Polymeric nanoparticles as vaccine adjuvants

Abstract

To provide a biocompatible nanoparticle which can be used as an adjuvant and can promote a cell-mediated CD8 and / or Th1 response without requiring a co-adjuvant.SOLUTION: The present invention is polymeric nanoparticles having diameters less than CD8 for use in inducing a Th1 and / or 80nm response to an immunogenic species in a subject.SELECTED DRAWING: Figure 1-1

Description

[Technical Field]

[0001] The present invention relates to polymer particles. In particular, the present invention relates to biocompatible and / or biodegradable polymeric nanoparticles having a diameter of 50 nm ± 10 nm and methods of using the same. In particular, the present invention relates to biocompatible and / or biodegradable polymeric nanoparticles having a diameter of 50 nm ± 10 nm as adjuvants for inducing cellular immunity in addition to antibody responses. The present invention also relates to polymeric nanoparticles having a diameter of 50 nm ± 10 nm that can stimulate caspase-11 and / or gasdermin D-dependent cellular immunity against antigens. [Background technology]

[0002] Vaccines are one of the most effective methods for preventing infectious diseases and the spread of pathogens that can cause disease. A vaccine is a preparation that provides a subject with acquired immunity to a disease, preventing or reducing the severity of the disease in the subject.

[0003] Traditionally, vaccines are made using small amounts of weakened or killed microorganisms, such as disease-causing viruses or bacteria. As vaccine research has progressed, it has been discovered that it is not always necessary to use the entire microorganism to provide protection; rather, small components of the microorganism, called antigens, can be used. Vaccines containing purified antigens provoke fewer adverse reactions and are safer than vaccines made from whole microorganisms. However, highly purified antigens are often too weak to activate the subject's immune system. To solve this problem, additional components, called adjuvants, are incorporated with the vaccine antigen. The function of this adjuvant is to "turn on" the immune system, helping it mount a stronger response to the antigen.

[0004] Only a few adjuvants are approved for use in human vaccines. Most approved adjuvants induce protection by inducing antibodies circulating in the subject's blood and neutralizing microorganisms. However, complex microorganisms, such as those causing influenza, tuberculosis, or malaria, can evade these antibodies, or antibodies alone are unable to mediate protection. Therefore, protection against complex microorganisms requires an additional defense mechanism that does not require antibodies, known as cell-mediated immunity. This involves the activation of phagocytes, cytotoxic T lymphocytes, and various cytokines in response to antigens. There are several types of effector CD4+ T helper cell responses that can be induced by antigen-presenting cells (APCs), including responses called Th1 and Th2 responses. Th1 responses are effective against intracellular pathogens (viruses and bacteria within host cells), destroying infected cells and providing a powerful defense against cancer. CD8+ T cells also play an important role in cell-mediated immunity against intracellular pathogens, including viruses and bacteria, and in tumor surveillance. Currently, there is a lack of effective adjuvants that can stimulate cellular immunity for these types of protection.

[0005] Polymer nanoparticles, including poly(D,L-lactide-co-glycolide)-PLGA polymer particles, have been tested as adjuvants in many preclinical scenarios. However, despite the efforts of many research groups, there have been no reports of PLGA nanoparticles that can promote the desired cellular immunity more effectively than adjuvants in clinical use. Furthermore, currently reported PLGA particles require coadjuvants or complex encapsulation to elicit such effects.

[0006] Although PLGA particles have been used effectively as drug carriers, they have not been used effectively in the vaccine industry. In general, the reported induction of cell-mediated immunity (CMI) is poor. This has led to alternative co-formulation strategies using Toll-like receptor (TLR) agonists and other coadjuvants, or to complex antigen encapsulation or complexation methods that complicate formulation stability, increase costs, and make these difficult to translate into clinically relevant formulations.

[0007] Several groups have investigated PLGA nanoparticles and microparticles and their use to modulate immune responses.

[0008] Canadian Patent Application Publication No. 2753567 (CA2753567) (Patent Document 1) discloses particles having a hydrophobic segment (PGLA) and a hydrophilic segment (a poorly soluble polysaccharide, such as dextran). The preferred diameter of these particles is stated to be 0.1 μm to 50 μm.

[0009] Oyewumi et al. (Expert Rev Vaccines, 2010) reviewed nanoparticles as immune adjuvants, correlating particle size with the resulting immune response. However, the authors concluded that overall, conflicting data prevented accurate prediction of the particle size range that would affect the outcome of a Th1 or mixed Th1 / Th2 immune response.

[0010] Canadian Patent Application Publication No. 02731995 (CA02731995) discloses a method for inducing a Th1 immune response using microparticles. The particles are sized so that at least 50% are less than 5 μm, preferably less than 3 μm. Furthermore, the mean diameter of the microparticles is described as being 2.2 μm or greater.

[0011] International Publication No. 2012 / 054425 (WO2012054425) (Patent Document 3) discloses a composition for tumor treatment, the aim of which was to induce a strong Th1 response. It discloses particles preferably made of PLGA. The average diameter is approximately 100 nm to 20 μm, 200 nm to 15 μm, and most preferably 500 nm to approximately 10 μm. The particles used also require a coadjuvant to elicit their effects.

[0012] O'Grady et al. (J Immunol, 2019) (Non-Patent Document 2) disclose polystyrene nanoparticles as drivers of antigen-specific cellular immunity following vaccination.

[0013] Chenxi Li, et al. (Applied Materials and Interfaces, 2018, 10, 2874-2889) (Non-Patent Document 3) disclose the amphiphilic diblock copolymer poly(2-ethyl-2-oxazolin)poly(D,L-lactide) (PEOz-PLA) combined with carboxyl-terminated pluronic acid F127 to construct nanoparticles for delivery of the antigen ovalbumin. This system includes a coadjuvant, namely, TLR7, to elicit a response.

[0014] International Publication No. WO 2017 / 151922 (Patent Document 4) discloses a vaccine comprising an antigen and a polymer, where the antigen is within the core of the polymer particle. Organic polymer nanoparticles as delivery agents are also disclosed in U.S. Patent Application Publication No. US 2015 / 0342883 (Patent Document 5).

[0015] Carmen Garcia Arevelo et al. (Molecular Pharmaceutics, 2013, 10, 586-597) (Non-Patent Document 4) discloses a generally modified elastin-like block core combinator (ELbcR) that can generate antigen particle-based constructs assembled with nanopores (nanovesicles). A Th2 / IL-5 response was reported in mice.

[0016] Fazren Azmi et al. (Bioorganic and Medicinal Chemistry) (Non-Patent Document 5) disclose conjugating lipid moieties to peptides to facilitate non-polymeric particle formation and use in vaccine constructs.

[0017] In addition to PLGA, several other particulate systems, such as those disclosed in Schiins et al. (Immunol Rev. 2020 Jul;296(1):169-190), have been shown to have specific adjuvant activity, but most are highly complex formulations that require multiple components in addition to antigen encapsulation or covalently bound antigens to work. Complex multi-component particulate products are impractical for mass production, which is a disadvantage for the vaccine industry. Another common problem with such systems is the lack of knowledge of their mechanism of action or the immune pathways they modulate that are necessary to be effective adjuvants. [Prior art documents] [Patent documents]

[0018] [Patent Document 1] Canadian Patent Application Publication No. 2753567 [Patent Document 2] Canadian Patent Application Publication No. 02731995 [License 3] International Publication No. 2012 / 054425 [License 4] International Publication No. 2017 / 151922 [Patent Document 5] U.S. Patent and Trademark Office Publication No. 2015 / 0342883 [License 6] U.S. Patent No. 4599230 [License 7] U.S. Patent No. 4601903 [Non-licensed literature]

