NOVEL METHODS FOR INDUCING AN IMMUNE RESPONSE

专利摘要:
The present invention provides methods for inducing an immune response, particularly methods for providing the immune response to an antigen comprising separately administering a saponin and a TLR4 agonist.
公开号:BE1025119B1
申请号:E2017/5958
申请日:2017-12-19
公开日:2018-11-05
发明作者:Margherita Coccia；Arnauld Michel Didierlaurent
申请人:Glaxosmithkline Biologicals Sa；
IPC主号:

专利说明:

(30) Priority data:
12/20/2016 GB 1621686.3 (73) Holder (s):
GLAXOSMITHKLINE BIOLOGICALS SA 1330, RIXENSART
Belgium (72) Inventor (s):
COCCIA Margherita
1330 RIXENSART
Belgium
DIDIERLAURENT Arnauld Michel
1330 RIXENSART
Belgium (54) NOVEL METHODS FOR INDUCING AN IMMUNE RESPONSE (57) The present invention relates to methods for inducing an immune response, in particular methods for adjuvanting the immune response to an antigen comprising separate administration of a saponin and a TLR4 agonist.
BELGIAN INVENTION PATENT
FPS Economy, SMEs, Classes
Medium & Energy
Intellectual Property Office
Publication number: 1025119
Filing number: BE2017 / 5958
International Classification: A61K 39/39
Date of issue: 05/11/2018
The Minister of the Economy,
Having regard to the Paris Convention of March 20, 1883 for the Protection of Industrial Property;
Considering the law of March 28, 1984 on patents for invention, article 22, for patent applications introduced before September 22, 2014;
Given Title 1 “Patents for invention” of Book XI of the Code of Economic Law, article XI.24, for patent applications introduced from September 22, 2014;
Having regard to the Royal Decree of 2 December 1986 relating to the request, the issue and the maintenance in force of invention patents, article 28;
Given the patent application received by the Intellectual Property Office on 19/12/2017.
Whereas for patent applications falling within the scope of Title 1, Book XI of the Code of Economic Law (hereinafter CDE), in accordance with article XI. 19, §4, paragraph 2, of the CDE, if the patent application has been the subject of a search report mentioning a lack of unity of invention within the meaning of the §ler of article XI.19 cited above and in the event that the applicant does not limit or file a divisional application in accordance with the results of the search report, the granted patent will be limited to the claims for which the search report has been drawn up.
Stopped :
First article. - It is issued to
GLAXOSMITHKLINE BIOLOGICALS SA, Rue de l'arstitut 89, 1330 RIXENSART Belgium;
represented by
PRONOVEM - Office Van Malderen, Avenue Josse Goffin 158, 1082, BRUXELLES;
a Belgian invention patent with a duration of 20 years, subject to the payment of the annual fees referred to in article XI.48, §1 of the Code of Economic Law, for: NEW PROCESSES FOR INDUCING AN IMMUNE RESPONSE.
INVENTOR (S):
COCCIA Margherita, c / o GlaxoSmithKline Biologicals SA, Rue de l'institut 89, 1330, RIXENSART;
DIDIERLAURENT Arnauld Michel, c / o GlaxoSmithKline Biologicals SA, Rue de l'institut 89, 1330, RIXENSART;
PRIORITY (S):
12/20/2016 GB 1621686.3;
DIVISION:
divided from the basic request:
filing date of the basic application:
Article 2. - This patent is granted without prior examination of the patentability of the invention, without guarantee of the merit of the invention or of the accuracy of the description thereof and at the risk and peril of the applicant (s) ( s).
Brussels, 05/11/2018,
By special delegation:
NEW METHODS FOR INDUCING AN IMMUNE RESPONSE
BE2017 / 5958
TECHNICAL AREA
The present invention relates to methods for inducing an immune response, in particular to methods for enhancing the immune response to an antigen comprising the separate administration of a saponin and a TLR4 agonist.
BACKGROUND OF THE INVENTION
Adjuvants are included in vaccines to improve humoral and cellular immune responses, particularly in the case of weakly immunogenic subunit vaccines. Similar to natural infections with pathogens, adjuvants rely on the activation of the innate immune system to promote lasting adaptive immunity. Because the simultaneous activation of multiple innate immune pathways is a feature of natural infections, adjuvants can combine multiple immunostimulants to promote adaptive immune responses to vaccination.
The adjuvant system 01 (AS01) is a liposome-based adjuvant that contains two immunostimulants, 3-O-deacyl-4'-monophosphoryl-lipid A (3D-MPL) and QS-21 (Garçon and Van Mechelen, 2011 ). The TLR4 agonist, 3D-MPL, is a non-toxic derivative of the lipopolysaccharide from Salmonella minnesota. QS21 is a natural saponin molecule extracted from the bark of the South American tree Quillaja saponaria Molina (Kensil et al., 1991; Ragupathi et al., 2011).
ASOl is included in the RTS, S vaccine recently developed against malaria (Mosquirix®) and the vaccine against Herpes zoster HZ / su (Shingrix®), and in multiple candidate vaccines in development against pathogens such as Herpes zoster (HZ / su), the
BE2017 / 5958 human immunodeficiency virus and Mycobacterium tuberculosis. During the preclinical and clinical evaluation of these candidate vaccines, both antigen-specific antibodies and immunity due to CD4 + T cells were constantly observed. The ability of ASOl to constantly produce cellular immune responses to vaccination distinguishes it from other adjuvants which generally favor mainly humoral responses to vaccination (Black et al., 2015; Garçon and Van Mechelen, 2011). Simultaneously, adjuvanted vaccines with ASOl have been effective in promoting
Immunogenicity and efficacy of vaccination in complex populations, such as infants (with RTS, S) and older adults (with HZ / su).
Injection of ASOl produces rapid and transient activation of innate immunity in animal models
Neutrophils and recruits to the lymph node after immunization. In monocytes are rapidly draining lymphatic (GLD) addition, ASOl induces the recruitment and activation of dendritic cells (CD) CMHII ^{hil h} , which are necessary for the activation of T lymphocytes (Didierlaurent AM et al., 2014). Some data are also available on the mechanism of action of the components of ASOl. The 3D-MPL sends signals via TLR4, in
BE2017 / 5958 stimulating the transcriptional activity of NF-κΒ and the production of cytokines and directly activates the antigen presenting cells (APC) both in humans and in mice (De Becker et al., 2000; Ismaili et al., 2002; Martin et al., 2003; Mata-Haro et al., 2007). QS-21 promotes high antigen-specific antibody and CD8 ⁺ T-cell responses in mice (Kensil and Kammer, 1998; Newman et al., 1992; Soltysik et al., 1995) and specific antibody responses antigen in humans (Livingston et al., 1994). Because of its physical properties, it is believed that QS-21 could act as an in vivo danger signal (Lambrecht et al., 2009; Li et al., 2008). Although QS-21 has been shown to activate the ASCNLRP3 inflammasome and the subsequent release of IL-1 / IL-18 (MartyRoix, R. et al., 2016), the exact molecular pathways involved in adjuvant effect of saponins are yet to be clearly defined.
3D-MPL and QS21 have been shown to act synergistically in the induction of immune responses (Coccia et al. 2016). In addition, it has been shown that the way in which the two immunostimulants are supplied is an important factor influencing the quality of the induced responses, with the liposomal presentation of 3D-MPL and QS21 in ASOl providing a power greater than 1 'AS02 based on oil in water emulsion (Dendouga et al. 2012).
Despite the flow of more than 20 years since combinations of the TLR4 agonist, 3D-MPL, and the saponin QS21 were first described
BE2017 / 5958 times (see, for example, international patent application WO 96/33739), to date, those skilled in the art have only obtained the synergistic benefits of the combination of a TLR4 agonist and a saponin as by the co-formulation of these immunostimulants.
There remains a need for new immunization approaches that are variants or provide benefits over current approaches, being highly effective, safe, conveniently formulated, cost-effective, durable and that induce a broad spectrum of immune responses exhibiting cross-reactivity.
SUMMARY OF THE INVENTION
It has now been surprisingly found that co-formulation of TLR4 agonist and saponin is not required, a TLR4 agonist and a saponin can be administered separately without appreciable compromise of the synergistic adjuvant effect which has been observed with co-formulation.
Therefore, a TLR4 agonist is provided for use as an adjuvant with a saponin, where the TLR4 agonist is administered separately from the saponin. A saponin is also provided for use as an adjuvant with a TLR4 agonist, where the saponin is administered separately from the TLR4 agonist.
The present invention further provides the use of a TLR4 agonist in the manufacture of an adjuvant for use with a saponin, where the TLR4 agonist is administered separately from the saponin. In addition, it is provided the use of a
BE2017 / 5958 saponin in the manufacture of an adjuvant for use with a TLR4 agonist, where the saponin is administered separately from the TLR4 agonist.
A method is also provided for inducing an immune response in a subject using a TLR4 agonist and a saponin, characterized in that the TLR4 agonist and the saponin are administered separately to the subject. There is provided a method for inducing an immune response in a subject using a TLR4 agonist and a saponin, said method comprising the following steps:
(i) administration about the TLR4 agonist;
(ii) administration regarding the saponin;
in which the steps can be taken in any order and the TLR4 agonist and the saponin are administered separately.
A method of adjuvanting a subject's immune response to an antigen using a TLR4 agonist and a saponin, characterized in that the TLR4 agonist and the saponin are administered separately to the subject also forms an aspect of the invention. A method is provided for adjuvanting a subject's immune response to an antigen using a TLR4 agonist and a saponin, said method comprising the following steps:
(i) administration about the TLR4 agonist;
(ii) administration regarding the saponin;
(iii) administration regarding the antigen;
in which steps can be taken in any order, the agonist of TLR4 and the
BE2017 / 5958 saponin are administered separately, and the antigen may be optionally co-formulated with the TLR4 agonist, saponin or co-formulated with each of the agonist of
TLR4 and saponin.
The present invention also provides a kit comprising: (i) a first composition comprising a TLR4 agonist; and (ii) a second composition comprising a saponin. The antigen may be present in the first or second composition, or in both the first and second compositions.
Kits are provided comprising: (i) a first composition comprising a TLR4 agonist; (ii) a second composition comprising a saponin; and (iii) instructions that the TLR4 agonist and the saponin should be administered separately. The antigen may be present in the first or second composition or in both the first and second compositions.
The present invention also provides a kit comprising: (i) a first composition comprising a TLR4 agonist; (ii) a second composition comprising a saponin; and (iii) a third composition comprising an antigen.
Kits are provided comprising: (i) a first composition comprising a TLR4 agonist;
(ii) a second composition comprising a saponin;
(iii) a third composition comprising an antigen; and (iv) instructions that the TLR4 agonist and the saponin should be administered separately.
There is also provided a multi-chamber syringe comprising a first compartment and a second
BE2017 / 5958 compartment, said first compartment comprising a first composition comprising a TLR4 agonist and said second compartment comprising a second composition comprising a saponin, said first and second compositions being administered to a subject either sequentially or simultaneously (although separately). The antigen may be present in the first or second composition, or in both the first and second compositions. Alternatively, the multi-chamber syringe may include a third compartment comprising a third composition comprising the antigen.
Also provided is a kit comprising: (i) a first syringe containing a composition comprising a TLR4 agonist; and (ii) a second syringe containing one comprising a saponin. The antigen may be present in the first or second composition, or in both the first and second compositions.
