV-2 after developing SARS-CoV-2 positive conjunctivitis.

Preferably, said immunogenic or vaccinal composition is administered to the ocular mucosa.

According to an even more preferred embodiment, the present invention relates to an immunogenic or vaccinal composition against SARS-CoV-2, for its use in the prevention of Covid-19, characterized in that it is administered to the ocular mucosa .

According to the invention, said immunogenic or vaccinal composition can be administered to one or both eyes, preferably to both eyes. Unlike intramuscular administration of a vaccine, which requires organization and increased medical effort (e.g. preparation of syringes, need for resuscitation equipment in the event of a serious reaction to vaccination, etc.), the injection on an ocular mucosa, typically by eye drops, is much simpler and quicker to implement. In fact, the serious risks are reduced (for example the risks of thrombosis as indicated above), as well as the serious allergic risks which are manifested mainly by itchy eyes, watery eyes or even red eyes. Vaccination by eye drops also makes it possible to vaccinate much more quickly and on a larger scale.

According to one embodiment, said immunogenic or vaccinal composition can be administered to one or both eyes as the first dose of the vaccination schedule, or else as a booster dose. According to one embodiment, said immunogenic or vaccinal composition is administered to the ocular mucosa and/or the urogenital mucosa before or after at least one administration of an immunogenic or vaccinal composition by intramuscular route. Preferably, said immunogenic or vaccinal composition is administered to the ocular mucosa after at least one administration of an immunogenic or vaccinal composition by intramuscular route. Preferably, said vaccine composition is administered to the ocular mucosa before or after at least one administration of a vaccine composition by the intramuscular route. Typically, this means that the subject is vaccinated a first time intramuscularly then that the booster dose or doses are carried out by administration into the mucous membranes, in particular the eyes. This may also mean that the subject is vaccinated a first time intramuscularly, that one or more booster doses are also given intramuscularly, then that one or more additional booster doses are given by administration into the mucous membranes, in particular eyepieces. The administration of an immunogenic or vaccine composition on the ocular mucosa and/or the urogenital mucosa after one or more intramuscular injections of a vaccine may be of interest if new booster doses are desirable to target specific variants of the SARS-CoV-2 virus. In general, the administration to the ocular mucosa and/or the urogenital mucosa of an immunogenic or vaccinal composition according to the invention after at least one administration of an immunogenic or vaccinal composition by intramuscular route aims to potentiate acquired immunity after the first intramuscular administration(s). This also means that the first administration of the immunogenic or vaccinal composition can be carried out on the ocular mucosa and/or the urogenital mucosa, and that the booster dose(s) can be carried out on these same mucous membranes or else intramuscularly.

According to one embodiment, said immunogenic or vaccine composition comprises one or more substances making it possible to provoke an immune reaction against SARS-CoV-2. Typically, said immunogenic or vaccine composition according to the invention comprises one or more SARS-CoV-2 antigens. Preferably, the said antigen(s) are specific for SARS-CoV-2.

According to one embodiment, the immune or vaccine composition makes it possible to provoke an immune response against SARS-CoV-2, provided that it comprises at least one SARS-CoV-2 antigen, a microorganism (for example a virus or bacterium) that can produce at least one SARS-CoV-2 antigen, or that it comprises the necessary genetic material allowing the expression of at least one SARS-CoV-2 antigen (typically an mRNA) . In the first case, the antigen will be in contact with the mucous membrane directly upon administration and in the second and third cases, a latency period may be expected, the time for the antigen to be produced after administration of the composition. Preferably, said microorganism is not pathogenic per se, the only immune reaction that it can cause is that linked to the SARS-CoV-2 antigen.