[0019] [Non-licensed Document 1] Oyewumi et al, (Expert Rev Vaccines, 2010) [Non-licensed Document 2] O'Grady et al., (J Immunol, 2019) [Non-licensed Document 3] Chenxi Li, et al (Applied materials and interfaces, 2018, 10, 2874-2889) [Non-licensed Document 4] Carmen Garcia Arevelo, et al.,(Molecular Pharmaceutics, 2013, 10, 586-597) [Non-licensed Document 5] Fazren Azmi, et al., (Bioorganic and Medicinal Chemistry) [Non-licensed Document 6] Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit.1985) [Non-licensed Document 7] “Handbook of Pharmaceutical Excipients”, 2nd Edition, (1994), edited by A Wade and PJ Weller [Non-patent document 8] McKee et al, International Immunology, 20, 659-669, 2008) [Non-Patent Document 9] Ben-Sasson et al., Journal of experimental medicine, 210, 491-502, 2013a [Non-Patent Document 10] Ben-Sasson et al., Cold Spring Harbor symposia on quantitative biology 78, 117-124, 2013b Summary of the Invention [Problem to be solved by the invention]

[0020] The present invention serves to address the problems of the prior art by providing biocompatible nanoparticles that can be used as adjuvants and enhance cell-mediated CD8 and / or Th1 responses without the need for a co-adjuvant. [Means for solving the problem]

[0021] The present invention provides pure, endotoxin-free polymeric nanoparticles that successfully and surprisingly induce effective Th1 and cytotoxic T lymphocyte (CTL) responses with mixed antigens. The nanoparticles are capable of inducing responses without coadjuvants, which has not been reported or predicted in the prior art. The present invention offers the advantage of ease of formulation.

[0022] We have demonstrated that PLGA particles are potent inducers of CD8 T cells and antigen-specific secretion of the cytokine IFN-γ, which is typical of Th1 responses. We further show that this response cannot be driven by larger biodegradable PLGA or larger biocompatible polystyrene particles.

[0023] Thus, the present invention provides a novel, previously undescribed strategy to induce strong CD8 or Th1 responses via vaccination using purified antigens and biodegradable PLGA particles.

[0024] The inventors have also shown that the ability of the 50 nm nanoparticles of the present invention to induce CD8 responses is dependent on a pyroptotic-related pathway, which can lead to programmed immunogenic cell death and / or cellular hyperactivation.

[0025] Prior to the present invention, limitations in the formulation process of PLGA particles limited their production to sizes between 80 nm and 250 μm. A new platform involving microfluidic technology applied to the production of the 50 nm particles used in the present invention allows for fine control of particle size, particles with a low polydispersity index, avoidance of the use of harmful solvents, and an amenable escalation for large-scale production of the adjuvant.

[0026] One aspect of the present invention provides polymeric nanoparticles having a diameter of less than 80 nm for use in inducing a CD8 and / or Th1 response in a subject.

[0027] The polymeric nanoparticles for use are as described herein and are referred to as "nanoparticles of the invention."

[0028] The response is directed against an immunogenic species of interest. Typically, the immunogenic species is an antigen.

[0029] Preferably, the use is to induce a CD8 response in a subject against an antigen. Typically, the nanoparticles induce or affect a pyroptosis-related pathway in the subject.

[0030] In one embodiment, a CD8 response and a Th1 response are induced.

[0031] Preferably, the polymeric nanoparticles have a diameter of about 30 nm to about 65 nm, preferably about 40 nm to about 60 nm.

[0032] Typically, in said uses, the polymeric nanoparticles are mixed with an immunogenic species, preferably adsorbed to the surface of the nanoparticles and / or free in a composition or formulation comprising said nanoparticles.

[0033] Most preferably, the polymeric nanoparticles for use are biocompatible.

[0034] Typically, the nanoparticles for use are endotoxin-free.

[0035] Of note, the nanoparticles for use are solid particles.

[0036] In one embodiment, the nanoparticles for use are provided in the form of a composition comprising the nanoparticles of the invention or a preparation of the nanoparticles of the invention. Typically, the composition also comprises an immunogenic species. The immunogenic species is mixed with the immunogenic species in the composition.

[0037] In one embodiment, the composition is a vaccine composition.

[0038] In one embodiment, the composition is an adjuvant.

[0039] There is also provided the use of the nanoparticles of the invention as an adjuvant.

[0040] One aspect of the present invention provides polymeric nanoparticles (herein referred to as "nanoparticles of the present invention") having a diameter of less than 80 nm, preferably 40 nm to 60 nm, more preferably about 52 nm to about 65 nm, or 52 nm to 60 nm.

[0041] The polymeric nanoparticles are biocompatible.

[0042] In one embodiment, the nanoparticles of the present invention comprise a biocompatible polymer. Preferably, the nanoparticles are polymer particles. Preferably, the nanoparticles are poly(D,L-lactide-co-glycolide) (PLGA) polymer nanoparticles. Preferably, the nanoparticles are polylactic acid (PLA) nanoparticles. Typically, the biocompatible polymer is selected from the group consisting of PLGA, PLA, polyphosphazene, and chitosan.

[0043] One aspect of the present invention provides a preparation of the biocompatible polymeric nanoparticles of the present invention ("the nanoparticle preparation of the present invention").

[0044] In one embodiment, at least 90% of the nanoparticles in the preparation have a diameter of less than 80 nm, preferably between 40 nm and 60 nm.

[0045] In one embodiment, at least 90% of the nanoparticles in the preparation have a diameter of about 52 nm to 65 nm, preferably 52 nm to 60 nm.

[0046] Preferably, the nanoparticles in the preparation are uniform or monodisperse.

[0047] In an aspect of the present invention, there is provided a composition comprising the nanoparticles of the present invention.

[0048] Typically, the composition comprises at least one immunogenic species.

[0049] In one embodiment, the composition is a vaccine.

[0050] In one embodiment, the composition is a pharmaceutical composition.

[0051] In one embodiment, the immunogenic species is an antigen.

[0052] Typically, the polymeric nanoparticles are mixed with the immunogenic species in the composition.

[0053] Preferably, the immunogenic species is adsorbed to the surface of the nanoparticles and / or free in the composition.

[0054] In one embodiment of the present invention, at least 90% of the nanoparticles in the composition have a diameter of less than 80 nm.

[0055] In an embodiment of the invention, at least 90% of the nanoparticles in the composition have a diameter of 40 nm to 60 nm, or about 52 nm to about 65 nm, preferably 52 nm to 60 nm.

[0056] In one embodiment, the composition comprises a plurality of nanoparticles.

[0057] In one embodiment, the composition further comprises a pharmaceutically acceptable carrier or salt.

[0058] One aspect of the present invention provides an adjuvant comprising the nanoparticles of the present invention.

[0059] One aspect of the invention provides a nanoparticle, preparation or composition of the invention and described herein for use in vaccine therapy to prevent or treat a condition or disease in a subject. In one embodiment, the disease or condition is a bacterial or viral infection. Preferably, the disease or condition is cancer.

[0060] The disease or condition may be a chronic infectious disease. The chronic infectious disease may be selected from, but is not limited to, tuberculosis, a viral infection, an intracellular bacterium, and an intracellular parasite.

[0061] One aspect of the invention provides a method of treating or preventing a disease or condition in a subject, the method comprising administering to the patient a therapeutically or prophylactically effective amount of a nanoparticle, preparation, or composition of the invention.

[0062] A further aspect of the invention provides a nanoparticle, preparation or composition of the invention and as described herein for use in inducing a Th1 and / or CD8 response in a subject to an antigen.

[0063] A further aspect of the invention is a method for generating a Th1 and / or CD8 response in a subject against an antigen, comprising administering to said subject a therapeutically effective amount of the nanoparticles, preparations or compositions of the invention.

[0064] A further aspect of the invention provides the nanoparticles, preparations or compositions of the invention as carriers for delivering an active agent to a subject. The active agent may be a drug, such as a drug for the treatment of cancer, preferably a chemotherapeutic drug.