There is further provided a kit comprising: (i) a first syringe containing a composition comprising a TLR4 agonist; (ii) a second syringe containing a composition comprising a saponin; and (iii) a third syringe containing a composition comprising an antigen.
To fully obtain the benefits of the present invention, the TLR4 agonist should be administered in a composition which is substantially free of saponin. In addition, the saponin should be administered in a composition which is substantially free of TLR4 agonist.
BE2017 / 5958
BRIEF DESCRIPTION OF THE FIGURES
Figure 1: IFNgamma level in GLD 6 hours after injection following administration of the gE antigen alone or the gE antigen with 3D-MPL and QS21 separated or co-formulated in mice.
Figure 2: IFNgamma level following administration of the gE antigen with 3D-MPL and QS21 separated or co-formulated in mice.
Figure 3: CD4 T cell responses following separate administration of gE antigen and 3D-MPL and QS21 separated or co-formulated in mice.
Figure 4: Ratio of CD4 T cell responses following administration of the gE antigen and 3D-MPL and QS21 separated or co-formulated in mice.
Figure 5: Antibody responses following administration of the gE antigen and 3D-MPL and QS21 separated or co-formulated in mice.
Figure 6: Report of antibody responses following administration of the gE antigen and 3D-MPL and QS21 separated or co-formulated in mice.
DESCRIPTION OF SEQUENCE IDENTIFIERS from de
SEQ ID NO:
SEQ ID NO:
strain M.
SEQ ID NO:
: polypeptide sequence polypeptide sequence tuberculosis H37Rv M. strain
SEQ ID NO: 4 de RTS de
Rvll96 H37Rv tuberculosis polypeptide sequence of
Rv0125 polypeptide sequence of the M72 fusion
BE2017 / 5958
SEQ ID NO: 5: sequence polypeptide of the merger of M72-2his SEQ ID NO: 6: sequence polypeptide of gE truncated from varicella zoster virus SEQ ID NO: 7: sequence polypeptideof
conformally constrained RSV PreF antigen
SEQ ID NO: 8: sequence polypeptide of lpg 2 0 of HIV TV1SEQ ID NO: 9: sequence polypeptide of lpg 2 0 of HIV 1086.C. DESCRIPTION DETAILED
It has now surprisingly been discovered that the co-formulation of TLR4 agonist and saponin is not required. The TLR4 agonist and the saponin can be administered separately without appreciable compromise of the adjuvant effect. Therefore, the present invention allows for alternative approaches to administration based on the separate formulation of the TLR4 and saponin components, which approaches are advantageous in circumstances such as when co-formulation difficulties exist for a particular vaccine (e.g., stability formulation or to overcome other antigen / adjuvant incompatibilities).
Therefore, a TLR4 agonist is provided for use as an adjuvant with a saponin, where the TLR4 agonist is administered separately from the saponin. A saponin is also provided for use as an adjuvant with
BE2017 / 5958 a TLR4 agonist, where saponin is administered separately from the TLR4 agonist.
The present invention further provides the use of a TLR4 agonist in the manufacture of an adjuvant for use with a saponin, where the TLR4 agonist is administered separately from the saponin. Additionally, the use of a saponin is provided in the manufacture of an adjuvant for use with a TLR4 agonist, where the saponin is administered separately from the TLR4 agonist.
A method is also provided for inducing an immune response in a subject using a TLR4 agonist and a saponin, characterized in that the TLR4 agonist and the saponin are administered separately to the subject. There is provided a method for inducing an immune response in a subject using a TLR4 agonist and a saponin, said method comprising the following steps:
(i) administration about the TLR4 agonist;
(ii) administration regarding the saponin;
in which the steps can be taken in any order and the TLR4 agonist and the saponin are administered separately.
A method of adjuvanting a subject's immune response to an antigen using a TLR4 agonist and a saponin, characterized in that the TLR4 agonist and the saponin are administered separately to the subject also forms an aspect of the invention. A method is provided for adjuvanting the immune response of a subject
BE2017 / 5958 to an antigen using a TLR4 agonist and a saponin, said method comprising the following steps:
(i) administration about the TLR4 agonist;
(ii) administration regarding the saponin;
(iii) administration regarding the antigen;
wherein the steps can be taken in any order, the TLR4 agonist and the saponin are administered separately, and the antigen can be optionally co-formulated with the TLR4 agonist, the saponin or co-formulated with each TLR4 and saponin agonist.
The present invention also provides a kit comprising: (i) a first composition comprising a TLR4 agonist; and (ii) a second composition comprising a saponin. The antigen may be present in the first or second composition, or in both the first and second compositions.
Kits are provided comprising: (i) a first composition comprising a TLR4 agonist; (ii) a second composition comprising a saponin; and (iii) instructions that the TLR4 agonist and the saponin should be administered separately. The antigen may be present in the first or second composition or in both the first and second compositions.
The present invention also provides a kit comprising: (i) a first composition comprising a TLR4 agonist; (ii) a second composition comprising a saponin; and (iii) a third composition comprising an antigen.
BE2017 / 5958
Kits are provided comprising: (i) a first composition comprising a TLR4 agonist;
(ii) a second composition comprising a saponin;
(iii) a third composition comprising an antigen; and (iv) instructions that the TLR4 agonist and the saponin should be administered separately.
There is further provided a multi-chamber syringe comprising a first compartment and a second compartment, said first compartment comprising a first composition comprising a TLR4 agonist and said second compartment comprising a second composition comprising a saponin, said first and second compositions being administrable to a subject either sequentially or simultaneously (although separately). The antigen may be present in the first or second composition, or in both the first and second compositions. Alternatively, the multi-chamber syringe may include a third compartment comprising a third composition comprising the antigen.
Also provided is a kit comprising: (i) a first syringe containing a composition comprising a TLR4 agonist; and (ii) a second syringe containing a composition comprising a saponin. The antigen may be present in the first or second composition, or in both the first and second compositions.
There is further provided a kit comprising: (i) a first syringe containing a composition comprising a TLR4 agonist; (ii) a second syringe containing one comprising a saponin; and (iii) a
BE2017 / 5958 third syringe containing a composition comprising an antigen.
Definitions
Topic
The methods and uses of the present invention are generally intended for mammalian subjects. The subject can be a wild or domesticated animal. Mammalian subjects include, for example, cats, dogs, pigs, sheep, horses, or cattle. In one embodiment of the invention, the subject is human.
The subject to be treated using the method of the invention can be of any age.
In one embodiment, the subject is a human infant (up to 12 months of age). In one embodiment, the subject is a human child (of an age less than 18 years old). In one embodiment, the subject is an adult human being (18 to 59 years of age). In one embodiment, the subject is an older human being (60 years of age or older).
Separate administration
The term "administered separately" excludes the co-formulation of the TLR4 agonist and saponin, but includes the simultaneous and sequential administration of the individually formulated TLR4 agonist and saponin.
Administration location
TLR4 agonist and saponin are desirably administered at nearby locations
BE2017 / 5958 sufficient space in such a way that their synergistic adjuvant effect is not appreciably compromised.
TLR4 agonist and saponin can be administered by a variety of suitable routes, including parenteral administration, such as intramuscular or subcutaneous. TLR4 agonist and saponin can be administered by different routes. Suitably, the TLR4 agonist and the saponin are administered by the same route, in particular, intramuscularly.
When the antigen is also supplied, the antigen,
The TLR4 agonist and saponin can be administered by different routes. Appropriately, the agonist of
TLR4, saponin and
The antigen are administered by the same route, in particular, intramuscularly.
The TLR4 agonist and saponin are desirably administered to a draining location to the same lymph node, as to the same member, in particular, to the same muscle.
Suitably, the TLR4 agonist and saponin are administered intramuscularly same muscle.
In some embodiments, the agonist at
TLR4 and saponin are administered in the same location.
If an antigen is also provided, appropriately, the TLR4 agonist, saponin and antigen are administered to a site draining to the same lymph node, such as to the same limb, in particular, to the same muscle and especially, to the same location.
BE2017 / 5958
When the antigen is also appropriately supplied, the TLR4 agonist, saponin and the antigen are administered intramuscularly to the same muscle.
The spatial separation of the administration sites can be at least 5 mm, as at least 1 cm. The spatial separation of the administration sites can be less than 10 cm, as less than 5 cm apart.
Time of administration
The TLR4 agonist and the saponin are desirably administered with sufficient temporal proximity so that their synergistic adjuvant effect is not significantly compromised. The TLR4 agonist and the saponin can be administered within 6 hours of each other. Suitably, the TLR4 agonist and the saponin are administered 2 hours apart, in particular 1 hour apart, as 30 minutes and especially 15 minutes (e.g. , 5 minutes).
If the TLR4 agonist and the saponin are administered with a delay, the TLR4 agonist may be administered first and the saponin administered second, alternatively, as appropriate, the saponin is administered first and the agonist of TLR4 administered second.
When an antigen is also appropriately supplied, the TLR4 agonist, saponin and the antigen are all administered within 6 hours of each other. Suitably, the TLR4 agonist, the saponin and the antigen are administered at 2 hours the
BE2017 / 5958 each other, in particular, at 1 hour, as at minutes and especially, at 15 minutes (for example, at 5 minutes).
Desirably, the TLR4 agonist and the saponin are administered without intentional delay (taking into account the practical modalities of an antigen is also the TLR4 agonist, the administered without delay practical modalities multiple administrations). If provided, appropriately, saponin and the antigen are intentional (taking into account multiple administrations).
The time between the administration of the agonist of
TLR4 and saponin can be at least 5 seconds, like 10 seconds, and in particular, at least seconds.
Administration regimes
Approaches to establishing strong and lasting immunity often include the addition of repeated vaccination, i.e., stimulating an immune response by administering one or more other doses of the antigen. These other administrations can be carried out with the same vaccine (homologous booster) or with a different vaccine (heterologous booster).
The present invention can be applied as part of a homologous or heterologous awareness / booster regimen, such as either awareness or booster vaccination.
Administration result
TLR4 agonist and saponin can be administered separately without significant compromise of
BE2017 / 5958 the synergistic adjuvant effect which has been observed with co-formulation. Suitably, the immunological response observed after a process of the present invention is at least 50% of the response observed with the co-formulation, such as at least 60%, suitably at least 70%, especially at least 80% and in particular at least 90%. Suitably, the immunological response observed after a method of the present invention is not less than the response observed with the co-formulation.
In one embodiment, the immune response observed is the innate production of IFNgamma (as described in Example 1). In another embodiment, the immune response observed is an antibody response (as described in Example 2). In a third embodiment, the immune response observed is a response of CD4 T lymphocytes (as described in Example 2).
TLR4 agonists
A suitable example of a TLR4 agonist is a lipopolysaccharide, suitably a non-toxic derivative of lipid A, particularly a monophosphoryllipid A and more particularly monophoshoryllipid A 3-de-O-acylated (3D-MPL).