According to a particular embodiment, the substances/antigens making it possible to provoke an immune reaction against SARS-CoV-2 are chosen from: (i) an inactivated SARS-CoV-2 virus, (ii) a SARS-CoV-2 virus attenuated, (iii) a modified, preferably inactivated or attenuated, SARS-CoV-2 virus expressing or capable of expressing one or more antigens (such as a SARS-CoV-2 viral protein) of SARS-CoV-2, ( iv) a genetically modified microorganism (eg bacterium or virus, preferably inactivated or attenuated) expressing or capable of expressing one or more SARS-CoV-2 antigens (such as a SARS-CoV-2 viral protein), ( v) a messenger ribonucleic acid (mRNA) encoding one or more SARS-CoV-2 antigens (such as a SARS-CoV-2 viral protein), (vi) a deoxyribonucleic acid (DNA) encoding one or more SARS-CoV-2 antigens (such as a SARS-CoV-2 viral protein), (vii) a SARS-CoV-2 viral protein or one or more fragments thereof, or (viii) a a fusion protein comprising a viral protein of SARS-CoV-2 or one or more fragments thereof, ... Substances/antigens capable of inducing an immune reaction against SARS-CoV-2 can also be (ix) a SARS-CoV-2 virus, i.e. the 'live', non-inactivated or non-attenuated virus, or (x) a recombinant cell (for example a dendritic cell) expressing or capable of expressing one or more antigens of SARS-CoV-2 or (xi) a DNA plasmid encoding one or more SARS-CoV-2 antigens (such as a SARS-CoV-2 viral protein) or (xii) virus-like particles comprising one or more SARS-CoV-2 antigens. According to a particular embodiment, the substances/antigens making it possible to cause a immune reaction against SARS-CoV-2 are a genetically modified microorganism (including an adenovirus) expressing or capable of expressing one or more SARS-CoV-2 antigens (such as a SARS-CoV-2 viral protein) or a SARS-CoV-2 virus, preferably a genetically modified microorganism (in particular an adenovirus) expressing or capable of expressing one or more SARS-CoV-2 antigens (such as a viral vector). A fragment of a viral protein preferably contains the immunodominant or biosimilar epitope(s) thereof. According to one embodiment, the fragment of a viral protein is an immunogenic fragment. Said fragment and said viral protein can be recombinant.

According to a particular embodiment, the substances/antigens making it possible to provoke an immune reaction against SARS-CoV-2 are chosen from: (i) an inactivated SARS-CoV-2 virus, (ii) a SARS-CoV-2 virus attenuated, (iii) a modified, preferably inactivated or attenuated, SARS-CoV-2 virus expressing or capable of expressing one or more antigens (such as a SARS-CoV-2 viral protein) of SARS-CoV-2, ( iv) a genetically modified micro-organism (e.g. bacterium or virus, preferably inactivated or attenuated) expressing or capable of expressing one or more SARS-CoV-2 antigens (such as a SARS-CoV-2 viral protein), ( v) a messenger ribonucleic acid (mRNA) encoding one or more SARS-CoV-2 antigens (such as a SARS-CoV-2 viral protein), (vi) a deoxyribonucleic acid (DNA) encoding one or more SARS-CoV-2 antigens (such as a SARS-CoV-2 viral protein) or (vii) a SARS-CoV-2 virus.

The composition according to the invention may comprise one or more substances (antigens) making it possible to provoke an immune reaction against SARS-CoV-2. According to one embodiment, the different substances (antigens) can target different parts of the same virus (for example different epitopes on viral proteins), different strains of the same virus, variants of the same virus, or even several different viruses (combined vaccine ). The immunogenic or vaccinal composition according to the invention can thus be monovalent or polyvalent. A monovalent immunogenic or vaccine composition protects against a single pathogen, while the polyvalent immunogenic or vaccine composition protects against multiple pathogens.

According to one embodiment, the expression “a viral protein of SARS-CoV-2” means more particularly the structural proteins and the accessory proteins of the virus, preferably the structural proteins. Thus, according to one embodiment, said viral protein is chosen from: the S, HE, M, N or E proteins or the ORF proteins, more particularly, ORF1a, ORF1b, ORF3, ORF6, ORF7a, ORF7b, ORF8a, ORF8b . Preferably, said viral protein is chosen from the S, HE, M, N or E proteins, more particularly the S, HE, M or E proteins, and even more particularly the S protein. An immunogenic fragment of the S protein is more particularly the receptor binding domain (RBD), the S1 subunit or the cleavage region between the S1 subunit and the S2 subunit. According to the invention, the viral proteins mean the native proteins of the virus (the proteins of the virus as found in nature), mutated proteins or variants with respect to the native proteins or synthesized proteins (modified, mutated or not) .