[0065] The invention will be more clearly understood from the following description of embodiments thereof, given by way of example only, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0066] [Figure 1-1]When administered with a protein antigen, 50 nm PLGA nanoparticles promoted CD8+ T cell responses and antigen-specific IFN-γ. C5 / BL / 6 mice were immunized intramuscularly with positively charged (PLGA-DOTAP) or negatively charged (PLGA-PhS) particles and ovalbumin (OVA) on days 0 and 14. On day 21, splenocytes were analyzed by flow cytometry, and the percentage (A) and number (B) of CD8+ T cells specific for the H-2Kb OVA epitope SIINFEKL were quantified using tetramers. In all cases, 50 nm polystyrene (PS) particles were used as a positive control. Results are shown as mean SEM, and symbols represent individual animals. (C) IFN-γ secretion was analyzed by ELISA after 72 h of restimulation with antigen. Asterisks indicate statistical differences calculated according to multiple comparison ANOVA and Dunnett's multiple comparison test to determine differences compared to OVA alone. [Figure 1-2] When administered with a protein antigen, 50 nm PLGA nanoparticles promoted CD8+ T cell responses and antigen-specific IFN-γ. C5 / BL / 6 mice were immunized intramuscularly with positively charged (PLGA-DOTAP) or negatively charged (PLGA-PhS) particles and ovalbumin (OVA) on days 0 and 14. On day 21, spleen cells were analyzed by flow cytometry, and the percentage (A) and number (B) of CD8+ T cells specific for the H-2Kb OVA epitope SIINFEKL were quantified using tetramers. In all cases, 50 nm polystyrene (PS) particles were used as a positive control. Results are shown as mean SEM, and symbols represent individual animals. (C) IFN-γ secretion was analyzed by ELISA after 72 h of restimulation with antigen. Asterisks indicate statistical differences calculated according to multiple comparison ANOVA and Dunnett's multiple comparison test to determine differences compared to OVA alone. [Figure 2-1]Administration of large PLGA particles with protein antigens fails to induce CD8+ T cell responses and IFN-γ. C57BL / 6 mice were immunized intramuscularly with particles and ovalbumin (OVA) on days 0 and 14. On day 21, spleen cells were analyzed by flow cytometry, and the percentage (A) and number (B) of CD8+ T cells specific for the H-2Kb OVA epitope SIINFEKL were quantified using tetramers. Results are shown as mean SEM, and symbols represent individual animals. (C) IFN-expression was analyzed by ELISA after 72 hours of restimulation with antigen. Polystyrene nanoparticles were used as a positive control (+Ctrl); anti-CD3 stimulation was used to confirm IFN-production in all groups (right panel). Asterisks indicate statistical differences calculated according to multiple comparison ANOVA and Dunnett's multiple comparison test to determine differences compared to OVA alone. [Figure 2-2] Administration of large PLGA particles with protein antigens fails to induce CD8+ T cell responses and IFN-γ. C57BL / 6 mice were immunized intramuscularly with particles and ovalbumin (OVA) on days 0 and 14. On day 21, spleen cells were analyzed by flow cytometry, and the percentage (A) and number (B) of CD8+ T cells specific for the H-2Kb OVA epitope SIINFEKL were quantified using tetramers. Results are shown as mean SEM, and symbols represent individual animals. (C) IFN-expression was analyzed by ELISA after 72 hours of restimulation with antigen. Polystyrene nanoparticles were used as a positive control (+Ctrl); anti-CD3 stimulation was used to confirm IFN-production in all groups (right panel). Asterisks indicate statistical differences calculated according to multiple comparison ANOVA and Dunnett's multiple comparison test to determine differences compared to OVA alone. [Figure 3]50 nm nanoparticles induce antigen-specific CD8+ T cell responses through immunogenic cell death pathways. Mice were immunized intramuscularly (i.m.) with OVA alone or with particles (PS) on days 0 and 14. Between priming and boosting, mice were treated intraperitoneally (i.m.) or i.m. with 250 μg of necrosulfonamide (NSF) to inhibit pyroptosis. On day 21, the frequency of splenic CD8+ T cells specific for the H-2 Kb OVA epitope SIINFEKL was determined using tetramers by flow cytometry. Results are shown as mean ± SEM, and symbols indicate individual animals. Asterisks indicate statistical differences calculated according to multiple comparison ANOVA and Dunnett's multiple comparison test to determine differences compared to OVA + 50 nm PS. [Figure 4] Caspase-11 is required for antigen-specific CD8 responses induced by nanoparticles. Caspase-11 knockout (Casp11 KO) or wild-type (WT) mice were inoculated intramuscularly with 50 nm particles and OVA, OVA alone as a vehicle control, or PBS on days 7 and 14. On day 21, antigen-specific responses in the spleen were quantified using H-2Kb / OVA(SIINFEKL) MHC tetramer. (A) Percentage of OVA-specific CD8+ T cells relative to total CD8+ T cells; (B) Total number of OVA-specific CD8+ T cells in the spleen. Statistical differences were determined by one-way ANOVA with Bonferroni post hoc test. Asterisks indicate significant differences. **P<0.01, *P<0.05. [Figure 5]Characterization of nanoparticles formulated using PLGA 50:50, PLGA:DDA (dioctadecylammonium bromide), or PLGA PhS (phosphatidylserine) 1:1 w / w. After formulation, nanoparticles were characterized using a Zetasizer Nano Z system, and zeta potential and electrophoretic mobility in aqueous dispersions were measured using laser Doppler microelectrophoresis. Size (nm), polydispersity index (PDI), and zeta potential (ZP) are shown in the table. Results are presented as mean and standard deviation (SD) and are representative of at least two separate batches of nanoparticles. [Figure 6] This figure illustrates the potent antigen-specific antitumor immunity driven by 50 nm PS nanoparticles. A) Tumor growth rates at the challenge site of tumor-bearing mice were expressed as mean tumor volume ± SEM. A two-way ANOVA with Tukey's multiple comparison test was used to determine statistical significance between PBS- and 50 nm-vaccinated mice (*p<0.05, **p<0.01, ***p<0.001). (n=7 mice / group). B) Kaplan-Meier survival analysis of challenged mice. A Mantel-Cox test was used to determine statistical significance between PBS- and 50 nm-vaccinated mice, where *p<0.05, **p<0.01, and ***p<0.001). [Figure 7]Injection of polymer particles enhances antigen-specific antibody responses independently of size. C57BL / 6 mice were immunized with endotoxin-free ovalbumin (OVA) or OVA and particles of different sizes on days 0 and 14 according to previously optimized doses: (A) intramuscularly (10 μg OVA + 1 mg particles or Alum (aluminum hydroxide gel)) or (B) intraperitoneally (1 μg OVA, 4 mg particles, or Alum (aluminum hydroxide gel)). For subcutaneous immunization, either OVA (C) or Staphylococcal agglutination factor A-ClfA (D, E) was used as the antigen (50 μg OVA or 1 μg ClfA, 4 mg particles). PBS was used as a vehicle control, and Alhydrogel® (Alum) was used as a gold standard control for both im and ip administration. Blood samples were collected on day 21, and antigen-specific IgG titers in the serum were measured by ELISA. Neutralizing antibodies were determined by testing the ability of sera from vaccinated mice to inhibit the adherence of Staphylococcus aureus (S. aureus) to fibrinogen at 37°C by measuring Abs570nm of fixed bacteria previously stained with crystal violet. Adherence is expressed as percent inhibition relative to the maximum (100%), calculated as the amount of bacterial adherence in the absence of serum. Results are shown as mean ± SEM, and each symbol represents an individual animal. Asterisks indicate statistical differences (p>0.0001) calculated according to multiple comparison ANOVA and Dunnett's test to determine differences compared to antigen alone. No specific antibodies were detected in mice injected with PBS alone. Values ​​are representative of two experiments administered by the i.p. and sc routes and one experiment administered by the im route. [Figure 8]Particle size affects antibody class switching but not antibody neutralizing activity. Mice were immunized intramuscularly (AC) with particles and OVA or subcutaneously (DG) with particles and ClfA on days 7 and 14. Serum was collected on day 21 for analysis of antigen-specific IgG isotypes by ELISA. Results are shown as mean ± SEM, and symbols represent individual animals. Asterisks indicate statistical differences calculated according to multiple comparison ANOVA and Dunnett's test to determine differences compared to antigen alone. Representative of two experiments. [Figure 9] 50 nm particles promote the secretion of antigen-specific IFN-γ, which is required for IgG2c class switching. Mice were immunized intramuscularly with particles and OVA on days 0 and 14. Spleens and serum were collected on day 21. (A) Spleen cells were restimulated with OVA, anti-CD3 (inner panel) as a positive control, or left unstimulated. IFN-γ production was determined by ELISA. Bars represent the mean ± SEM for n = 4 / group. (B) OVA-specific total IgG titers and isotypes in serum were determined by ELISA. Results are shown as the mean ± SEM, and symbols represent individual animals. In all panels, asterisks indicate statistical differences calculated according to multiple comparison ANOVA and Dunnette's multiple comparison test to determine differences compared to OVA alone. [Figure 10-1]CD8+ T cell responses are exclusively induced by 50 nm particles, independent of the route of administration or polymer. Mice were immunized on days 0 and 14 with OVA alone or in combination with Alum or particles via the im (A) or i.p. (B) route. On day 21, spleen cells were analyzed by flow cytometry, and the number of CD8+ T cells