The 3D-MPL is sold under the name "MPL" by GlaxoSmithKline Biologicals N.A. and is called throughout the 3D-MPL document. See, for example, US Patents 4,436,727; 4,877,611; 4,866,034 and 4,912,094. 3D-MPL can be produced according to the methods described in document GB 2 220 211 A. From a chemical point of view, it is a mixture of monophosphoryl18
BE2017 / 5958 lipid A 3-deacylated with 4, 5 or 6 acylated chains. In the context of the present invention, small particle 3D-MPL can be used to prepare the aqueous adjuvant composition. The small particle 3D-MPL has a particle size such that it can be sterilized by filtration through a 0.22 μm filter. Such preparations are described in document WO 94/21292. Suitably, powdered 3D-MPL is used to prepare aqueous adjuvant compositions used in the present invention
Other TLR4 agonists that can be used are alkyl glucosaminide phosphates (AGP) such as those described in WO 98/50399 or US Patent No. 6,303,347 (methods for preparing AGP are also described ). Other AGPs are as described in US Patent No. 6,764,840. Some AGPs are TLR4 agonists, and some are TLR4 antagonists.
Other TLR4 agonists which can be used in the present invention include
The glucopyranosyl-lipid adjuvant (GLA) as described in the documents
WO 2009/143457 or articles from the Coler literature
RN and
Development and
Characterization of
Synthetic
Glucopyranosyl Lipid Adjuvant
System as a Vaccine
Adj uvant.
PLoS
ONE e! 6333.
do i: 10.1371 / journal.pone.0016333 and Arias
MA et al.
(2012) Glucopyranosyl Lipid Adjuvant (GLA),
TLR4 Agonist, Promotes Potent Systemic a Synthetic and Mucosal
Responses to Intranasal Immunization with HIVgpl40. PLoS
ONE 7 (7): e41144. doi: 10.1371 / journal.pone .0041144. The
BE2017 / 5958 documents WO 2008/153541 or WO 2009/143457 are incorporated here by reference with the aim of defining TLR4 agonists which can be used in the present invention.
A TLR4 agonist, such as a lipopolysaccharide, such as 3D-MPL, can be used in amounts between 1 and 100 pg per human dose. 3D-MPL can be used at a rate of approximately 50 pg. Examples of suitable ranges are 40 to 60 pg, suitably 45 to 55 pg or 49 to 51 pg, such as 50 pg. In another embodiment, the human dose comprises 3D-MPL at a rate of about 25 pg. Examples of lower ranges include 20 to 30 pg, suitably 22 to 28 pg or 24 to 26 pg, such as 25 pg. The human doses for children may be reduced compared to those for an adult (for example, 50% reduction).
Suitably, the TLR4 agonist for use in the invention is isolated. An "isolated" naturally occurring TLR4 agonist is one that is removed from its original environment. For example, separated from some or all of the materials coexisting in the natural system (for example, being supplied in at least 50% pure form, as at least 80%, in particular at least 90%, and especially at at least 95% (like at least 99%) w / w.
Saponins
A saponin suitable for use in the present invention is Quil A and its derivatives. Quil A is a saponin preparation isolated from the South American tree Quillaj a saponaria Molina and described
BE2017 / 5958 for the first time as having an adjuvant activity by Dalsgaard et al. in 1974 (Saponin adjuvants, Archiv, für die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, p 243-254). It was isolated by HPLC from the purified fractions of Quil A which retain the adjuvant activity without the toxicity associated with Quil A (see, for example, document EP 0 362 279). Fractions of general interest include QS7, QS17, QS18 and QS21, for example, QS7 and QS21 (also known as QA7 and QA21). QS21 is a saponin of particular interest.
In certain embodiments of the present invention, saponin is a quil A derivative of Quillaj a saponaria Molina, suitably an immunologically active fraction of Quil A, such as QS7, QS17, QS18 or QS21, in particular QS21.
Saponin is optimally supplied in a less reactogenic composition where it is neutralized with an exogenous sterol, such as cholesterol, and it is supplied in a liposomal formulation as defined herein. Several particular forms of less reactogenic compositions in which QS21 is neutralized with exogenous cholesterol exist. The saponin / sterol is presented in a liposomal formulation structure. Methods for obtaining saponin / sterol in a liposomal formulation are described in document WO 96/33739, in particular example 1.
A saponin, such as QS21, can be used in amounts between 1 and 100 pg per human dose. The QS21 can be used at a rate of approximately
BE2017 / 5958 gg. Examples of suitable ranges are 40 to 60 gg, suitably 45 to 55 gg or 49 to 51 gg, such as 50 gg. In another embodiment, the human dose comprises QS21 at a rate of about 25 gg. Examples of lower ranges include 20 to 30 gg, suitably 22 to 28 gg or 24 to 26 gg, such as 25 gg The human doses for children may be reduced compared to those for an adult (e.g. reduction in 50%).
The weight ratio of the TLR4 agonist to the saponin is suitably between 1/5 and 5/1, suitably 1/1. For example, when 3D-MPL is used in an amount of 50 gg or 25 gg per dose, then, suitably, QS21 can also be used in an amount of 50 gg or 25 gg per dose (for example, dose human).
Suitably, the saponin used in the invention is isolated. A naturally occurring "isolated" saponin is one that is removed from its original environment. For example, separated from some or all of the materials coexisting in the natural system (for example, being supplied in at least 50% pure form, as at least 80%, in particular at least 90%, and especially at at least 95% (like at least 99%) w / w.
Liposomes
The TLR4 saponin and / or agonist can be formulated with any suitable carrier. The saponin and the agonist of TLR4 can be formulated with different supports, or one can be formulated with a support and the other unformulated with a support.
BE2017 / 5958
Saponin is appropriately formulated with liposomes. Optionally, the TLR4 agonist and the saponin are formulated with liposomes.
The size of the liposomes can vary from 30 nm to several μm depending on the composition of phospholipids and the process used for their preparation. In particular embodiments of the invention, the size of the liposomes will be in the range of 50 nm to 500 nm and in other embodiments, of 50 nm to 200 nm.
Optimally, the liposomes should be stable and have a diameter of -100 nm to allow sterilization by filtration.
The term "liposome" is well known in the art and defines a general category of vesicles which include one or more lipid bilayers surrounding an aqueous space. Liposomes thus consist of one or more bilayers of lipids and / or phospholipids and can contain other molecules, such as proteins or carbohydrates, in their structure. Because both lipid and aqueous phases are present, liposomes can encapsulate or trap water-soluble material, lipid-soluble material, or amphiphilic compounds.
As used herein, "neutral liposome adjuvant" means that the adjuvant includes neutral liposomes for the presentation of the included immunopotentializing agents.
The structural integrity of liposomes can be estimated by methods such as dynamic light scattering (DDL) measuring the size and
BE2017 / 5958 polydispersity of the liposomes, or, by electron microscopy for the analysis of the structure of the liposomes. In one embodiment, the average particle size (by photon correlation spectroscopy) is between 95 and 120 nm, and / or, the polydispersity index (by photon correlation spectroscopy) is not greater than 0.2. In another embodiment, the average particle size (by photon correlation spectroscopy) is between 95 and 120 nm, and / or, the polydispersity index (by photon correlation spectroscopy) is not greater at 0.3.
The liposomes provided for the present invention may contain a neutral lipid or consist essentially of neutral lipid. By "neutral lipid" it should be understood that the overall net charge of the lipid is (approximately) zero. Therefore, the lipid can be generally nonionic or it can be zwitterionic. In one embodiment, the liposomes include a zwitterionic lipid. Examples of suitable lipids are phospholipids such as phosphatidylcholine species. In one embodiment, the liposomes contain phosphatidylcholine as the liposome-forming lipid which is suitably at room temperature. Examples of non-crystalline phosphatidylcholine lenses with such non-lipids include egg yolk phosphatidylcholine, dioleoylphosphatidylcholine (DOPC) or dilaurylphosphatidylcholine (DLPC). In a particular embodiment, the liposomes of the present invention
BE2017 / 5958 contain DOPC, or, consist essentially of DOPC. Liposomes may also contain a limited amount of a charged lipid which increases the stability of the liposome-saponin structure for liposomes composed of saturated lipids. In these cases, the amount of loaded lipid is suitably from 1 to 20% w / w, preferably from 5 to 10% w / w of the liposomal composition. Suitable examples of these charged lipids include phosphatidylglycerol and phosphatidylserine. Suitably, the neutral liposomes will contain less than 5% w / w of loaded lipid, such as less than 3% w / w or less than 1% w / w.
The liposomes provided for the present invention may further comprise a sterol. Suitable sterols include β-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. In a particular embodiment, the liposomal formulation comprises cholesterol as a sterol. These sterols are well known in the art, for example, cholesterol is disclosed in the Merck index, 11th Edn., Page 341, as a naturally occurring sterol found in animal fat. The ratio of sterol to phospholipid is 1 to 50% (mol / mol), suitably 20 to 25%.
The saponin / sterol ratio will generally be of the order of 1/100 to 1/1 (w / w), suitably between 1/10 and 1/1 (w / w), and preferably 1/5 at 1/1 (w / w). Suitably, an excess of sterol is present, the saponin / sterol ratio being at least 1/2 (w / w). In one embodiment, the saponin /
BE2017 / 5958
sterol East 1/5 (w / w). In one fashion of achievement, the sterol East cholesterol. The amount of liposomes (lipid weight and of sterol ) will generally locate in the range of 0.1 mg at 10 mg per human dose of a composition, in
particular 0.5 mg to 2 mg per human dose of a composition.
In a particularly suitable embodiment, the liposomes used in the invention comprise (as essentially consist of) DOPC and a sterol, in particular cholesterol. Thus, in a particular embodiment, a composition used in the invention comprises QS21 in the form of a liposome, where said liposome comprises DOPC and a sterol, in particular cholesterol.
If the TLR4 agonist is formulated with liposomes, these can be the same as or different from the liposomes used with saponin. Therefore, the TLR4 agonist (such as 3D-MPL) can be formulated with liposomes comprising DOPC and a sterol, in particular cholesterol.
Antigen
The compositions, kits and methods of the present invention may include an immunogen or an antigen. In certain embodiments, the compositions, kits and methods of the present invention may include a polynucleotide encoding the immunogen or the antigen.
By the term "immunogenic" is meant a polypeptide which is capable of triggering an immune response. Suitably, the immunogen is a
BE2017 / 5958 antigen which comprises at least one epitope of B or T lymphocyte. The triggered immune response can be a response of B lymphocytes specific for the antigen, which produces neutralizing antibodies. The triggered immune response may be an antigen-specific T cell response, which may be a systemic response and / or a local response. The antigen-specific T cell response may include a CD4 + T cell response, such as a response involving CD4 + T cells expressing a plurality of cytokines, for example, IFNgamma, TNFalpha and / or IL2 . Alternatively, or in addition, the antigen-specific T cell response includes a CD8 + T cell response, such as a response involving CD8 + T cells expressing a plurality of cytokines, for example, IFNgamma, TNFalpha and / or IL2.
The antigen may be derived (as obtained from) from a human or non-human pathogen including, for example, bacteria, fungi, parasitic microorganisms or multicellular parasites that infect human and non-human vertebrates, or from a cancer cell or a tumor cell.
In one embodiment, the antigen is a recombinant protein, such as a recombinant prokaryotic protein.
In one embodiment, the antigen is derived
of Plasmodium spp. (such as Plasmodium falciparum), from Mycobacterium spp. (such as Mycobacterium tuberculosis (TB)), virus varicella zoster (VZV), virus
BE2017 / 5958 respiratory syncytial, human immunodeficiency virus (HIV), Moraxella spp. (such as Moraxella catarrhalis) or non-typable Haemophilus influenzae (ntHi).
The antigen can include or consist of preparations derived from parasites that cause malaria such as Plasmodium falciparum or Plasmodium vivax.