The immunogenic or vaccinal composition according to the invention is administered to a subject. According to one embodiment, said immunogenic or vaccine composition is used in the prevention and/or treatment of Covid-19 in humans. According to one embodiment, said composition immunogen or vaccine is used in the prevention and/or treatment of Covid-19 in a child or an adult, in particular an adult. According to the invention, an adult means a person from the age of 16, preferably a person from the age of 18. Even more preferably, the adult has an age greater than or equal to 18 years and up to 55 years. According to one embodiment, said immunogenic or vaccine composition can be administered to a subject who is at least 12 years old. According to one embodiment, said immunogenic or vaccine composition can be administered to a subject who is at least 5 years old. According to one embodiment, said immunogenic or vaccine composition is also used in the prevention and/or treatment of Covid-19 in an infant and a person over the age of 55.

According to another embodiment, the veterinary applications of said immunogenic or vaccinal composition according to the invention are envisaged. In such a case, the subject is thus an animal.

According to one embodiment, said immunogenic or vaccine composition is administered to a subject in an immunologically effective amount. Such an amount can be determined by the practitioner. The expression “an immunologically effective amount” means an amount sufficient to be effective from a preventive and/or therapeutic point of view, in particular in a subject in need of such prevention or such treatment. By way of example, the dosage of the vaccine composition could be between 0.005 ml and 0.05 ml, for each of 1 to several epiocular drops or subconjunctival injections spaced at least one week apart, preferably between 2 to 12 weeks, for the non-replicating viral vector vaccine from AstraZeneca, the Sputnik V vaccine, the vaccine from Johnson & Johnson, the inactivated anti-COVID-19 vaccine from Sinovac, or the Nuvaxovid vaccine from Novavax.

According to one embodiment, said immunogenic or vaccine composition is administered to a subject who has never been contaminated, or even infected, with SARS-CoV-2 or else to a subject who has already been contaminated, or even infected, with SARS- CoV-2 (whether the person developed symptoms (mild or severe) or was asymptomatic).

According to one embodiment, said immunogenic or vaccinal composition is administered once or several times, preferably one, two or three times, to the ocular mucosa and/or the uro-genital mucosa. According to a particular embodiment, said immunogenic or vaccinal composition is administered at least twice to the ocular mucosa and/or the urogenital mucosa (in other words two doses of immunogenic or vaccinal composition are administered). According to a particular embodiment, when the immunogenic or vaccine composition is administered several times, it is the same mucosa which is targeted: for example two or three administrations to the ocular mucosa or two or three administrations to the urogenital mucosa. Preferably, a period of at least 1 week separates the first and the second administration, more particularly a period of between 4 to 12 weeks. The expression "between 1 to 12 weeks" means (days from 7 to 84 days) 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks, i.e. between 7 to 84 days.

According to one embodiment, said immunogenic or vaccine composition is administered in the form of a drop, lyophilisate or other dry form. According to one embodiment, said immunogenic or vaccine composition can also be administered in the form of a dried composition, powder, gel, nanoparticle or ointment. Typically, when the administration is in the form of a drop, this means that said immunogenic or vaccine composition is a liquid formulation, and when the administration is in the form of a lyophilisate or dried composition, said immunogenic or vaccine composition is in powder form. Said powder can be applied directly to the mucous membranes, or else be applied to a filter paper, a polymer, a gel, a nanoparticle or an ointment or any other suitable substance or support which will then be brought into contact with the ocular mucosa and/or or the urogenital mucosa. The immunogenic or vaccine composition will thus be transferred from the filter paper, polymer, gel or any other suitable substance or support to the ocular and/or uro-genital mucosa. Said dried composition can be obtained after freezing the immunogenic or vaccinal composition according to the invention and then dried using any appropriate technique (with the exception of freeze-drying which makes it possible to obtain a lyophilisate).

According to one embodiment, said immunogenic or vaccine composition is administered using an applicator, for example which facilitates the instillation of the drops (of the pouring aid type) or of the powder, of the lyophilisate. The applicator can also be an ampoule, a syringe, a filter paper (for example with a fluid or a lyophilisate), a drop bottle, a single dose applicator, a spiral or vaginal sponge type applicator or even a tissue, a sanitary pad comprising a modified outer surface (i.e. said surface comprising one or more substances making it possible to provoke an immune reaction against SARS-CoV-2, such as a protein, a viral vector, etc.), or a condom comprising a modified outer surface, ...