specific for the H-2Kb OVA epitope SIINFEKL was quantified using tetramers. Asterisks indicate statistical differences calculated according to multiple comparison ANOVA and Dunnette's multiple comparison test to determine differences compared to OVA alone. Figure 1 shows a representative example of one experiment administered im and two experiments administered i.p. The efficacy of 50 nm polystyrene (PS) and 50 nm poly(lactic-co-glycolic acid) (PLGA) was compared after im injection following the same vaccination scheme as before. (C) On day 21, splenocytes were analyzed by flow cytometry to quantify the number of CD8+ T cells specific for the H-2 Kb OVA epitope SIINFEKL (mean ± SEM shown) using tetramers, and (D) a representative dot plot. (E) The mean ± SEM of SIINFEKL-specific CD8+ and CD44+ T cells and the corresponding dot plots (F) after vaccination with 50 nm PS, 100 nm PLGA, or 500 nm PLGA were shown. Antigen-specific IFN-γ responses were measured after ex vivo stimulation of splenocytes by ELISA, comparing 50 nm PS vs. 50 nm PLGA (G) and 50 nm PS vs. 100 nm and 500 nm PLGA (H). Asterisks indicate statistical differences calculated according to multiple comparison ANOVA and Dunnette's multiple comparison test to determine differences compared to OVA alone. Representative example of one experiment via im and two experiments via ip. [Figure 10-2]CD8+ T cell responses are exclusively induced by 50 nm particles, independent of the route of administration or polymer. Mice were immunized on days 0 and 14 with OVA alone or in combination with Alum or particles via the im (A) or i.p. (B) route. On day 21, spleen cells were analyzed by flow cytometry, and the number of CD8+ T cells specific for the H-2Kb OVA epitope SIINFEKL was quantified using tetramers. Asterisks indicate statistical differences calculated according to multiple comparison ANOVA and Dunnette's multiple comparison test to determine differences compared to OVA alone. Figure 1 shows a representative example of one experiment administered im and two experiments administered i.p. The efficacy of 50 nm polystyrene (PS) and 50 nm poly(lactic-co-glycolic acid) (PLGA) was compared after im injection following the same vaccination scheme as before. (C) On day 21, splenocytes were analyzed by flow cytometry to quantify the number of CD8+ T cells specific for the H-2 Kb OVA epitope SIINFEKL (mean ± SEM shown) using tetramers, and (D) a representative dot plot. (E) The mean ± SEM of SIINFEKL-specific CD8+ and CD44+ T cells and the corresponding dot plots (F) after vaccination with 50 nm PS, 100 nm PLGA, or 500 nm PLGA were shown. Antigen-specific IFN-γ responses were measured after ex vivo stimulation of splenocytes by ELISA, comparing 50 nm PS vs. 50 nm PLGA (G) and 50 nm PS vs. 100 nm and 500 nm PLGA (H). Asterisks indicate statistical differences calculated according to multiple comparison ANOVA and Dunnette's multiple comparison test to determine differences compared to OVA alone. Representative example of one experiment via im and two experiments via ip. [Figure 11]50 nm particles induce antigen-specific CD4+ T cells and long-term CD8+ T cell memory. (A) Wild-type (WT) mice were immunized i.p. on days 7 and 14 with the I-Ab-restricted influenza nucleoprotein peptide NP311-325 alone or in combination with different sizes of PS particles or Alum as a positive control. Antigen-specific CD4+ responses were assessed in the spleen by FACS using class II (C-II) tetramers (Tmers) on day 21. Vaccination with 50 nm PS particles effectively increased the number of antigen-specific CD4+ T cell responses. (B) 50 nm PS also enhanced NP311-325CD T cell responses 7 days after a booster i.m. vaccination with a fusion antigen (OVA-NP311) generated by complexing NP311-325 to OVA. (C) The number and percentage of H-2 Kb OVA epitope SIINFEKL CD8+ T cells were also upregulated 7 days after im prime / boost vaccination with 50 nm PS and fusion NP311-OVA antigen. (D) The upper left graph shows that Ag-specific CD8+ memory T cells were detected in the spleen 100 days after im prime / boost vaccination with PS + NP311-OVA. The dot plot shows the CI Tmer+ CD8+ T cells relative to total CD8+ lymphocytes. The blue histogram shows surface marker expression of CD69, CD103, and CD62L on CI Tmer+ memory CD8+ T cells in the spleen, and the gray histogram shows unstained controls for each marker. The lower left graph shows IFN-γ production measured by ELISA in splenocytes harvested 100 days after vaccination and restimulated with the fusion antigen for 72 hours. Bars represent mean ± SEM. Asterisks indicate significant differences by one-way analysis of variance and multiple comparison post-hoc Dunnett's test, *P≦0.05, *P≦0.01. (A) Representative of one experiment with n=5 / group, (B) Representative of two experiments with n=3 / group, (C) Representative of two experiments with n=3 / group, (D) Representative of one experiment with n=3 / group. [Figure 12]50 nm PS nanoparticles confer potent antigen-specific antitumor immunity. Mice were vaccinated with PBS, OVA (10 μg) alone, or 50 nm PS particles (1 mg) plus OVA (10 μg) on ​​days 0 and 14 and then challenged sc with 3.5 × 10 B16-OVA tumor cells on day 28 as in (A). Tumors were measured daily. Spider plots (B) show early tumor development, measured as tumor volume in mm. Each line represents an individual mouse. Graphs of the percentage of tumor-bearing mice and Kaplan-Meier survival at the early stage of challenge are shown in (C). Expanded analyses of the percentage of tumor-bearing mice and survival analysis are shown in (D). For tumor-bearing mice analysis, n = 19 for the PBS and OVA groups and 17 for the 50 nm + PS and 50 nm + OVA groups. For survival, n = 14 were used for PBS and OVA, and n = 12 for 50 nm + OVA. Statistical significance of survival analysis was determined using the Mantel-Cox test: *p<0.05, **p<0.01, ***p<0.001. CP indicates the number of animals completely protected from tumor challenge relative to the total for each group. [Figure 13]IL-1 and IL-18 signaling regulate nanoparticle-induced IFN-γ and CD8+ T cell responses, respectively. WT or IL1-R1- or IL-18-deficient mice were conventionally immunized with OVA alone or in combination with 50 nm particles by the im route. (A and E) Antigen-specific humoral responses were measured by ELISA on sera obtained on day 21. (B, C, and F, G) Graphs showing the percentage and number of OVA H-2Kb CD8+ T cells in the spleens of vaccinated mice 7 days after the booster. (D and H) IFN-secretion was analyzed by ELISA in supernatants from splenocytes harvested 7 days after vaccination and stimulated with OVA for 72 hours. Asterisks indicate statistical differences calculated using an unpaired t-test with Welch's correction for comparisons of OVA vs. 50 nm + OVA within each strain for ELISA, or according to ANOVA and Dunnett's multiple comparison test to determine differences between WT 50 nm + OVA vs. knockout mice receiving the same treatment. Quantification of tetramer+ cells is shown as Tmer+, CD8+, CD44+ relative to total viable CD8+. Asterisks indicate significant differences relative to OVA alone; ns: not significant by one-way ANOVA and Dunnett's post-hoc test. Quantification of IFN- is shown as mean ± SEM. Significant differences are indicated for 50 nm + OVA vs. OVA alone. *p<0.05, **p<0.01, ***p<0.001. [Figure 14]Caspase-11 is required for antigen-specific CD8 responses. Caspase-11 knockout (Casp11 KO) or wild-type (WT) mice were vaccinated intramuscularly with 1 mg of 50 nm particles and 10 mg of OVA, or with particles, OVA, and the caspase-1 inhibitor Y-VAD (0.25 mg) intramuscularly on days 7 and 14. Antigen-specific responses were quantified in the spleen on day 21 using H-2Kb / OVA (SIINFEKL) MHC tetramers. PBS or OVA alone was used as a vehicle or antigen-only control. (A) Percentage of OVA-specific CD8+ T cells relative to total CD8+ T cells; (B) Total number of OVA-specific CD8+ T cells in the spleen. Statistical differences were determined by one-way analysis of variance and Bonferroni post hoc test. Asterisks indicate significant differences; **P<0.01, *P<0.05. [Figure 15]Polymer nanoparticles induce antibody responses independent of caspase-1, caspase-11, or gasdamin D. C57BL / 6 mice were immunized intramuscularly with endotoxin-free ovalbumin (OVA) or OVA plus 50 nm polystyrene particles (10 μg OVA + 1 mg particles) on days 0 and 14. PBS was used as a vehicle control. Blood samples were collected on day 21, and antigen-specific IgG titers in serum were measured by ELISA on day 28. (A) To assess the role of caspase-1, a group of mice was administered the caspase-1 inhibitor Ac-YVAD-cmk along with the vaccine at prime and boost. (B) To examine the role of caspase-11, a group of caspase-11-deficient mice was vaccinated alongside wild-type mice using the same formulation. (C) The role of gasdermin D in humoral responses was determined by assessing antibodies in mice administered the gasdermin D inhibitor necrosulfonamide (NSF) intramuscularly with the formulation or intraperitoneally at the time of vaccination between prime and boost. Results are shown as mean ± SEM, and each symbol represents an individual animal. Asterisks indicate statistical differences (p>0.0001) calculated according to multiple comparison ANOVA and Dunnette's test to determine differences compared to antigen alone. Antibodies in the PBS group served as baseline to define the limit of detection. [Figure 16] 50 nm particles induce cell death in myotubes. C2C12 mouse myoblasts were differentiated into myotubes and stimulated overnight with the indicated concentrations of 50 nm or 1 μm polymer particles. Cell death was measured by LDH release assay and expressed as percentage cytotoxicity. Nigericin and LPS were used as positive controls for cell death. DETAILED DESCRIPTION OF THE INVENTION