In one embodiment, the antigen may be the circumsporozoite (CS) protein of Plasmodium falciparum or one of its variants. A suitable variant of the CS protein can be a variant in which parts of the CS protein are in the form of a protein hybrid with the surface antigen S originating from hepatitis B (Ag HBs). The CS variant antigen can be, for example, in the form of a hybrid protein comprising substantially the entire C-terminal part of the CS protein, four or more tandem repeats of the immunodominant region of the CS protein, and 1 'Ag HBs. The hybrid protein can comprise a sequence which contains at least 160 amino acids and which is substantially homologous to the C-terminal part of the CS protein, but which lacks the hydrophobic anchoring sequence. The CS protein can be devoid of at least 12 of the last amino acids of the C-terminal part. In addition, it may contain 4 or more, for example, 10 or more repeating units of the Asn-Ala-AsnPro tetrapeptide (NANP).
The hybrid protein for use in the invention may be a protein which comprises a portion of the corresponding P. falciparum CS protein
BE2017 / 5958 substantially at amino acids 207 to 395 of the clone 3D7 of P. falciparum, derived from the strain NF54 fused in the frame via a linear linker at the N-terminal end of the HBsAg. The linker can comprise a part of preS2 originating from Ag HBs. CS constructs suitable for use in the present invention are presented in WO 93/10152, which was issued in the United States as US Pat. Nos. 5,928,902 and 6,169,171, both of which are incorporated in reference for the purpose of describing proteins suitable for use in the present invention.
A particular hybrid protein for use in the invention is the hybrid protein known as RTS (SEQ ID NO: 1, also disclosed in documents WO 2015/150568, WO 93/10152 (in which it is indicated by RTS * ) and in document WO 98/05355, which consists of:
- a methionine residue
- three amino acid residues, Met Ala Pro
a series of 189 amino acids representing amino acids 207 to 395 of the CS protein of the 3D7 strain of P. falciparum
- a residue of glycine
- four amino acid residues, Pro Val Thr Asn, representing the four carboxy-terminal residues of the preS2 protein of the hepatitis B virus (serotype adw), and
- a series of 226 amino acids, coded by nucleotides 1653 to 2330, and specifying protein S of the hepatitis B virus (serotype adw).
BE2017 / 5958
RTS can be in the form of mixed particles of
RTS, S. The particles of RTS, S comprise two polypeptides, RTS and S, which can be synthesized simultaneously and spontaneously form the composite particle structures (RTS, S).
The antigen can comprise or consist of preparations derived from Mycobacterium spp., Such as Mycobacterium bovis or Mycobacterium tuberculosis, in particular Mycobacterium tuberculosis.
Antigens of interest in the field of tuberculosis include Rv1196 and Rv0125. Rv1196 (described, for example, by the name of Mtb39a in Dillon et al. Infection and Immunity 1999 67 (6): 2941-2950) is highly conserved, with 100% sequence identity among the strains H37Rv, C, Haarlem , CDC1551, 94-M4241A, 98-R604INH-RIF-EM, KZN605, KZN1435, KZN4207, KZNR506, the Fil strain with a single point mutation Q30K (most other clinical isolates have 90% excess identity with H37Rv ). Rv0125 (described, for example, by the name of Mtb32a in Skeiky et al. Infection and Immunity 1999 67 (8): 3998-4007) is also highly conserved, with 100% sequence identity among many species. Full-length Rv0125 includes an N-terminal signal sequence which is cleaved to provide the mature protein.
In one embodiment, the antigen is derived from Rv1196, as comprises, as consists of, a sequence having at least 70% identity with SEQ ID NO: 2, as at least 80%, in particular at least 90 %, especially at least 95%, for example at least 98%, as at least 99%. In general, antigens associated with Rvll96 will understand (as will
BE2017 / 5958 consisting of) a derivative of SEQ ID NO: 2 comprising a small number of deletions, insertions and / or substitutions.
Examples are those with deletions of up to 5 residues at location, insertions of up to 5 residues at 0 to 5 five locations, and substitutions of up to 20 residues. Other derivatives of Rv1196 are those comprising (as consisting of) a fragment of
SEQ ID NO: 2 which has a length of at least 200 amino acids, such as a length of at least 250 amino acids, in particular a length of at least 300 amino acids, especially a length of at least 350 amino acids .
In one embodiment, the antigen is derived from Rv0125, as comprises, as consists of, a less sequence having the identity with
SEQ ID NO: 3, as in less 80 oGold in particular at less 90%, specially at least 95 oθ r for example at less 98%, like at less 99 o0 · In general, the
(as will be antigens associated with Rv0125 will consist of) a derivative of SEQ ID NO: 3 comprising a small number of deletions, insertions and / or substitutions.
Examples are those with deletions of up to 5 residues at location, insertions of up to 5 residues at 0 to 5 five locations, and substitutions of up to 20 residues. Other derivatives of Rv0125 are those comprising (as consisting of) a fragment of
SEQ ID NO: 3 which has a length of at least 150 amino acids, such as a length of at least 200 amino acids, in particular a length of at least 250 amino acids,
BE2017 / 5958 specially a length of at least 300 amino acids. Particular derivatives of Rv0125 are those comprising (as consisting of) the fragment of SEQ ID NO: 3 corresponding to residues 1 to 195 of SEQ ID NO: 3. Other immunogenic derivatives of Rv0125 are those comprising (as consisting of) the fragment of SEQ ID NO: 3 corresponding to residues 192 to 323 of SEQ ID NO: 3. Particularly preferred antigens associated with Rv0125 are derivatives of SEQ ID NO: 3 in which at least one (for example, one, two , or even all three) of the catalytic triad have been substituted or deleted, so that the protease activity has been reduced and the protein more easily produced - the catalytic residue of serine can be deleted or substituted (for example, substituted by alanine) and / or the catalytic residue of histidine can be deleted or substituted and / or the catalytic residue of aspartic acid can be deleted or substituted. Especially of interest are derivatives of SEQ ID NO: 3 in which the catalytic residue of serine has been substituted (for example, substituted by alanine). Also of interest are antigens associated with Rv0125 which comprise, as consist of, a sequence having at least 70% identity with SEQ ID NO: 3, such as at least 80%, in particular at least 90%, especially at least 95%, for example at least 98%, as at least 99% and in which at least one of the catalytic triad has been substituted or deleted or those comprising, as consisting of, a fragment of SEQ ID NO: 3 which is at least 150 amino acids in length, such as at least 200 amino acids in length,
BE2017 / 5958 in particular a length of at least 250 amino acids, especially a length of at least 300 amino acids and in which at least one of the catalytic triad has been substituted or deleted. Other immunogenic derivatives of Rv0125 are those comprising (as consisting of) the fragment of SEQ ID NO: 3 corresponding to residues 192 to 323 of SEQ ID NO: 3 in which at least one (for example, one, two, or even the three) of the catalytic triad has been substituted or deleted. Specific immunogenic derivatives of Rv0125 are those comprising (as consisting of) the fragment of SEQ ID NO: 3 corresponding to residues 1 to 195 of SEQ ID NO: 3 in which the catalytic residue of serine (position 176 of SEQ ID NO: 3 ) has been substituted (for example, substituted by alanine).
Suitably, the antigen will comprise, as will consist of, a sequence having at least 70% identity with SEQ ID NO: 4, such as at least 80%, in particular at least 90%, especially at least 95%, like at least 98%, for example at least 99%. In general, the antigens associated with M72 will comprise, as will consist of, a derivative of SEQ ID NO: 4 comprising a small number of deletions, insertions and / or substitutions. Examples are those with deletions of up to 5 residues at 0 to 5 locations, insertions of up to 5 residues at 0 to 5 locations and substitutions of up to 20 residues. Other derivatives of M72 are those comprising, as consisting of, a fragment of SEQ ID NO: 4 which has a length of at least 450 amino acids, such as a length of at least
BE2017 / 5958
500 acids amines, as a length of less 550 acids amines, as a length of less 600 acids amines, as a length of less 650 acids amines or a length at least 700 acids amines. Of make that M72 is a protein of fusion
derived from the two individual antigens Rv0125 and Rv1196, any fragment of at least 450 residues will comprise a plurality of epitopes derived from the full length sequence (Skeiky et al. J. Immunol. 2004 172: 7618-7628; Skeiky Infect. Immun. 1999 67 (8): 39984007; Dillon Infect. Immun. 1999 67 (6): 2941-2950).
An antigen associated with M72 will comprise, as will consist of, a sequence having at least 70% identity with SEQ ID NO: 4, as at least 80%, in particular at least 90%, especially at least 95%, as at minus 98%, for example at least 99%.
In general, the antigens associated with M72 will comprise, as will consist of, a derivative of SEQ ID NO: 4 comprising a small number of deletions, insertions and / or substitutions. Examples are those with deletions of up to 5 residues at 0 to 5 locations, insertions of up to 5 residues at 0 to 5 locations and substitutions of up to 20 residues.
In particular embodiments, an antigen associated with M72 will comprise residues 2 to 723 of SEQ ID NO: 4, for example, will comprise (or will consist of) SEQ ID NO: 4 or will comprise (or will consist of) SEQ ID NO: 5.
Another antigen which can be used in accordance with the present invention is the antigen of
BE2017 / 5958 tuberculosis Rvl753 and its variants, as described in document WO 2010010180, for example, a sequence of Rvl753 chosen from SEQ ID NO: 1 and 2 to 7 of document WO 2010010180, in particular SEQ ID NO: 1. Another antigen of interest in the field of tuberculosis is Rv2386 and its variants, as described in document WO 2010010179, for example, a sequence of Rv2386 chosen from SEQ ID NO: 1 and 2 to 7 from document WO 2010010179, in particular SEQ ID NO: 1. Other antigens of interest in the field of tuberculosis include Rv3616 and its variants, as described in document WO 2011092253, for example, a natural sequence of Rv3616 chosen from SEQ ID NO: 1 and 2 to 7 of document WO 2011092253 or a modified sequence of Rv3616 such as those chosen from SEQ ID NO: 161 to 169, 179 and 180 of document WO 2011092253, in particular SEQ ID NO: 167. Another antigen of interest is HBHA, as described in WO 97044463, WO 03044048 and WO 2010149657. The aforementioned patent applications WO 2010010180, WO 2010010179, WO 2011092253, WO 97044463, WO 03044048 and WO 2010149657 are incorporated herein by reference in their entirety for the purpose of defining antigens which may be used in the present invention.
Other antigens of interest are those comprising (or consisting of): Rv174, also known under the name of DPV, as described by SEQ ID NO: 8 of document WO 2010010177; Rvl793, also known as MTI or Mtb9.9, as described by SEQ ID NO: 10 from WO 2010010177; Rv2087, also known as
BE2017 / 5958 name of MSL or Mtb9.8, as described by SEQ ID NO: 9 of document WO 2010010177; Rv3616, also known as HTCC1 or Mtb40, as described by SEQ ID NO: 1 and 2 to 7 of document WO 2010010177 or SEQ ID NO: 161 to 169, 179 or 180 of document WO 2011092253; and / or Rv3874, also known as CFP10 or Tb38.1, as described by SEQ ID NO: 9 from document WO 2010010177; or one of their immunogenic parts (as at least 20, 50, 75 or 100 residues resulting therefrom) or one of their variants (as having at least 70%, 80%, 90% or 95% d 'identity with them). (Documents WO 2010010177 and WO 2011092253 are incorporated herein by reference in their entirety for the purpose of defining antigens which can be used in the present invention).