According to one embodiment, the immunogenic composition according to the invention comprises or consists of one or more substances (antigens) making it possible to cause an immune reaction against SARS-CoV-2. According to one embodiment, it may be considered that the immunogenic composition according to the invention is included in a vaccine composition (a vaccine). The term "vaccine" or "vaccine against Covid-19" can also be used instead of the expression "vaccine composition". According to one embodiment, the immunogenic or vaccine composition according to the invention comprises (1) the Pfizer/BioNTech mRNA vaccine, generally called Bnt162b2 or Comirnaty®; (2) Moderna's mRNA vaccine, generally referred to as Moderna mRNA-1273 or COVID-19 Vaccine Moderna; (3) AstraZeneca's non-replicating viral vector vaccine, commonly referred to as Vaxzevria®, ChAdOx1-S or COVID-19 Vaccine AstraZeneca, or its equivalent Covishield®; (4) Janssen's non-replicating viral vector vaccine, commonly referred to as Ad26COV2.S, JMJ Vaccine, or J&J COVID-19; (5) Gamaleya's non-replicating viral vector vaccine, generally referred to as Sputnik V or Gam-COVID-Vac or its light version called Sputnik light; (6) the whole inactivated Covid-19 vaccine with adjuvant from Sinovac R&D, generally referred to as CoronaVac; (7) the adjuvanted recombinant nanoparticle subunit vaccine (Matrix M) from Novavax, generally referred to as Nuvaxovid, Covovax or NVX-CoV2373; (8) Medicago's SARS-CoV-2 spiked protein virus-like particle vaccine, commonly referred to as CoviFenz®; (9) Baylor College of Medicine/BioE Limited protein subunit vaccine, commonly referred to as Corbevax® or BioE COVID-19; (10) Cadila Healthcare's DNA plasmid vaccine, generally referred to as ZyCoV-D; or (11) Bharat Biotech's inactivated virus vaccine, generally referred to as Covaxin®. According to a preferred embodiment, said vaccine composition comprises an adjuvant and/or an excipient.

According to one embodiment, said immunogenic or vaccine composition comprises a pharmaceutically acceptable vehicle.

The adjuvant and/or the excipient and/or the pharmaceutically acceptable vehicle are those conventionally used. For example, the pharmaceutically acceptable vehicle can be a solvent or a solution making it possible to dilute the composition before administration by the ocular route or on the uro-genital mucosa.

According to one embodiment, an immunogenic or vaccine composition according to the invention may also comprise one or more additional substances. Preferably, said additional substance is chosen from: a preservative, a marker, such as a dye, a surfactant, an additive.

According to one embodiment, the immunogenic or vaccine composition according to the invention is characterized in that it is administered after the administration of a first marker (such as a dye) and before the administration of a second marker (such as a dye). The immunogenic or vaccinal composition according to the invention is thus administered between two markers. The administration of these two markers, preferably different, makes it possible to verify that the immunogenic or vaccinal composition according to the invention has been correctly administered to the mucosa, in particular ocular.

By way of example, a preservative can be an antimicrobial preservative which aims to prevent microbial contamination of the immunogenic or vaccine composition. According to the invention, the preservative is a preservative compatible with the mucous membranes.

According to the invention, a “marker” means any measurable or indicator substance which can be administered in an immunogenic or vaccine composition. It is therefore a pharmaceutically acceptable marker. The marker also makes it possible to verify the quantity administered and/or the site of administration, in order to ensure safe and effective application of the immunogenic or vaccine composition.

According to a particular embodiment, the marker makes it possible to quantify and/or visualize the application of said immunogenic or vaccinal composition to the ocular mucosa and/or the uro-genital mucosa. More precisely, the quantification means the measurement of the quantity of immunogenic or vaccinal composition administered. According to one embodiment, the marker is an indicator substance such as a dye. By way of example, dyes of the fluorescein or indocyanine type can be used. This marker serves in particular to monitor the application of the immunogenic or vaccinal composition to the ocular and/or uro-genital mucosa. The dye can also make it possible to follow the dissolution of the immunogenic or vaccinal composition on the ocular and/or uro-genital mucosa. Visualization of the marker (such as a dye) on the ocular and/or urogenital mucosa makes it possible to ensure that the immunogenic or vaccine composition has indeed been applied to the ocular and/or urogenital mucosa, or even the quantity of immunogenic or vaccinal composition administered.

In order to obtain a high antigenicity and therefore a high immunological response, substances, for example surfactants, can be added to the immunogenic or vaccine composition, in order to prolong the exposure time on the ocular and/or urogenital mucosa. , and optionally make it possible to calculate the exposure time of said composition on the mucosa.