[0067] Definitions and general precedence As used herein, unless otherwise specified, the following terms are intended to have the following meanings in addition to any broader (or narrower) meaning that such terms may enjoy in the art.

[0068] All documents cited herein are incorporated by reference in their entirety.

[0069] Unless otherwise required by context, the use of the singular herein shall be read to include the plural and vice versa. The term "a" or "an," when used in reference to an entity, shall be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0070] As used herein, the term "comprise," or variations thereof, such as "comprises" or "comprising," shall be read to indicate the inclusion of any stated integer (e.g., feature, element, characteristic, property, method / process step, or limitation) or group of integers (e.g., features, elements, characteristics, properties, method / process step, or limitation), but not the exclusion of other integers or groups of integers. Thus, as used herein, the term "comprising" is inclusive or open-ended and does not exclude additional, unstated integers or method / process steps.

[0071] As used herein, the term "cell mediated immunity" refers to the production of cytokines, chemokines and other such molecules produced by activated T cells and / or other leukocytes, including those derived from CD4+ and CD8+ T cells.

[0072] As used herein, the term "nanoparticle" refers to a particle having a diameter of less than 1,000 nm. Nanoparticles may be solid or hollow. Nanoparticles may also be porous.

[0073] The term "immune response" should be understood to mean a humoral and / or cellular response induced in a subject.

[0074] As used herein, the term "antigen" means a substance that induces an immune response in the body when introduced into the body. This term is sometimes used interchangeably with the term "immunogen."

[0075] As used herein, the term "vaccine" should be understood to mean a composition or formulation comprising the nanoparticles of the present invention and at least one antigen. The preparation of vaccines is well described in the literature, for example in U.S. Pat. No. 4,599,230 (US4599230) and U.S. Pat. No. 4,601,903 (US4601903), the entire contents of which are incorporated herein by reference.

[0076] The term "adjuvant" should be understood to mean an agent that enhances the recipient's immune response to an antigen.

[0077] The term "therapeutically effective amount" should be understood to mean an amount that results in a clinically significant treatment, alleviation, or prevention of a disease or condition. This amount will vary from subject to subject, depending on the individual's age and overall condition, the mode of administration, and other factors. Thus, an exact effective amount cannot be specified, but one of ordinary skill in the art would be able to determine an appropriate "effective" amount in any individual case using routine experimentation and background general knowledge. Therapeutic results include eradication or alleviation of symptoms, relief of pain or discomfort, prolongation of survival, improved mobility, and other indicators of clinical improvement. A therapeutic result need not be a complete cure.

[0078] In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans.

[0079] As used herein, the term "polymeric material" is a material that includes one or more polymers.

[0080] "PLGA" is generally a copolymer synthesized by ring-opening copolymerization of two different monomers, namely the cyclic dimer of glycolic acid and lactic acid (1,4-dioxane-2,5-dione). PLGA is usually specified with respect to the molar ratio of the monomers used; for example, PLGA 75:25 defines a copolymer whose composition is 75% lactic acid and 25% glycolic acid.

[0081] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic is administered. Typically, it is a pharmaceutically acceptable carrier. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the formulation or composition is administered intravenously. Saline solutions, aqueous dextrose, and aqueous glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. The carrier may be specifically for human therapeutic use. Therapeutically acceptable carriers are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro, editor, 1985) (Non-Patent Document 6). Examples of suitable carriers include sucrose, lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, and surfactants such as DOTAP and phosphatidylserine. Examples of suitable diluents include ethanol, glycerol, water, PBS, Tris-buffered saline, and other physiological buffers. The choice of pharmaceutical carrier can be selected based on the intended route of administration and standard pharmaceutical practice. Preferably, any carrier included is present in trace amounts. The nature and amount of any carrier should not unacceptably alter the benefits of the antigen of the present invention.

[0082] The term "excipient" refers to any essential auxiliary substance that may be present in the finished formulation of the present invention. Typically, it is a pharmaceutically acceptable excipient. For example, the term "excipient" includes, but is not limited to, vehicles, binders, disintegrants, fillers (diluents), lubricants, suspending / dispersing agents, coating agents, stabilizers, dyes, emulsifiers, softeners, preservatives, and / or surfactants. Suitable excipients include, but are not limited to, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The excipient may be specifically intended for human therapy. Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein can be found in "Handbook of Pharmaceutical Excipients", 2nd Edition, (1994), edited by A Wade and PJ Weller (Non-Patent Document 7). The choice of pharmaceutical excipient can be selected taking into consideration the intended route of administration and standard pharmaceutical practice.

[0083] The term "surfactant" includes, inter alia, nonionic surfactants, cationic surfactants, anionic surfactants, and zwitterionic surfactants. Surfactants may be added, for example, to ensure that lyophilized nanoparticles can be resuspended without unacceptable size increase (e.g., without significant aggregation). Surfactants may be particularly useful for human therapy.

[0084] Preservatives, stabilizers, or dyes may be provided in the composition or formulation. Examples of preservatives include sodium benzoate, sorbic acid, and esters of hydroxybenzoic acid. Antioxidants and suspending agents can also be used.

[0085] The term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans.