The tuberculosis antigens are most suitably used in the form of a polypeptide, but alternatively they can be provided in the form of a polynucleotide encoding said polypeptide.
Another antigen which can be used in accordance with the present invention is derived from the varicella zoster virus (VZV). The VZV antigen for use in the invention may be any suitable VZV antigen or one of its immunogenic derivatives, suitably being a purified VZV antigen.
In one embodiment, the VZV antigen is the VZV glycoprotein gE (also known as gpl) or one of its immunogenic derivatives. The wild-type or full-length gE protein consists of 623 amino acids comprising a signal peptide, the main part of the protein, an anchoring region
BE2017 / 5958 hydrophobic (residues 546 to 558) and a C-terminal tail. In one aspect, a C-terminal truncated form of gE (also called truncated gE or truncated form of gE) is used, whereby truncation removes 4 to 20 percent of the total amino acid residues at the carboxyl terminus. terminal. In another aspect, in truncated gE, the carboxyterminal anchoring region is lacking (suitably, approximately amino acids 547 to 623 of the wild-type sequence). In another aspect, gE is a truncated gE having the sequence of SEQ ID NO: 6.
The gE antigen, its anchorless derivatives (which are also immunogenic derivatives) and their production are described in document EP 0 405 867 and the references therein [see also, Vafai A., Antibody binding sites on truncated forms of varicellazoster virus gpl (gE) glycoprotein, Vaccine 1994 12: 12659]. Document EP 192 902 also describes gE and its production. Truncated gE is also described by Haumont et al. Virus Research (1996) vol 40, p 199-204, incorporated here in its entirety. An adjuvanted VZV gE composition suitable for use in accordance with the present invention is described in WO 2006/094756, i.e., carboxy-terminal truncated VZV gE in combination with an adjuvant comprising QS21, 3D-MPL and liposomes additionally containing cholesterol. Leroux-Roels I. et al. (J. Infect. Dis. 2012, 206: 1280-1290) reported in a phase I / II clinical trial the evaluation of the truncated gE VZV subunit vaccine.
BE2017 / 5958
The antigen may include or consist of preparations derived from the human respiratory syncytial virus (RSV). In certain favorable embodiments, a polypeptide antigen is an F protein polypeptide antigen from RSV. Particularly suitable as the polypeptide antigen component in context, conformally constrained polypeptide F antigens.
Proteins
Conformationally constrained have been described before in both the prefusion (PreF) and postfusion (PostF) conformations.
Such conformally constrained proteins generally include a modified protein ectodomain of the
RSV. A protein ectodomain polypeptide is a part of the protein
RSV F which comprises all or part of the extracellular domain of the RSV F protein and which lacks a functional transmembrane domain (for example, by deletion or substitution), which can be expressed, for example, in soluble form ( not attached to a membrane) in cell culture.
Examples of protein F antigens conformally constrained in the prefusion conformation have been described in the art and are disclosed in detail, for example, in US Patent No. 8,563,002 (WO 2009079796); published patent application US No. US 2012/0093847 (WO 2010/149745); US 2011/0305727 (WO 2011/008974); US 2014/0141037, WO 2012/158613 and WO 2014/160463, each being incorporated herein by reference for the purpose of illustrating prefusion F polypeptides (and nucleic acids), and methods
BE2017 / 5958 for their production. Typically, the antigen is in the form of a trimer of polypeptides. Other publications providing examples of F proteins in the prefusion conformation include: McLellan et al., Science, Vol. 340: 1113-1117; McLellan et al., Science, Vol 342: 592-598, and Rigter et al., PLOS One, Vol. 8: e71072, each of them can also be used in the context of the immunogenic combinations disclosed here.
For example, a protein F polypeptide stabilized in the prefusion conformation generally comprises an ectodomain of an F protein (for example, a soluble protein F polypeptide) comprising at least one modification which has stabilized the prefusion conformation of the F protein. example, the modification can be chosen from an addition of a trimerization domain (generally at the C-terminal end), the deletion of one or more of the furin cleavage sites (at amino acids -105 at 109 and -133 at 136), a deletion of the pep27 domain, the substitution or addition of a hydrophilic amino acid in a hydrophobic domain (for example, HRA and / or HRB). In one embodiment, the conformally constrained preF antigen comprises an F2 domain (for example, amino acids 1 to 105) and a Fl domain (for example, amino acids 137 to 516) of an F protein polypeptide of the RSV without any intervening furin cleavage site, where the polypeptide further comprises a heterologous trimerization domain in the C-terminal position with respect to the Fl domain. Optionally, the PreF antigen comprises
BE2017 / 5958 also a modification which changes glycosylation (for example, increases glycosylation), such as a substitution of one or more amino acids at the positions corresponding to amino acids -500 to 502 of an F protein of RSV. When an oligomerization sequence is present, it is preferably a trimerization sequence. Appropriate oligomerization sequences are well known in the art and include, for example, the leukine zipper protein zipper GCN4 from yeast, the trimerization sequence from fibritin from bacteriophage T4 ("foldon"), and trimeric domain of HA flu. Additionally or alternatively, the conformally constrained F polypeptide in the prefusion conformation may comprise at least two introduced cysteine residues, which are in close proximity to each other and form a disulfide bridge which stabilizes the F polypeptide in pre- merger of the RSV. For example, the two cysteines can be about 10 Å apart. For example, cysteines can be introduced at positions 165 and 296 or at positions 155 and 290. An example of PreF antigen is represented by SEQ ID NO: 7.
The antigen may include or consist of preparations derived from HIV. The antigen can be an HIV protein such as the HIV envelope protein. For example, the antigen may be an HIV envelope gp120 polypeptide or one of its immunogenic fragments.
A suitable antigen is the HIV clade B gpl20 polypeptide of SEQ ID NO: 8 of the published application
BE2017 / 5958
WO 2008/107370 (or an immunogenic fragment of this polypeptide). SEQ ID NO: 8 of document WO 2008/107370 is incorporated by reference in this application.
Suitable antigens also include a polypeptide comprising the V1V2 region of SEQ ID NO: 1 of published application WO 2015/036061, or an immunogenic derivative or fragment of the V1V2 region of SEQ ID NO: 1. In addition, a polypeptide comprising V1V2 region of SEQ ID NO: 5 of document WO 2015/036061 or an immunogenic derivative or fragment of the V1V2 region of SEQ ID NO: 5 can be used as an appropriate antigen. SEQ ID NO: 1 and SEQ ID NO: 5 of document WO 2015/036061 are incorporated by reference.
In another embodiment, the antigen may comprise two or more different polypeptide antigens from the HIV envelope gp120 (or immunogenic fragments of these polypeptides). Suitable antigens include HIV clade C gpl20 and polypeptide antigens including TV1 gpl20 (SEQ ID NO: 8) and 1086C gpl20 (SEQ ID NO: 9).
Other suitable HIV antigens include the HIV proteins Nef, Gag and Pol and their immunogenic fragments.
The composition may comprise one or more non-typable Haemophilus influenzae antigens, for example chosen from: the fimbrin protein [(US 5766608 - Ohio State Research Foundation)] and fusions comprising peptides derived therefrom [for example, fusions LB1 (f) peptide; US 5,843,464 (OSU) or WO 99/64067]; OMP26 [WO 97/01638 (Cortecs)]; P6 [EP 281 673 (State University of New York)]; TbpA and / or
BE2017 / 5958
TbpB; Hia; Hsf; Hin47; Hif; Hmwl; Hmw2; Hmw3; Hmw4; Hap; D15 (WO 94/12641); protein D (EP 594,610); P2; and P5 (WO 94/26304); protein E (WO 07/084053) and / or PilA (WO 05/063802). The composition can comprise one or more protein antigens of Moraxella catarrhalis, for example, chosen from: OMP106 [WO 97/41731 (Antex) and WO 96/34960 (PMC)]; OMP21; LbpA and / or LbpB [WO 98/55606 (PMC)]; TbpA and / or TbpB [WO 97/13785 and WO 97/32980 (PMC)]; CopB [Helminen
ME, et al. (1993) Infect. Immun. 61: 2003-2010]; UspAl and / or UspA2 [WO 93/03761 (University of Texas)]; OmpCD; HasR (PCT / EP 99/03824); PilQ (PCT / EP 99/03823); OMP85 (PCT / EP 00/01468); lipo06 (GB 9,918 208.1); lipoil (GB 9,918,038.2); P6 (PCT / EP 99/03822); OmplAl (PCT / EP 99/03257); and OmpE.
(GB 9,917,977.2); lipolO (GB 9,918 302.2); lipol8 (PCT / EP 99/03038); D15 (PCT / EP 99/06781); Hly3
In one embodiment, the composition may include one or more non-typeable H. influenzae protein (S) antigens (NTHi) and / or one or more M. catarrhalis protein (s) antigens. The composition may include protein D (PD) from H. influenzae. Protein D can be as described in document WO 91/18926. The composition may further comprise protein E (PE) and / or pilin A (PilA) from H. Influenzae. Protein E and pilin A can be as described in document WO 2012/139225. Protein E and pilin A can be in the form of a fusion protein; for example, LVL735 as described in document WO 2012/139225. For example, composition can
BE2017 / 5958 include three NTHi antigens (PD, PE and PilA, with the latter two combined in the form of a PEPilA fusion protein). The composition may further comprise UspA2 of Μ. catarrhalis. UspA2 can be as described in document WO 2015125118, for example, MC009 ((Μ) (UspA2 31-564) (HH)) described in document WO 2015125118. For example, the composition can comprise three antigens of NTHi (PD , PE and PilA, with the latter two combined as a fusion protein PEPilA) and an antigen of Μ. catarrhalis (UspA2).
A plurality of antigens can be provided. For example, a plurality of antigens can be provided to enhance the triggered immune response (e.g., to provide strong protection), a plurality of antigens can be provided to enhance the immune response (e.g., to provide strong protection protection against a wide range of pathogenic strains or in a large proportion of a population of subjects) or a plurality of antigens can be provided to currently trigger immune responses to a number of disorders (thereby simplifying the administration protocols). When a plurality of antigens are provided, these may be in the form of separate proteins, or they may be in the form of one or more fusion proteins.
The antigens can be supplied separately from the TLR4 agonist and saponin or the antigens can be supplied in co-formulation with the TLR4 agonist, saponin or with the TLR4 agonist and also
BE2017 / 5958 with saponin. When a plurality of antigens are provided, some or all of these may be provided separately from the TLR4 agonist and saponin or some or all of them may be provided in a co-formulation with the TLR4 agonist , saponin or with the agonist of TLR4 and also with saponin. Optionally, one or more antigens from the antigen or antigens coformulated with saponin can be coformulated with the agonist of TLR4.
The antigen can be supplied in an amount of 0.1 to 100 µg per human dose.
Other excipients
The TLR4 agonist, saponin and (as appropriate) the antigen are suitably provided in the form of liquid preparations which are substantially aqueous.
Suitably, the compositions used in the present invention have a human dose volume of between 0.05 ml and 1 ml, such as between 0.1 and 0.5 ml, in particular a dose volume of about 0.5 ml, or 0.7 ml. The volumes of the compositions used may depend on the route and location of administration, with smaller doses being given intradermally or if both the TLR4 agonist and the saponin are administered at the same location.