Other substances, such as additives, can be added to confer advantageous properties on the immunogenic or vaccine composition, for example a substance improving the penetration of the immunogenic or vaccine composition into the mucosa or a substance making it possible to prolong the time of contact between the mucosa and the composition (for example hyaluronic acid, one or more polymers to form a hydrogel (e.g. carbomers), cellulose derivatives, etc.).

The present invention also relates to a method for preventing and/or treating Covid-19 in a subject comprising the administration of an immunogenic or vaccinal composition against SARS-CoV-2 on the ocular mucosa and/or the uro mucosa. -genital. More particularly, the present invention relates to a method for inducing a protective immune response against SARS-CoV-2, comprising the administration of an immunogenic or vaccine composition to the ocular mucosa and/or the urogenital mucosa.

The present invention also relates to the use of an immunogenic or vaccinal composition against SARS-CoV-2, for the preparation of a medicament intended for the prevention and/or treatment of Covid-19, and said medicament being intended to be administered to the ocular mucosa and/or the urogenital mucosa.

FIGURES

Figure 1 represents the results of group 1 of hamsters.

Figure 2 represents the results of group 2 of hamsters.

Figure 3 depicts the Penh whole body plethysmographic data of all groups prior to viral exposure (12 hours).

Figure 4 depicts the Penh whole body plethysmographic data of all groups 3 days after exposure to the SARS-CoV-2 virus.

Figure 5 depicts the Penh whole body plethysmographic data of all groups 5 days after exposure to the SARS-CoV-2 virus.

Figure 6 depicts the Penh whole body plethysmographic data of all groups 7 days after exposure to the SARS-CoV-2 virus.

Figure 7 depicts the Penh whole body plethysmographic data of all groups 12 days after exposure to the SARS-CoV-2 virus.

Figure 8 depicts the Penh whole body plethysmographic data of all groups 14 days after exposure to the SARS-CoV-2 virus. Figure 9 represents the maximum dilution which always inhibits SARS CoV-2 infection in 50% of cases (NT50) in the serum of the different groups of hamsters.

Figure 10 represents the average weight of the hamsters in the four groups tested in Example 5.

EXAMPLES

Example 1 Examples of Liquid Compositions Table 1 below summarizes compositions according to the invention which can be used.

Table 1: Liquid compositions

Example 2: Examples of freeze-dried compositions

Table 2 below summarizes compositions according to the invention which can be used.

Table 2: Lyophilized compositions

Example 3: Immunization of hamsters against SARS-CoV-2 by the ocular route

Hamsters have the ACE2 receptor. They can thus be contaminated with SARS-CoV-2 and become ill.

1. Comparison of two groups of hamsters after two virus injections Two groups of hamsters were studied.

In group 1, n=7 hamsters were contaminated with SARS-CoV-2 by nasal injection of 3x10 ⁶ of a SARS-CoV-2 virus (strain B.1.214) and in group 2, n=7 hamsters were contaminated with eye drops containing 3x10 ⁶ SARS-CoV-2 virus (strain B.1.214).

During the observation, the hamsters of group 1 fell ill, recognizable by significant weight loss, while the hamsters of group 2 did not fall ill.

Indeed, we can observe in Figure 1 that the hamsters of group 1 began to lose weight from day 2 with a maximum on day 5 and a normalization on day 12, which indicates that the hamsters fell ill following contamination/infection with SARS-CoV-2 by nasal injection. On the contrary, it can be observed in Figure 2 that the hamsters of group 2 do not lose weight after inoculation of SARS-CoV-2 by the ocular route. This therefore indicates that the animals remain in good health.

After 14 days, both groups were again exposed to a nasal injection containing a 3x10 ⁶ viral dose of SARS-CoV-2.

In both groups the hamsters continue to gain weight (see Figures 1 and 2). Specifically, at day 21, group 1 animals did not fall ill again after inhalation of the second viral dose of SARS-CoV-2. This means that they became immune due to the disease developed during the first 10 days of the experiment.

Also, on day 21, group 2 hamsters did not become ill after inhalation of the second viral dose of SARS-CoV-2. This means that hamsters became immune through the first ocular application of SARS-CoV-2. Group 2 hamsters thus acquired immunity with epi-ocular application, which, unlike group 1, was acquired without developing severe systemic disease.