[0086] The formulation or composition of the present invention can be prepared with a carrier that protects the compound from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.Methods for preparing such compositions are known.Desirably, a pharmaceutically acceptable substance is provided that can increase the shelf life or effectiveness of the formulation or composition.

[0087] In practicing the present invention, the amount or dose range of an antigen of the present invention typically used is one that effectively induces, promotes, or enhances a physiological response associated with a nanoparticle, formulation, or composition of the present invention.

[0088] As used herein, the term "cancer" includes gastrointestinal cancer, head and neck cancer, cancer of the nervous system, kidney cancer, renal cell carcinoma, retinal cancer, melanoma, stomach cancer, liver cancer, genitourinary tract cancer, colon and bladder cancer, multiple myeloma, glioblastoma, lymphoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangioendothelial sarcoma, synovial tumor, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colorectal cancer, pancreatic cancer, breast cancer, ER-positive breast cancer, ovarian cancer, squamous cell carcinoma. The cancer may be selected from the group including, but not limited to, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular tumor, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, retinoblastoma, and leukemia, epithelial carcinoma, solid tumors and hematological malignancies, metastatic cancer. Typically, treating cancer involves one or more of reducing cancer cell survival, proliferation and migration, or invasion by cancer cells.

[0089] As used herein, the term "composition" should be understood to mean something made by man and not to include naturally occurring compositions. The composition may be for human or veterinary use.

[0090] The term "mammal" should be understood to mean higher mammals, especially humans. However, the term also includes non-mammalian animals, such as fish. A human may be an infant, toddler, child, adolescent, adult, or elderly human. A mammal may also be an animal.

[0091] As used herein, the terms "treat," "treatment," and "treating" refer to an approach for obtaining beneficial or desired results, e.g., clinical results.

[0092] As used herein, the term "prevention" is used interchangeably with "prophylaxis" and can mean the complete prevention of an infection or disease, or the prevention of the progression of symptoms of that infection or disease; a delay in the onset of an infection or disease or symptoms thereof; or a reduction in the severity of an infection or disease or symptoms thereof that subsequently develops.

[0093] As used herein, the term "vaccine" refers to an immunogenic composition, such as a formulation of the invention, used to induce an immune response that provides protective immunity (e.g., immunity that protects a subject against infection by a pathogen and / or reduces the severity of a disease or condition caused by infection by a pathogen, or immunity that prevents the survival, spread, or proliferation of cancer cells).

[0094] As used herein, an "immunogenic composition" is a composition comprising the nanoparticles of the invention and an antigen, such that administration of the composition to a subject leads to the development in the subject of a cellular immune response to the antigen. The term may be used interchangeably with "vaccine formulation" in this context.

[0095] As used herein, "polydispersity index" (PDI) is a measure of the heterogeneity of a sample based on size. Polydispersity can arise from the same size distribution. Methods for measuring polydispersity index are known in the art. One way to calculate PDI is by cumulative analysis performed on a machine called a Zetasizer, which uses dynamic light scattering. PDI can assume values ​​between 0 and 1. The lower the polydispersity index, the more homogeneous the nanoparticles. The closer the value is to 1, the greater the variation in particle size. In this context, the nanoparticles of the present invention, or the nanoparticles in the preparations of the present invention, typically have a PDI of 0.26 + / - 0.10 or less.

[0096] As used herein, "CD8 T cell response" refers to the induction of cytotoxic T cells that express the CD8 coreceptor and are capable of killing cells, proliferating, and producing cytokines upon recognition of cells expressing a target antigen.

[0097] As used herein, "Th1 response" refers to the induction of Th1 cells, a lineage of CD4+ T cells that promotes cell-mediated immunity against intracellular pathogens and cancer. Th1 cells produce certain cytokines, particularly interferon-γ, that are important for their function.

[0098] Other advantages and features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention. All documents cited herein, including the specifications, are incorporated herein by reference in their entirety.

[0099] Detailed Description of the Invention Cellular immunity relies on the generation of cells called CD8 T cells, which have cytotoxic activity, and Th1 cells, which secrete IFN-γ. Certain acute and chronic diseases require a Th1 response. However, many commonly used adjuvants, including alum, bias the immune response toward a Th2 response, primarily promoting antibody responses.

[0100] Currently, there is a lack of effective adjuvants that can promote Th1 cell-mediated responses. Similarly, current clinical adjuvants are unable to induce functional CD8 T cell responses because these responses typically require a high activation threshold that involves the simultaneous activation of CD4 T cells and dendritic cells.

[0101] CD8+ T cells are necessary for defense against intracellular pathogens such as viruses, bacteria, mycobacteria, and parasites, but can also contribute to defense against cancer. CD8+ T cells have the ability to directly kill infected or malignant cells and can also provide cytokines that activate the immune system. Pyroptosis is a form of programmed cell death associated with the formation of plasma membrane pores, cytoplasmic swelling, membrane rupture, and the release of cytosolic components such as IL-1β and IL-18 into the extracellular environment, amplifying local or systemic inflammatory responses. Pyroptosis is thought to be mediated by gasdermin. Activation of the pyroptosis molecular pathway involves activation of caspase-11, which subsequently activates the pore-forming protein gasdermin D (GSDMD). Activation of pyroptosis pathway components does not necessarily lead to cell death but has been shown to promote a hyperactivated state of cells through the generation of reactive oxygen species. Cell death or cell hyperactivation both activate the immune system and induce the release of endogenous immunogenic molecules, which promote a strong immune response. This pathway has been shown to promote cellular immunity during infectious diseases, but particulate adjuvants have not previously been shown to promote it. The present inventors surprisingly found that the ability to induce a pathway involved in a type of cell death called pyroptosis is key to the ability of particles to promote CD8 responses.

[0102] The present invention solves the problems of the prior art by providing adjuvants that are surprisingly capable of enhancing a subject's CD8 T cell and / or Th1 cell-mediated immune response against an immunogenic species.

[0103] In one embodiment, the CD8+ T cell response is mediated by caspase-11 or gasdermin D, or by both caspase-11 and gasdermin D. In one embodiment, the CD8+ T cell response is mediated by IL-1 and / or IL-18.

[0104] In one embodiment, the CD8 T cell response is mediated by caspase 11, gasdermin D, IL-1, and IL-18.

[0105] In one embodiment, the Th1 response is mediated by caspase-11 or gasdermin D, or by both caspase-11 and gasdermin D, and in one embodiment, the Th1 response is mediated by IL-1 and / or IL-18.

[0106] In one embodiment, the Th1 response is one mediated by caspase-11, gasdermin D, IL-1, and IL-18.

[0107] The adjuvant can achieve this effect without the need for a coadjuvant, in contrast to prior art polymer particles that require a coadjuvant for use, simplifying formulation and limiting the unwanted inflammatory response or toxicity associated with effective, yet highly reactogenic, coadjuvants such as Toll-like receptor agonists.

[0108] The present inventors have demonstrated the precise size of nanoparticles that are capable of inducing protective Th1 and CD8 responses in subjects, which has not been reported previously.

[0109] The adjuvant is a polymeric nanoparticle (herein referred to as "nanoparticles of the invention").

[0110] The present invention provides biocompatible polymeric nanoparticles (i.e., nanoparticles of the present invention) for use in inducing a CD8 and / or Th1 response in a subject. The response is directed against an immunogenic species. In one embodiment, both a CD8 and a Th1 response are elicited. This is particularly beneficial for use in cancer and viral vaccines.

[0111] The immunogenic species are as described herein.

[0112] The nanoparticles of the present invention comprise a biocompatible polymer. Any suitable polymer may be used. The biocompatible polymer may be selected from the group including, but not limited to, PLGA, PLA, polyphosphazene, and chitosan. Preferably, the nanoparticles are poly(D,L-lactide-co-glycolide) (PLGA) polymer nanoparticles.

[0113] The nanoparticles of the present invention are small, preferably having a diameter (i.e., particle size) of about 30 nm to about 80 nm. Preferably, the particles have a diameter of about 40 nm to 60 nm, or 50 nm to 60 nm in size, typically 35 nm, 40 nm, 45 nm, 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 63 nm, 64 nm, 65 nm, 70 nm, or 75 nm in size. It may be smaller than these values.