In another embodiment, a buffer is added to the composition. The pH of a liquid preparation is adjusted in view of the components of the composition and of a suitability necessary for administration to the subject. Suitably, the pH of a liquid mixture is at least 4, at least 5, at least 5.5, at least
BE2017 / 5958 of the liquid mixture may be less than
9, less than less than 7.5 or less than 7. In other embodiments,
The pH of the liquid mixture is between and 9, between and as between 5.5 and
A suitable buffer can acetate, citrate, succinate, buffer
Na / KcPCt or in being chosen from tartrates is a
K / K2PO4.
The buffer can histidine, maleate, phosphate, and TRIS. In one embodiment phosphate buffer be present in an amount of at least 6 mM, minus 40 mM. The buffer may such as Na / Na2PC> 4, liquid mixture minus 10 mM or be present in the liquid mixture in an amount less than 100 mM, less than
It is parenteral, mM well or less than known as osmolality solutions pharmaceutically distortion or the pharmaceutically as solutions
0 mM.
for an acceptable administration one should present to avoid cell lysis.
acceptable will exhibit approximately isotonic or
Osmolality will generally mean osmolality which is slightly hypertonic.
Suitably, the compositions of the present invention, when reconstituted, will exhibit osmolality in the range of 250 to 750 mOsm / kg, by
500 example, the osmolality can be in the range
250 to 550 mOsm / kg, as in the mOsm / kg range.
Osmolality can be known in the art, to be measured using techniques such as the use of a commercially available osmometer, for example,
BE2017 / 5958 the Advanced® Model 2020 instrument available from
Advanced Instruments Inc. (United States).
An "isotonic agent" is a compound which is physiologically tolerated and imparts an appropriate tone to a formulation to prevent the net flow of water through the cell membranes which are in contact with the formulation. In certain embodiments, the isotonicity agent used for the composition is a salt (or mixtures of salts). However, in other embodiments, the composition comprises a nonionic isotonic agent and the concentration of sodium chloride in the composition is less than 100 mM, as less than 80 mM, for example, less than 50 mM, as less than 40 mM, less than 30 mM and especially less than 20 mM. The ionic strength in the composition can be less than 100 mM, as less than 80 mM, for example, less than 50 mM, as less than 40 mM or less than 30 mM.
In a particular embodiment, the nonionic isotonicity agent is a polyol, such as sorbitol. The concentration of sorbitol can be, for example, between about 3% and about 15% (w / v), such as between about 4% and about 10% (w / v). Adjuvants comprising an immunologically active saponin fraction and a TLR4 agonist in which the isotonicity agent is a salt or a polyol have been described in document WO 2012/080369.
The present invention can be applied for use in the treatment or prophylaxis of a disease or disorder associated with one or more of the antigens described above. In one embodiment,
BE2017 / 5958 the disease or disorder is chosen from malaria, tuberculosis, COPD, HIV and herpes.
In order to fully obtain the benefits of the present invention,
The agonist of
TLR4 should be administered in a composition which is substantially free from saponin. Suitably, the composition comprising the agonist of
TLR4 will contain less than 1 pg of saponin per human dose, in particular less than 0.5 pg of saponin, especially less than 0.1 pg of saponin (for example, devoid of saponin). In certain embodiments, 3D-MPL is provided in a composition which contains less than 1 pg of saponin per human dose, in particular less than 1 pg of saponin, especially less than 0.1 pg of saponin (for example, devoid of saponin). In some embodiments, 3D-MPL is provided in a composition which contains less than 1 pg of QS21 per human dose, in particular less than 0.5 pg of QS21, especially less than 0.1 pg of QS21
In addition, the saponin should be administered in a composition which is substantially free of TLR4 agonist. Suitably, the composition comprising the saponin will contain less than 1 pg of TLR4 agonist per human dose, in particular less than 0.5 pg of TLR4 agonist, especially less
0.1 µg TLR4 agonist (for example, devoid of TLR4 agonist). In certain embodiments, QS21 is provided in a composition which contains less than 1 pg of TLR4 agonist per human dose, in particular less than 0.5 pg of TLR4 agonist,
BE2017 / 5958 especially less than 0.1 pg of TLR4 agonist (for example, devoid of TLR4 agonist). In some embodiments, QS21 is provided in a composition which contains less than 1 pg of 3D-MPL per human dose, in particular less than 0.5 pg of 3D-MPL, especially less than 0.1 pg of 3D- MPL (for example, devoid of 3D-MPL).
Suitably, the compositions used in the present invention do not include aluminum salt particles.
Multi-chamber syringes
The use of multi-chamber syringes provides a convenient method for the separate administration of the TLR4 agonist and the saponin. Multi-chamber syringes can be configured to provide simultaneous but separate administration of the TLR4 agonist and saponin, or they can be configured to provide sequential administration (in one order or the other).
In other configurations, an element may be supplied in dry form (for example, lyophilized) in one chamber and reconstituted by the diluent contained in the other before administration.
Examples of multi-chamber syringes can be found in disclosures such as
WO 2016172396, although a variety of other configurations are possible.
The teachings of all of this application, including references in the patent application and granted patents, are incorporated herein by reference in their entirety. A composition or method or
BE2017 / 5958 a process defined as “comprising” certain elements is understood to include a composition, a method or a process (respectively) made up of these elements. As used herein, "consisting essentially of" means that additional components may be present provided that they do not change the overall properties or the function.
The invention will be further described with reference to the following nonlimiting examples.
Example 1 - Study of the impact of the separate formulation of the agonist of TLR4 and of saponin on the innate immune responses in mice
Goals)
The AS01 liposomal adjuvant system induces a specific signature of the draining lymph nodes very early after the injection. The innate IFNg (i-IFNg) is the IFNg produced by the innate cells of the draining lymph nodes which, in mice, peaks around 6 hours after injection with ASOl and results from the synergy between 3D -MPL and QS21 when co-formulated in liposomes. Consequently, the IFNg detected 6 h after the injection in mice may be a key parameter for distinguishing the integrity of the AS01 adjuvant system. It is believed that the innate signature is essential for the effect of the adjuvant AS01. In human sera, the signature of the innate IFNg is present in patients immunized with an adjuvanted vaccine with ASOIb and it is the mark of protection (MAL-027).
The objective of this study was to estimate whether the
BE2017 / 5958 coformulation of the TLR4 agonist, 3D-MPL, and the saponrne, QS21, is necessary to maintain the observed signature of
1'i-IFNg.
For this, sourrs were immunized by your intramuscular with
Varicella zoster virus gE antigen adjuvanted with 3DMPL and
QS21 co-formulated or with separate 3D-MPL and QS21 formulated in liposomes. The iliac lymph nodes were removed 6 hours after the injection, the IFNg content was evaluated by a test
ELISA.
Materials and processes
Study design overview
The mice were housed in a pathogen-free establishment and the animal studies were conducted according to protocols approved by the GSK Committee on Use and Care of Animals.
The mice used in this study were females 6 to 8 weeks old of the C57BL / 6 strain, they were separated into 3 groups. The mice all received intramuscular injections into the gastrocnemius muscles on the right and left. For group 1 (“combined” liposomal formulation), each mouse (n = 20) received 5 μg of recombinant gE (Haumont et al. 1996; Dendouga et al. 2012; Fochesato et al. 2016) formulated with 1/125 of the human dose of ASOlß, corresponding to 0.4 μg of 3D-MPL and 0.4 μg of QS21 in a total volume of 40 μl (volume injected per muscle = 20 μl, administered using syringes each with 10 μl ). In the group evaluating the “separate” liposomal formulations (group 2), each mouse (n =
BE2017 / 5958
20) received in each muscle (right and left), 10 μΐ syringe containing 3D-MPL (0.4 gg of 3DMPL) and gE (1.25 gg) and 1 syringe of 10 gl containing the QS21 (0.4 gg) and gE (1.25 gg). In group 3 (“antigen” formulation), each mouse (n =
5) received the gE antigen in buffer alone (5 gg / animal) in a total volume of 40 gl / animal, administered using four syringes each with 10 gl.
In order to inject with the two syringes at the same site, the mice were stained at the injection site. The time between each injection was kept to a minimum, generally less than 30 seconds.
See Table 1 for a summary of the study design.
Table 1
Study design summary
Group Injected volume(gl) Antigen(pg) MPL(pg) QS(pg)1 Byanimal 40 5 0.4 0.4 Bymu saw 20 2.5 0.2 0.2 By 10 1.25 0.1 0.1 Syringe 1INJ 10 1.25 0.1 0.1 Syringe 2 2 Byanimal 40 5 0.4 0.4
BE2017 / 5958
By 20 2.5 0.2 0.2 mu saw By 10 1.25 0.2 0.2 Syringe 1INJ 10 1.25 Syringe 2 3 By 40 5 animal By 20 2.5 mu saw By 10 1.25 Syringe 1INJ 10 1.25 Syringe 2
Immunological readings
- GLD sampling and processing of frozen samples instantly
For estimation of proinflammatory IFNg production, the left and right iliac lymph nodes were removed 6 hr after immunization.
Tissue samples dissected from the draining lymph were individually homogenized in 1 ml (0.5 ml / GLD) of PBS containing a cocktail of anti-protease inhibitors (Sigma-Aldrich). The homogenates were clarified by centrifugation and stored. at -70 ° C until analysis.
- mouse IFNg by ELISA test
The Quantikine Mouse IFN-gamma immunoassay (R&D systems MIF00) is designed to measure mouse IFNg in mouse serum and tissue homogenate.
BE2017 / 5958
The results obtained using natural mouse IFNg showed dose-response curves which were parallel to the calibration curves obtained using the recombinant standards from the kit. The entire assay procedure has been followed carefully and can be summarized as described below.
A presensitized microplate with the capture antibody was provided. Serum or tissue homogenate samples (or standards) were added. The IFNg present in the sample is bound by the immobilized antibody after incubation at room temperature for 2 hours. Unbound materials were washed away (5 washes, with the wash buffer provided). A second antibody labeled with HRP (detection antibody) was added and incubated at room temperature for 2 hours to bind to the captured IFNg. The unbound detection antibody was washed away with 5 more washes. The tetramethylbenzidine (TMB) substrate solution was then added to the wells and incubated at room temperature for 30 minutes (protected from light). The development of the color was stopped and the absorbance of the color at 450 nm was measured (E-Max Reader). The results were calculated using softmax-pro software.
For the statistical analysis, an analysis of variance model including the group factor was adapted on the IFNg measurements of groups 1 and 2 transformed into log. Least squares means and group difference were derived from the model. After retrotransformation, the geometric means with
BE2017 / 5958 95% confidence intervals as well as the ratio of geometric means have been reported.
Results
The data are shown graphically in Figures 1 and 2. The values of the unadjuvanted group were all below the detection limit. The geometric means estimated for each group are summarized in Table 2. The comparison between co-formulation and separate administration of 3D-MPL and QS21 is summarized in Table 3.
Table 2
Geometric means (with confidence intervals at
%) of the IFNg estimated by the ANOVA model
Average 95% CI Group Description geometric Limit Limit in e inferior superior(pg / ml) e e 1 Co-formulated 155.0 119, 8 200.4 2 Separate 149, 2 115.4 192, 9Table 3 Comparison between responses s in IFNg obtained Report of 90% confidence intervals Average comparison Limit Limit geometric *lower superior Administration separated against 0.96 0.71 1.30 co-formulation * The denominator of reports of medium geometric is always associated to the coformu lation.
Conclusion
It has been shown that the separate formulation is not less than the co-formulation under the test conditions.