2. Study of hamsters after an ocular injection of virus

In addition, the study of n=10 other hamsters which received a single ocular application of a composition containing a viral dose of 3x10 ⁶ SARS-CoV-2 showed that they developed a strong production of antibodies neutralizing against the virus (of at least 1:640 in the serum). These results mean that by epi-ocular application of the virus, hamsters do not develop disease, and have acquired immunity against SARS-CoV-2 which is detectable in serum.

3. Plestymographic analyzes

The lung function of groups 1 and 2 mentioned above (see point 1.) was measured using four single-camera whole-body plethysmographs from EMKA Technologies (model #PLT-UNR-RT-3). Each plethysmographic chamber was equipped with the same wire pneumotachograph and the same differential pressure transducer (EMKA Technologies, model#USB_DP_T). An opening in each room allowed the continuous extraction of stale air at a constant flow rate (500 ml/min) and the continuous replacement with fresh air from the room using a dedicated pump (model #VENT_4_PLT). The measurement campaign per animal tested lasted approximately 20 minutes. The following procedures were then used to record the flow signal generated in the plethysmographic chambers by quiet, awake breathing. The flow rate was systematically calibrated before and after the experiment. If a deviation of more than 5% was detected, the collected data was rejected and the measurement campaign was restarted.

The raw flow curves were acquired by sampling the signals at 2 kHz. The regularity of the respiratory pattern was first assessed manually by controlling the constancy of the flow peaks. Based on this criterion, with the exception of a few cases of coughing and preening, the majority of hamsters breathed regularly between the fifth and twentieth minute spent in the chamber. Unlike laboratory mice, hamsters are animals that can transition from awake state to deep sleep state and vice versa in seconds. Since the breathing pattern is very different between these two states, it is crucial to select episodes of regular breathing during the waking state. On the other hand, the exploratory behavior of these animals is much more intense than in mice or rats and is accompanied by a specific respiratory pattern consisting of intense sniffing. The selection of a single episode of regular breathing is therefore not sufficient. For this reason, the selection of episodes to be analyzed must be systematically carried out manually by experienced personnel, and the resulting flow curves were then processed using IOX2 software (IIOX_1 PULMO_4a from EMKA Technologies). The thoraco-abdominal flow curves are then analyzed to determine inspiratory time (Tl), expiratory time (TE), peak inspiratory flow (PIF), peak expiratory flow (PEF) and tidal volume (TV). This last parameter was systematically measured twice per cycle, once during the inspiratory part (T1) and once during the expiratory part of the respiratory cycle (TE). Two other parameters were calculated: the time required to exhale the first 64% of VT, called relaxation time (RT), and the bronchoconstriction index (Penh) proposed by Hamelmann et al., according to the formula Penh = [( TE/RT) - 1]*(PEF/PIF) (Hamelmann E, Schwarze J, Takeda K, Oshiba A, Larsen GL, Irvin CG, Gelfand EW. Noninvasive measurement of airway responsiveness in allergy mice using barometric plethysmography. Am J Respir Crit Care Med. 1997 Sep;156(3 Pt 1):766-75.doi:10.1164/ajrccm.156.3.9606031.PMID:9309991).

Finally, respiratory rate [RR = 60/(TI + TE)], minute volume (MV = RR * TV), mean inspiratory flow (MIF = TV/Tl), mean expiratory flow (MEF = TV/TE ) and the duty cycle [%TI = TI/(TI+TE)] were also calculated from the parameters measured above and the body weight (BW). From the quantitative values obtained, the median value was systematically determined and used to calculate a mean value representative of the hamster and the day on which the analysis is carried out.

Results :

The Penh is a composite, dimensionless value that can be used to screen for pulmonary distress in laboratory animals.

The results are presented in the form of 4 groups, as shown in Table 3 below.

Table 3: Description of the hamsters analyzed

The results are presented in Figures 3 to 8.

Prior to infection, all animals exhibited a similar ventilation pattern, regardless of group (Figure 3). After infection with the SARS-CoV-2 virus, two different patterns were observed.

First, animals in groups G2, G3, and G4 showed a pattern very similar to that seen before infection, with no significant difference, unlike group G1 (Figures 4-6).