[0114] The nanoparticles of the present invention may be biodegradable. The nanoparticles may also be non-degradable.

[0115] The nanoparticles of the present invention may have a PDI of 0.36 or less, preferably 0.26 or less. It may be 1.6 or more and 3.6 or less. The advantage of having low polydispersity is that most of the particles are of an ideal size for enhancing CD8 and / or Th1 responses. This also has the potential to reduce the dose of adjuvant required, as all particles are in the range that drives this beneficial response.

[0116] The nanoparticles of the present invention can have a positive, neutral, or negative charge. Typically, the effect of a nanoparticle is independent of its charge.

[0117] The nanoparticles of the present invention are uniform in size and shape. Typically, the nanoparticles are substantially spherical.

[0118] The nanoparticle of the present invention may be a plurality of nanoparticles.

[0119] The nanoparticles of the present invention may further comprise a coadjuvant. Coadjuvants are known in the art and all suitable coadjuvants are encompassed herein.

[0120] The nanoparticles of the present invention are endotoxin-free. Prior art reported methods for producing PLGA often introduce contaminants such as endotoxins, which act as coadjuvants. Endotoxin-freeness also makes the particles of the present invention suitable for human use. It is noteworthy that the nanoparticles may be solid or hollow.

[0121] The nanoparticles used in the method or use of the invention may be a preparation of nanoparticles of the invention or a composition comprising nanoparticles of the invention, both as described herein.

[0122] In one embodiment, the invention provides a preparation of nanoparticles of the invention. Typically, the nanoparticles in the preparation have a size distribution with an average size, or mean, of about 30 nm to about 80 nm, preferably about 40 nm to about 60 nm, or 52 nm and 65 nm, typically about 52 to 60 nm. Preferably, at least 80% of the nanoparticles in the preparation have a diameter of about 30 nm to about 80 nm, preferably 40 nm to 60 nm. Preferably, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nanoparticles have a diameter of about 30 nm to about 80 nm, preferably 40 nm to 60 nm. Preferably, the particles have a diameter of about 50 nm to about 60 nm in size, typically 35 nm, 40 nm, 45 nm, 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 63 nm, 64 nm, 65 nm, 70 nm or 75 nm in size, although they may be smaller than these values.

[0123] The present invention provides a composition comprising the nanoparticles of the present invention. The composition may further comprise at least one immunogenic species.

[0124] Typically, the immunogenic species is an antigen. Examples of antigens include polypeptide-containing antigens, polysaccharide-containing antigens, and polynucleotide-containing antigens, among others. Antigens can be derived from, for example, tumor cells and pathogenic organisms such as viruses, bacteria, fungi, and parasites, among other sources. It will be understood that the antigen can be any suitable antigen as required by the situation.

[0125] The antigen may be any suitable antigen, including, among others, tuberculosis antigens such as H56, pneumococcal antigens such as pneumolysin, PspA, conjugated pneumococcal polysaccharide, influenza antigens such as influenza nucleoprotein, viral antigens including neuraminidase, hemagglutinin, or human papillomavirus proteins and peptides, tumor-derived antigens such as carcinoembryonic antigen (CEA), MelanA / Mart-1, MUC-1, NY-ESO-1, viability, HER2 / neu, GM2, or combinations thereof.

[0126] It will be appreciated that the composition may contain multiple nanoparticles. Typically, the nanoparticles in the formulation have a size distribution with an average size, or mean, of about 30 nm to 80 nm, preferably 40 nm to 60 nm, or about 52 nm to 65 nm, typically about 52 to 60 nm. Preferably, at least 80% of the nanoparticles in the formulation have a diameter in the range of about 30 nm to about 80 nm. Preferably, at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the nanoparticles have a diameter in the range of about 30 nm to about 80 nm, preferably 40 nm to 60 nm. Preferably, the particles have a diameter of about 50 nm to about 60 nm in size, typically 35 nm, 40 nm, 45 nm, 50 nm, 51 nm, 52 nm, 53 nm, 54 nm, 55 nm, 56 nm, 57 nm, 58 nm, 59 nm, 60 nm, 61 nm, 62 nm, 63 nm, 64 nm, 65 nm, 70 nm or 75 nm in size, which may be less than, equal to or greater than these values.

[0127] The nanoparticles and the immunogenic species, which may be at least one antigen, may be combined in the composition by any suitable means known in the art. The particles may, for example, be mixed with the immunogenic species. In such instances, the nanoparticles and the antigen are not irreversibly bound and / or are not encapsulated. In contrast to the prior art, in this embodiment, the immunogenic species does not need to be encapsulated or irreversibly bound to the nanoparticles. In this embodiment, the antigen may be adsorbed to the nanoparticles and / or mixed with them in the composition, i.e., the antigen may be free in the composition.

[0128] In one embodiment, the immunogenic species may be entrapped or encapsulated within the nanoparticles.

[0129] The immunogenic species may be attached to a nanoparticle.

[0130] The invention provides a composition comprising the preparation of the invention and at least one antigen.

[0131] The composition of the present invention may be a vaccine. The vaccine of the present invention may be prophylactic. The vaccine of the present invention may be therapeutic. It is noteworthy that the composition of the present invention is suitable for anti-cancer vaccines and anti-viral vaccines, or vaccines against intracellular bacteria and parasites.

[0132] The compositions of the present invention may further comprise a pharmaceutically acceptable carrier or salt.

[0133] The composition of the present invention may be a pharmaceutical composition.

[0134] In one embodiment, the composition may further comprise an adjuvant or co-adjuvant(s). Such adjuvants are known in the art and all suitable adjuvants are encompassed herein.

[0135] The composition may further comprise an active agent. The active agent may be any known active agent, such as a drug. The active agent may be an agent for treating cancer, such as a chemotherapeutic agent.

[0136] The present invention also provides a formulation comprising the nanoparticles of the present invention. All features disclosed herein in relation to the compositions of the present invention apply equally to the formulation.

[0137] A composition comprising the nanoparticles of the present invention or the preparation of the present invention is provided. The composition may be an immunogenic composition or a pharmaceutical composition. In this particular embodiment of the present invention, in use, rather than mixing the nanoparticles with antigens before administration, the composition may be injected into a tumor and interact with endogenously released cancer antigens to elicit Th1 and CD8 T cell responses.

[0138] Adjuvants are provided comprising the nanoparticles of the invention, which may comprise at least one antigen.

[0139] Also provided is a method of vaccinating a subject, comprising administering to said subject a composition of the invention or a nanoparticle of the invention. The subject may be a human or an animal. Delivery can be by any known method or methods described herein.

[0140] Also provided is a method of delivering an immunogenic species or antigen to a subject to induce an immune response against the species or antigen, the method comprising administering to the subject a nanoparticle, preparation, or composition of the invention.

[0141] Also provided are methods for delivering the nanoparticles, preparations, or compositions of the invention to a subject. This may be for therapeutic, prophylactic, or diagnostic purposes. The subject may be a human or an animal. Delivery may be by any known method or methods described herein.

[0142] The present invention also provides a method of treating or preventing a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of the nanoparticles, preparations or compositions of the present invention. The subject may be a human or an animal. The disease and condition are as disclosed herein.

[0143] The present invention also provides a method for inducing a Th1 and / or CD8 T cell response in a subject, the method comprising administering to the subject a therapeutically effective amount of a nanoparticle, preparation or composition of the invention.

[0144] The invention also provides a nanoparticle, preparation or composition of the invention for use as an adjuvant in the treatment or prevention of a disease or condition in a subject.

[0145] The present invention also provides the nanoparticles, preparations or compositions of the present invention for use in treating or preventing a disease or condition in a subject. The subject may be a human or an animal. The disease or condition may be a viral infection or a bacterial infection. The disease or condition may be any target cancer.