BE2017 / 5958
Example 2 Study of the Impact of the Separate Formulation of the TLR4 Agonist and the Saponin on Adaptive Immune Responses in Mice
Goals)
In light of the positive discovery in Example 1 that the innate immune responses observed following administration of the separately formulated components were not inferior to the co-formulated components, work has been extended to study any impact on the adaptive immune responses. For this, mice were immunized intramuscularly with the varicella zoster virus gE antigen adjuvanted with co-formulated 3D-MPL and QS21 or with separate 3D-MPL and QS21 in a dose range. The triggered T-cell and antibody responses were then quantified.
Materials and processes
Study design overview
The mice were housed in a pathogen-free establishment and the animal studies were conducted according to protocols approved by the GSK Committee on Use and Care of Animals.
The mice used in this study were females 6 to 8 weeks old of the C57BL / 6 strain, they were separated into 11 groups of 5 mice. The
BE2017 / 5958 all mice received intramuscular injections into the gastrocnemius muscles on the right and left on day 0 and on day 14. Samples of serum and spleen tissue were taken on day 21, to measure the specific responses of humoral gE and T lymphocytes (CD4 and CD8 Thl) respectively.
Table 4
Study design summary
Group Materialtested Formulation approach Dose ofThe antigen Dose of each immunostimulant 1 gE / 3D-MPL+ QS21 Co-formulation 5 gg 1 pg 2 Separated 3 Co-formulation 0.4 pg 4 Separated 5 Co-formulation 0.2 pg 6 Separated 7 Co-formulation 0.1 pg 8 Separated 9 Co-formulation 0.05 pg 10 Separated 11 gE / PBS - -
For each group, a total volume of 20 μΐ was administered on each occasion. This consisted of 2 injections into the muscle to the left of either of the separate formulations (for separate 3D-MPL and QS21) or identical formulations (co-formulated and PBS control). In order to inject with the two syringes at
BE2017 / 5958 same site, the mice were stained at the injection site. The time between the two injections was kept to a minimum, less than 30 seconds.
Immunological readings
- Collection of spleens and treatment of samples for cell suspension and intracellular staining of cytokines (ICS) on day 7PII
The spleens were collected in RPMI medium and dissociated using a Potter-type tissue grinder (homogenizer) using two strokes up and down. The homogenized samples were transferred to 15 ml round bottom polypropylene tubes. The fibrous material was removed by filtration through a 100 μm nylon cell screen. The cells were then washed, counted and resuspended at 10 * 7 cells per ml.
ICS (intracellular cytokine staining) is the technology that allows the quantification of antigen-specific T cells based on the production of cytokines.
Lymphoid cells are restimated overnight in vitro with gE peptides in the presence of a secretion inhibitor (brefeldine A). These cells are then treated by a conventional immunofluorescence procedure using fluorescent antibodies (extracellular staining: CD4, CD8, intracellular staining: TNFa, IFNg and IL2).
The results are expressed in frequency of cells positive for a cytokine within a population of CD4 and CD8 T lymphocytes. The analysis was
BE2017 / 5958 concentrated on cells expressing at least two cytokines.
- Determination of gE by an ELISA test on day 7PII on sera
Total anti-gE IgG (VZV) was measured by an ELISA test. 96-well plates were sensitized with the antigen overnight at 4 ° C. The plates were then washed and saturated with saturation buffer for 1 hour at 37 ° C. Then, 100 μΐ of mouse or standard or diluted control serum was added and incubated for 1 hour at 37 ° C. After washing, the plates were incubated for 1 hour at 37 ° C with 100 μΐ of anti-mouse IgG antibody-HRP. After washing, 100 μΐ of TMB per well were added and the plates were kept in the dark at room temperature for 15 minutes. To stop the reaction, 100 μΐ of 0.4 N H2SO4 was added per well. The absorbance was read at a wavelength of 450/630 nm by an Elisa plate reader. The results were calculated using Softmax-pro software.
Results
1. T cell response: g4 specific CD4 + T cell responses
The data are represented graphically in FIGS. 3 and 4. The geometric means of each group are summarized in table 5. The comparisons of co-formulation against separate formulation are summarized in table 6.
Table 5
CD4 + expressing at least 2 cytokines: geometric means and their 95% CI by group
BE2017 / 5958
Process Dose NOT Geometric mean IC at95% -Lower limit IC at95% -Upper limit Co-formulated 0.05 5 0.33 0.21 0.54 Co-formulated 0.1 5 0.40 0.29 0.56 Co-formulated 0.2 5 0.52 0.42 0.64 Co-formulated 0.4 5 0.44 0.32 0.60 Co-formulated 1 5 0.48 0.31 0.75 Separated 0.05 5 0.23 0.12 0.42 Separated 0.1 5 0.29 0.16 0.53 Separated 0.2 5 0.43 0.36 0.52 Separated 0.4 5 0.45 0.36 0.57 Separated 1 5 0.45 0.34 0.61
Table 6
CD4 + expressing at least 2 cytokines: ratios of geometric means and their 95% CI (comparison of formulation dose by dose)
Comparison Ratio of geometric means s IC at95% -Lower limit IC at95% -Upper limit e Separated / Co-formulated at a dose of 0.05 0.68 0.35 1.31
BE2017 / 5958
Separated / Co-formulated at a dose of0.1 0.72 0.40 1.31 Separated / Co-formulated at a dose of0.2 0.83 0.66 1.05 Separated / Co-formulated at a dose of0.4 1.03 0.74 1.42 Separated / Co-formulateds at a dose of 1 0.94 0.60 1.48
In this experiment, the frequencies of gE-specific CD4 + T cells increase with the dose of immunostimulant up to the dose of 0.2 pg (1 / 250HD per animal) then reaching a plateau. This was observed for the separate formulations and co-formulations (see Table 6). Furthermore, when looking at the ratio of the geometric means of the frequencies of CD4 + T lymphocytes for the separate / co-formulated components, the two formulation approaches induce responses comparable to the dose of 1, 0.4 and 0.2 μg. . A slight difference appears with the lower doses (0.1 and 0.05 pg) (see Table 6).
2. Antibody response: anti-gE IgG level
The data are represented graphically in FIGS. 5 and 6. The geometric means of each group are summarized in table 7. The comparisons of coformulation against separate formulation are summarized in table 8.
Table 7
Antibody response: geometric means and their
95% CI per group
BE2017 / 5958
Process Dose NOT Geometric mean IC at95% -Lower limit IC at95% -Upper limit Co-formulated 0.05 5 5,642.86 3,223.63 9877.64 Co-formulated 0.1 5 9824.45 4,768.72 20,240.18 Co-formulated 0.2 5 8,708.63 5712.27 13,276.73 Co-formulated 0.4 5 13,041.60 10,126.89 16,795.23 Co-formulated 1 5 17,620.54 15,231.37 20,384.46 Separated 0.05 5 3514.16 2200.79 5611.32 Separated 0.1 5 5920.71 3603.14 9728.96 Separated 0.2 5 7,436.33 5600.54 9873.86 Separated 0.4 5 13,624.91 8850.08 20,975.86 Separated 1 5 17,129.23 11,845.40 24,770.00
Table 8
Antibody response: ratios of geometric means and their 95% CI (comparison of formulation dose by dose)
Comparison Ratio of geometric means s IC at95% -Lower limit IC at95% -Upper limit e Separated / Co-formulated at a dose of 0.05 0.62 0.34 1.15
BE2017 / 5958
Separated / Co-formulated at a dose of 0.1 0.60 0.29 1.27 Separated / Co-formulated at a dose of 0.2 0.85 0.55 1.32 Separated / Co-formulated at a dose of 0.4 1.04 0.68 1, 61 Separated / Co-formulateds at a dose of 1 0.97 0.68 1.40
The results suggest that the specific IgE levels of gE increase with the dose of the immunostimulants. This was observed for the 5 administrations which were both separate and co-formulated (see Table 8). Furthermore, when looking at the geometric mean ratios of the IgG levels for the separate / co-formulated components, it appears that the two formulations induce a similar antibody response at the dose of 1, 0.4 and 0, 2 pg. A slight difference appears with the lower doses (0.1 and 0.05 pg) (see Table 9).
Conclusion
Overall, separate administration of the TLR4 agonist and saponin did not produce a significant change in the cellular and antibody responses observed.
BE2017 / 5958
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权利要求:
Claims (69)
[1]
1. TLR4 agonist for use as an adjuvant with a saponin, where the TLR4 agonist is administered separately from the saponin.
[2]
2. Saponin for use as an adjuvant with a TLR4 agonist, where the saponin is administered separately from the TLR4 agonist.
[3]
3. Use of a TLR4 agonist in the manufacture of an adjuvant for use with a saponin, where the TLR4 agonist is administered separately from the saponin.
[4]
4. Use of a saponin in the manufacture of an adjuvant for use with a TLR4 agonist, where the saponin is administered separately from the TLR4 agonist.
[5]
5. Method for adjuvanting the immune response to an antigen using a TLR4 agonist and a saponin, characterized in that the TLR4 agonist and the saponin are administered separately.
[6]
6. Method for adjuvanting the immune response of a subject to an antigen using a TLR4 agonist and a saponin, said method comprising the following steps:
(i) administration about the TLR4 agonist;
(ii) administration regarding the saponin;
(iii) administration regarding the antigen;
in the leg the steps can be taken in any order, the TLR4 agonist and the saponin are administered separately, and the antigen can be optionally co-formulated with the TLR4 agonist,
BE2017 / 5958 saponin or co-formulated with each of the agonist of
TLR4 and saponin.
[7]
7. Method for inducing an immune response in a subject using a TLR4 agonist and a saponin, characterized in that the TLR4 agonist and the saponin are administered separately to the subject.
[8]
8. Method for inducing an immune response in a subject using a TLR4 agonist and a saponin, said method comprising the following steps:
(i) administration about the TLR4 agonist;
(ii) administration regarding the saponin;
in which the steps can be undertaken in any order, the TLR4 agonist and the saponin are administered separately.
[9]
9. Kit including:
(i) a first composition comprising a TLR4 agonist; and
(ii: ) a second composition including a saponin • 10. Kit according to the claim 7, including besides a antigen. 11. Kit according to the claim 10, in which
the antigen is present in the first and / or second compositions.
[10]
12. Kit including:
(i) a first composition comprising a TLR4 agonist;
(ii) a second composition comprising a saponin; and (iii) a third composition comprising a
BE2017 / 5958 antigen.
[11]
13. Multi-chamber syringe comprising a first compartment and a second compartment, said first compartment comprising a first composition comprising a TLR4 agonist and said second compartment comprising a second composition comprising a saponrne, said first and second compositions being administrable on a su j and either sequentially or simultaneously.
[12]
14. The multi-chamber syringe according to claim 13, further comprising at least one antigen.
[13]
15. A multi-chamber syringe according to claim 14, comprising a third compartment, said third compartment comprising a third composition comprising the at least one antigen.
[14]
16. Multi-chamber syringe according to claim 14, in which at least one antigen is present in the first and / or second compositions.
[15]
17. TLR4 agonist, saponin, use, method, kit or syringe according to any one of claims 1 to 16 for administration to a human subject.
[16]
18. TLR4 agonist, saponin, use, method, kit or syringe according to any one of claims 1 to 17 wherein the TLR4 agonist is a lipopolysaccharide.