A return to normal is observed on day 12 (post-infection) in the hamsters of group G1 (Figures 7 and 8). 4. Evaluation of Neutralizing Antibodies in Serum

A whole blood sample from the hamsters was collected in a cryotube, clotted at room temperature for 1-2 hours and stored at 4°C for 24 hours. The blood was then centrifuged, the serum was placed in cryotubes and decomplemented at 56°C for 45 minutes. The decomplemented sera were then stored at −20° C. until their use for the determination of the neutralizing antibody titer.

Virus stock was titrated in serial log dilutions to achieve a 50% tissue culture infectious dose (TCID50) on 96-well culture plates. The plaques were observed daily using an inverted optical microscope for five days to assess the presence of cytopathic effect (CPE) and the final titer was calculated according to the Reed & Muench method.

A serum sample was retained from all hamsters for quantification of neutralizing antibody titers. Virus neutralization assay was performed with BetaCov/Belgium/SartTilman/2020/1 strain of SARS-CoV-2 in 96-well plates containing confluent Vero E6 cells (ATCC CRL-1586).

Nine dilutions of each serum inactivated by heat (40 minutes at 56°C) were used (1:10 to 1:1280 - corresponding to the final dilutions of the test 1:20 to 1:2580). Dilutions were made in triplicate in DMEM/FBS on 96-well culture plates.

The sera (50 µl/well) were mixed with an identical volume (vol/vol) of a solution containing 100 TCID50 (Tissue Culture Infection Dose Fifty) of SARS-CoV-2 virus.

The serum-virus mixture was then incubated at 37°C for one hour in a humidified atmosphere with 5% CO2.

After incubation, 100 µl of a Vero cell suspension was added so as to deposit 20,000 cells in each well. The plates were then re-incubated for 5 days. For each serum, the process was repeated twice. After 5 days, the ECP was evaluated under the light microscope. Serum dilutions showing CPE were considered non-neutralizing (negative), while those showing no CPE were considered neutralizing/positive.

The virus neutralization titer was reported as the highest dilution of serum that neutralized ECP in 50% of the wells (NT50). For all sera with an NT50>1:320, a second process can be performed using higher dilutions (up to 1:20480).

Positive (NT50=1: 160, from the Belgian National Reference Center) and negative (saline solution) controls were inserted into each plate.

The results are shown in Figure 9.

Example 4: Study of the spread of SARS-CoV-2 during infection of the nasal cavity

The inventors have analyzed the spread of the virus in hamsters which have been contaminated with SARS-CoV-2 via a nasal injection, as described in Example 3. The inventors have thus observed that in the first 48 hours following the entry of the virus into the nose, the infection progresses slowly from an infection of the nasal cavity alone to the mucous membranes, with the formation of aerosols. These aerosols can then infect the bronchi and alveoli, especially during inspiration, and contaminate other people during expiration.

Example 5: Vaccination using different immunogenic or vaccine compositions

Four new groups of hamsters were studied.

In Group 1, 20 µL of Janssen/Johnson & Johnson's non-replicating viral vector vaccine (usually referred to as Ad26COV2.S), taken directly from the vial, without any modification, was administered to the left eye of n=8 hamsters at J=1 . After 14 days (D=14), 20 pL of this same vaccine, taken directly from the vial, without any modification, were again administered in the left eye of the n=8 hamsters.

In Group 2, 50 µL of Janssen/Johnson & Johnson's non-replicating viral vector vaccine (usually referred to as Ad26COV2.S), taken directly from the vial, without any modification, was administered intramuscularly into both hips of n=8 hamsters at D=1. After 14 days (D=14), 50 μL of this same vaccine, taken directly from the vial, without any modification, were again administered intramuscularly in the two hips of the n=8 hamsters.

In group 3, 20 µL of PBS (phosphate buffered saline) was administered in the left eye of n=8 hamsters on D=1. After 14 days (D=14), 20 µL of PBS (phosphate buffered saline) was again administered in the left eye of n=8 hamsters.

In group 4, 20 pL of a solution containing 3x 10 ^® of a SARS-CoV-2 virus (Wuhan-like variant of the SARS-CoV-2 virus), (non-attenuated virus, non-inactivated virus) were administered in both eyes of n=8 hamsters at 14 days (D=14) of the experiment.

After 14 additional days (D=28 compared to the start of the experiment) the four groups were contaminated intranasally with 200 μL of a solution containing 6x10 ^® TCID50 (Tissue Culture Infection Dose Fifty) of a SARS- CoV-2 (Wuhan-like variant of the SARS-CoV-2 virus).