[17]
19. TLR4 agonist, saponin, use, method, kit or syringe according to any one of
BE2017 / 5958 claims 1 to 17 wherein the TLR4 agonist is a non-toxic derivative of lipid A.
[18]
20. TLR4 agonist, saponin, use, method, kit or syringe according to claim 19 wherein the TLR4 agonist is monophosphoryl-lipid A.
[19]
21. TLR4 agonist, saponin, use, method, kit or syringe according to claim 20 wherein the TLR4 agonist is a 3-deO-acylated monophosphoryl-lipid A.
[20]
22. TLR4 agonist, saponin, use, method, kit or syringe according to claim 18 wherein the TLR4 agonist is a glucopyranosyl lipid adjuvant
[21]
23. TLR4 agonist, saponin, use, method, kit or syringe according to any of claims 1 to 22, wherein the amount of TLR4 agonist in a single dose for human adults is 1 to 100 pg, such as 40 at 60 pg or 20 to 30 pg.
[22]
24. TLR4 agonist, saponin, use, method, kit or syringe according to any one of claims 1 to 23 wherein the saponin can be obtained from Quillaja saponaria.
[23]
25. TLR4 agonist, saponin, use, method, kit or syringe according to claim 24 wherein the saponin comprises one or more of QS7, QS17, QS18 and QS21.
[24]
26. TLR4 agonist, saponin, use, method, kit or syringe according to claim 25 wherein the saponin comprises QS21.
[25]
27. TLR4 agonist, saponin, use, method, kit or syringe according to claim 25 wherein the saponin consists of QS21.
BE2017 / 5958
[26]
28. TLR4 agonist, saponin, use, method, kit or syringe according to one of claims 1 to 27, wherein the amount of saponin in a single dose for human adults is from 1 to 100 pg, such as 40 to 60 pg or 20 to 30 pg.
[27]
29. TLR4 agonist, saponin, use, method, kit or syringe according to claim 28, wherein the amount of TLR4 agonist in a single dose for a human adult is 40 to 60 µg, such as about 50 µg, and the amount saponin is 40 to 60 pg, like about 50 pg.
[28]
30. TLR4 agonist, saponin, use, method, kit or syringe according to claim 28, wherein the amount of TLR4 agonist in a single dose for human adults is 20 to 30 µg, such as about 25 µg, and the amount saponin is 20 to 30 pg, like about 25 pg.
[29]
31. TLR4 agonist, saponin, use, method, kit or syringe according to one of claims 1 to 30, wherein the TLR4 agonist and the saponin are administered intramuscularly.
[30]
32. TLR4 agonist, saponin, use, method, kit or syringe according to any of claims 1 to 31, wherein the TLR4 agonist and the saponin are administered at locations spaced at least 5 mm apart, as spaced apart at least 1 cm.
[31]
33. TLR4 agonist, saponin, use, method, kit or syringe according to any of claims 1 to 32, wherein the TLR4 agonist and the saponin are administered at locations spaced less than 10 cm apart, less than 5 cm.
[32]
34. TLR4 agonist, saponin, use, method, kit or syringe according to any of claims 1 to 33, wherein the TLR4 agonist and the saponin are administered at a draining location to the same lymph node.
BE2017 / 5958
35. Agonist of TLR4, saponin, use, process, kit or syringe depending 1 'a of claims 1 at 34, where the agonist of TLR4 and the
saponin are administered intramuscularly to the same muscle.
[33]
36. TLR4 agonist, saponin, use, method, kit or syringe according to any of claims 1 to 35, wherein the TLR4 agonist and the saponin are administered at the same location.
[34]
37. TLR4 agonist, saponin, use, method, kit or syringe according to any one of claims 1 to 36, wherein the TLR4 agonist and the saponin are administered at 6 hours apart, as in 2 hours apart, in particular 1 hour apart, such as 30 minutes and especially 15 minutes (for example, 5 minutes).
[35]
38. TLR4 agonist, saponin, use, method, kit or syringe according to any one of claims 1 to 37, in which the TLR4 agonist and the saponin are administered at least 5 seconds apart, such as 10 seconds, and especially at least 30 seconds.
[36]
39. TLR4 agonist, saponin, use, method, kit or syringe according to any of claims 1 to 38, wherein the TLR4 agonist is administered before the saponin.
BE2017 / 5958
[37]
40. TLR4 agonist, saponin, use, method, kit or syringe according to one of claims 1 to 39, wherein the saponin is administered before the TLR4 agonist.
[38]
41. TLR4 agonist, saponin, use, method, kit or syringe according to claim 37, wherein the TLR4 agonist and saponin are administered without delay.
[39]
42. TLR4 agonist, saponin, use, method, kit or syringe according to any of claims 1 to 41, wherein an antigen is provided and the TLR4 agonist, saponin and antigen are all administered within 6 hours , as in 2 hours, in particular in 1 hour, as in 30 minutes and especially in 15 minutes (for example, in 5 minutes).
[40]
43. TLR4 agonist, saponin, use, method, kit or syringe according to claim 42, wherein an antigen is provided and the TLR4 agonist, saponin and antigen are all administered without delay.
[41]
44. TLR4 agonist, saponin, use, process, kit or syringe according to one of claims 1 to 43, for use as a primary vaccination course.
[42]
45. TLR4 agonist, saponin, use, method, kit or syringe according to one of claims 1 to 43, for use as a booster vaccination.
[43]
46. TLR4 agonist, saponin, use, method, kit or syringe according to one of claims 1 to 45, wherein the TLR4 agonist is not formulated with liposomes.
BE2017 / 5958
[44]
47. TLR4 agonist, saponin, use, method, kit or syringe according to one of claims 1 to 45, wherein the TLR4 agonist is formulated with liposomes.
[45]
48. TLR4 agonist, saponin, use, method, kit or syringe according to one of claims 1 to 47, wherein the saponin is formulated with liposomes.
[46]
49. TLR4 agonist, saponin, use, method, kit or syringe according to claim 47 or 48, wherein each dose contains 0.1 to 10 mg, such as 0.5 to 2 mg of liposomes.
[47]
50. TLR4 agonist, saponin, use, method, kit or syringe according to claim 47 or 48, wherein each dose contains 0.1 to 10 mg, such as 0.5 to 2 mg of liposomes.
[48]
51. TLR4 agonist, saponin, use, method, kit or syringe according to one of claims 1 to 50, where one or more antigens are also provided.
[49]
52. TLR4 agonist, saponin, use, method, kit or syringe according to claim 51, wherein one or more antigens are co-formulated with the TLR4 agonist.
[50]
53. TLR4 agonist, saponin, use, method, kit or syringe according to claim either 51 or 52, wherein one or more antigens are co-formulated with the saponin.
[51]
54. TLR4 agonist, saponin, use, method, kit or syringe according to one of claims 1 to 53, wherein the TLR agonist is provided
BE2017 / 5958 in a composition substantially devoid of saponin, as comprising less than 1 μg of saponin per human dose.
[52]
55. TLR4 agonist, saponin, use, method, kit or syringe according to any one of claims 1 to 54, wherein the saponin is provided in a composition substantially free of TLR4 agonist, as comprising less than 1 µg of TLR4 agonist per human dose.
[53]
56. TLR4 agonist, saponin, use, method, kit or syringe according to any one of claims 1 to 55, wherein the 3D-MPL is supplied in a composition substantially devoid of saponin, as comprising less than 1 pg of saponin per human dose.
[54]
57. TLR4 agonist, saponin, use, method, kit or syringe according to any one of claims 1 to 56, wherein QS21 is provided in a composition substantially free of TLR4 agonist, as comprising less than 1 pg of TLR4 agonist per human dose.
[55]
58. TLR4 agonist, saponin, use, method, kit or syringe according to any of claims 1 to 57, wherein the TLR4 agonist is provided in a composition substantially free of QS21, as comprising less than 1 µg of saponin per human dose.
[56]
59. TLR4 agonist, saponin, use, method, kit or syringe according to any one of claims 1 to 58, wherein the saponin is supplied in a composition substantially devoid of 3D-MPL, as comprising less than 1 pg of 3D- MPL per human dose.
BE2017 / 5958
[57]
60. TLR4 agonist, saponin, use, method, kit or syringe according to any one of claims 1 to 59, wherein the 3D-MPL is provided in a composition substantially free of QS21, as comprising less than 1 pg of saponin per human dose.
[58]
61. TLR4 agonist, saponin, use, method, kit or syringe according to any one of claims 1 to 60, in which the QS21 is supplied in a composition substantially devoid of 3D-MPL, as comprising less than 1 pg of 3D- MPL per human dose.
[59]
62. TLR4 agonist, saponin, use, method, kit or syringe according to any one of claims 1 to 61, wherein the immunological response observed is at least 50% of the response observed with the co-formulation, as at at least 60%, suitably at least 70%, especially at least 80% and in particular at least 90%.
[60]
63. TLR4 agonist, saponin, use, method, kit or syringe according to any one of claims 1 to 62, wherein the immunological response observed is not less than the response observed with co-formulation.
[61]
64. TLR4 agonist, saponin, use, method, kit or syringe according to any of claims 1 to 63, wherein the antigen is derived from a human or non-human pathogen including, for example, bacteria, fungi, parasitic microorganisms or multicellular parasites that infect human and non-human vertebrates, or from a cancer cell or tumor cell.
BE2017 / 5958
[62]
65. TLR4 agonist, saponin, use, method, kit or syringe according to claim 64, wherein the antigen is derived from Plasmodium falciparum or Plasmodium vivax, such as the antigen of SEQ ID NO: 1.
[63]
66. TLR4 agonist, saponin, use, method, kit or syringe according to claim 64, wherein the antigen derived from Mycobacterium spp., As a derivative of any of SEQ ID NO: 2 to 5.
[64]
67. TLR4 agonist, saponin, use, method, kit or syringe according to claim 64, wherein the antigen is derived from the varicella zoster virus, such as the antigen of SEQ ID NO: 6.
[65]
68. TLR4 agonist, saponin, use, method, kit or syringe according to claim 64, wherein the antigen is derived from the human respiratory syncytial virus, such as the antigen of SEQ ID NO: 7.
[66]
69. TLR4 agonist, saponin, use, method, kit or syringe according to claim 64, wherein the antigen is derived from HIV, such as the antigen of SEQ ID NO: 8 or 9.
[67]
70. TLR4 agonist, saponin, use, method, kit or syringe according to claim 64, wherein the antigen is derived from non-typable H. influenzae (NTHi) and / or from M. catarrhalis.
[68]
71. TLR4 agonist, saponin, use, method, kit or syringe according to any of claims 64 to 70, wherein the antigen is provided in the form of a polypeptide.
[69]
72. TLR4 agonist, saponin, use, method, kit or syringe according to any one of the
BE2017 / 5958 claims 64 to 70, wherein there is provided a polynucleotide which codes for the polypeptide antigen.
BE2017 / 5958
OSti 'ïm. ί® iTiiîÂi î-i <ί; ΐϊ.®: · ίίί
BE2017 / 5958 “O W CfO s” c o
E ioo o LU separate
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BE2017 / 5958
BE2017 / 5958

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法律状态:
2018-12-07| FG| Patent granted|Effective date: 20181105 |

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2020-08-27| MM| Lapsed because of non-payment of the annual fee|Effective date: 20191231 |

优先权:

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GBGB1621686.3A|GB201621686D0|2016-12-20|2016-12-20|Novel methods for inducing an immune response|

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GB1621686.3|2016-12-20|

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