Animal weight loss (i.e. an indicator that the hamster is sick) was analyzed for each of the four groups. The results are presented in Figure 10. The curves represent the average weight of the hamsters in the four groups.

The results show that the hamsters of groups 1, 2 and 4 are protected and do not get sick unlike the hamsters of group 3.

Example 6: Ocular vaccination in man

Ocular vaccination is being tested in humans.

Two people who had previously been vaccinated intramuscularly (two injections into the musculus deltoideus of the left arm of a vaccine against Covid-19) received 20 pL of a new dose of a vaccine against Covid-19, administered to the ocular mucosa of the right eye, more than 6 months after the second intramuscular injection.

The determination of IgG in the serum and IgA in the oral and pharyngeal mucous membranes is then carried out, by blood sampling and by oral and pharyngeal washing with 10 ml of 0.9% NaCl for 2 minutes, on the day of administration. of the new dose of vaccine on the ocular mucosa, then 3 weeks later.

The results are shown in Table 4 below.

Table 4: Levels of IgG, IgM and IgA in subjects

Claims

1. Immunogenic or vaccinal composition against SARS-CoV-2, for its use in the prevention and/or treatment of Covid-19, characterized in that it is administered to the ocular mucosa and/or the uro- genital.

2. Immunogenic or vaccine composition according to claim 1, characterized in that it is administered to the ocular mucosa.

3. Immunogenic or vaccine composition according to any one of the preceding claims, characterized in that the composition comprises one or more substances making it possible to cause an immune reaction against SARS-CoV-2.

4. Immunogenic or vaccine composition according to claim 3, characterized in that the substance making it possible to provoke an immune reaction against SARS-CoV-2 is chosen from: a SARS-CoV-2 virus, an inactivated SARS-CoV-2 virus , an attenuated SARS-CoV-2 virus, a modified SARS-CoV-2 virus expressing or capable of expressing one or more SARS-CoV-2 antigens, a genetically modified micro-organism expressing or capable of expressing one or more SARS- CoV-2, a messenger ribonucleic acid (mRNA) encoding one or more SARS-CoV-2 antigens (such as a SARS-CoV-2 viral protein), a deoxyribonucleic acid (DNA) encoding one or more antigens SARS-CoV-2 (such as a SARS-CoV-2 viral protein), a SARS-CoV-2 viral protein or one or more fragments thereof, a fusion protein comprising a SARS viral protein -CoV-2 or one or more fragments thereof, a recombinant cell expressing or capable of expressing one or more several SARS-CoV-2 antigens, a DNA plasmid encoding one or more SARS-CoV-2 antigens or virus-like particles.

5. Immunogenic or vaccine composition according to claim 4, in which said viral protein is chosen from: the S, HE, M, N or E proteins or the ORF proteins, preferably chosen from the S, M, N or E proteins.

6. Immunogenic or vaccine composition according to any one of Claims 4 or 5, in which the said viral protein is the S protein or a fragment thereof, such as the receptor binding domain (RBD), the subunit S1 or the cleavage region between the S1 subunit and the S2 subunit.

7. Immunogenic or vaccine composition according to any one of the preceding claims, characterized in that it is administered in the form of a drop, lyophilisate, dried composition, powder, gel, nanoparticle or ointment.

8. Immunogenic or vaccine composition according to any one of the preceding claims, characterized in that it is administered using an applicator.

9. Immunogenic or vaccine composition according to any one of the preceding claims, for its use in the prevention and/or treatment of Covid-19 in an animal or in humans, in particular an adult.

10. Immunogenic or vaccine composition according to any one of the preceding claims, characterized in that it further comprises one or more additional substances.

11. Immunogenic or vaccine composition according to claim 10, characterized in that the additional substance is chosen from: - a preservative, a marker, in particular an indicator substance, such as a dye, a surfactant, an additive.

12. Immunogenic or vaccine composition according to any one of claims 1-11, characterized in that it is administered after the administration of a first marker and before the administration of a second marker.

13. Immunogenic or vaccinal composition according to any one of the preceding claims, characterized in that the administration to the ocular mucosa and/or the uro-genital mucosa is carried out before or after at least one administration of an immunogenic or vaccinal composition intramuscularly.