[0146] These diseases include, but are not limited to, viral infections such as SARS-CoV-2, influenza, hepatitis, shingles, dengue fever, and Zika fever; intracellular bacteria, e.g., Listeria monocytogenes, Brucella abortus, and bacteria that require effective macrophage activation for intracellular killing, particularly group B streptococci, Staphylococcus aureus, and Streptococcus pneumoniae; mycobacteria, including but not limited to Mycobacterium tuberculosis; infections caused by intracellular or intracellular parasites, such as Toxoplasma gondii and Leishmania spp.; and cancer. Th1 responses are required for the treatment and prevention of these diseases.

[0147] Methods of introduction or administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, mucosal, subcutaneous, intranasal, e.g., aerosol, epidural, intracerebral, sublingual, intratumoral, and oral routes. Typically, the nanoparticles or formulations of the present invention are formulated for administration by injection. Of course, oral or sublingual administration does not require injection. Intradermal administration may involve the use of a patch equipped with microneedles. The formulation or composition may be administered by any convenient route, e.g., infusion or bolus injection, via absorption through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa). Administration may be systemic or local. In addition, it may be desirable to introduce the formulation or composition of the present invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated, for example, by an intraventricular catheter attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration may also be employed, for example, by use of an inhaler or nebulizer and formulation with an aerosolizing agent. Preferably, the method of administration is by intramuscular administration.

[0148] Methods for measuring particle size are known in the art. The particle size of polymeric nanoparticles can be measured by dynamic light scattering (DLS) using a Malvern nano ZS. The method can be as described herein in the accompanying examples.

[0149] The nanoparticles of the present invention can be prepared using any suitable method known in the art, such as microfluidic techniques, which may be as described in the accompanying examples.

[0150] In use, the composition of the present invention is used to vaccinate a subject. After vaccination in the subject, the nanoparticles of the present invention promote the generation of CD8 T cells and / or Th1 cells in said subject. In particular, the small size of the particles makes this technical effect possible.

[0151] The present inventors have demonstrated that the PLGA particles of the present invention are potent inducers of CD8 T cells and antigen-specific secretion of the cytokine IFN-γ, which is a hallmark of a Th1 response. These results are shown in Figures 1A, B, and C. The present inventors further demonstrated that this response could not be driven by larger PLGA particles, as shown in Figures 2A and 2B. The present invention provides a novel, previously undescribed strategy for inducing potent CD8 and Th1 responses through vaccination with purified antigens and biodegradable PLGA particles.

[0152] The inventors have also shown that the ability of the 50 nm nanoparticles of the present invention to induce CD8 responses is dependent on pathways related to pyroptosis (programmed immunogenic cell death). This is shown in Figures 3 and 4. The inventors provide a novel mechanism for adjuvant-induced cellular immunity.

[0153] The inventors have surprisingly found that the ability of the particles to induce a type of cell death called pyroptosis is key to their ability to promote CD8 responses. [Example]

[0154] The present invention will now be described with reference to specific examples, which are merely exemplary and illustrative. They are not intended to limit in any way the scope of the claimed exclusive rights or the described invention. These examples constitute the best modes presently contemplated for carrying out the invention.

[0155] Example 1 50 nm PLGA nanoparticles inhibit CD8 when administered with protein antigens + Promotes T cell responses and IFN-γ Materials and Methods C57BL / 6 mice were immunized intramuscularly with 2 mg of 50 nm particles and 20 μg of ovalbumin (OVA) on days 0 and 14. On day 21, splenocytes were analyzed by flow cytometry and CD40 specific for the H-2Kb OVA epitope SIINFEKL was isolated using tetramers. + The percentage (Fig. 1A) and number (Fig. 1B) of 8T cells were quantified, and IFN-γ secretion in splenocytes after restimulation with antigen was assessed (Fig. 1C).

[0156] Other mice were immunized im on days 1 and 14 with 1 mg of biocompatible, non-degradable polystyrene particles with a positive or negative charge (according to prior derivatization with carboxylic acid or amine groups) mixed with 10 μg of the antigen OVA-NP311 (a fusion protein consisting of ovalbumin and the influenza nucleoprotein peptide NP311-325). On day 21, splenocytes were analyzed by flow cytometry and CD40 specific for the H-2Kb OVA epitope SIINFEKL was isolated using tetramers. + The percentage of 8T cells was quantified (Figure 1D).

[0157] Results and Conclusions Results are shown as mean ± SEM, and symbols represent individual animals. Figure 1(C) shows IFN-γ expression analyzed by ELISA after 72 h restimulation with antigen.

[0158] These results demonstrate that the PLGA particles of the present invention are potent inducers of CD8 T cells and the cytokine IFN-γ, which is indicative of a Th1 response (FIGS. 1A, B and 1C).

[0159] These results indicate that nanoparticles can induce antigen-specific CD8 responses regardless of the charge or degradability of the polymer (FIG. 1D).

[0160] Example 2 Larger PLGA nanoparticles (100 nm and 500 nm) inhibited CD8 + It failed to induce T cell responses and IFN-γ.

[0161] Materials and Methods C57BL / 6 mice were immunized intramuscularly with particles (100 nm and 500 nm) and ovalbumin (OVA) on days 0 and 14. On day 21, splenocytes were analyzed by flow cytometry to identify CD8+ cells specific for the H-2 Kb OVA epitope SIINFEKL. + The percentage (A) and number (B) of T cells were quantified using tetramers.

[0162] Results and Conclusions Results are shown as mean ± SEM, and symbols represent individual animals. Figure 2(C) shows IFN-γ expression analyzed by ELISA after 72 hours of restimulation with antigen. Polystyrene nanoparticles were used as a positive control (+Ctrl).

[0163] The response shown by FIG. 1 using the particles of the present invention cannot be driven by other PLGA particles of larger size (FIGS. 2A-B).

[0164] Example 3 50 nm nanoparticles induce antigen-specific CD8+ T cell responses via a pyroptosis-dependent pathway.

[0165] Materials and Methods Mice were immunized intramuscularly (i.m.) with OVA alone or with 50 nm particles on days 0 and 14. Between priming and boosting, mice were treated intraperitoneally or i.m. with 250 μg of necrosulfonamide (NSF) to inhibit pyroptosis. On day 21, splenic (FIG. 3) CD8 markers specific for the H-2 Kb OVA epitope SIINFEKL (SEQ ID NO. 1) were identified by flow cytometry using tetramers. + The percentage of T cells was measured.

[0166] Results and Conclusions Results are shown as mean ± SEM, symbols represent individual animals, and asterisks indicate statistical differences calculated according to multiple comparison ANOVA and Dunnett's multiple comparison test to determine differences compared to OVA + 50 nM PS.

[0167] We showed that the ability of these small PLGA nanoparticles to induce a CD8 response is dependent on necroptosis (Figure 3). Figure 4 shows that 50 nm particles require caspase 11, a protein involved in pyroptosis, to drive a CD8 response.

[0168] Example 4 Preparation of 50nm Anionic Polymer Nanoparticles and Calculation of Their Size Materials and Methods

[0169] [Table 1]

[0170] Nanoparticle formulations Preparation of lipid and polymer stocks and buffers STEP 1. Lipid and polymer were weighed together and dissolved in DMSO. Heat and bath sonication can optionally be used to achieve complete dissolution of the materials, if necessary.

[0171] The final concentration of polymer and lipid was 0.5 mg / mL in total, thus 0.25 mg / mL for PLGA and 0.25 mg / mL for PS. 1:1 weight-to-weight ratio

[0172] STEP 3. Prepare Tris buffer to 10 mM, pH 7.4. Note: pH is adjusted using 0.1 M HCl.

[0173] STEP 4. Filter the buffer solution through a 0.22 μm filter to remove impurities.

[0174] Microfluidics STEP 5. The desired final concentration of polymer NPs is 20 mg / mL, therefore, 160 mL of particles are produced using a Nanoassemblyr™ (Precision Nanosystems, Inc.) at a flow rate ratio of 1:1 and a total flow rate of 10 mL / min. The initial and final waste volumes are 0.35 and 0.05, respectively.

[0175] Tangential Flow Filtration (TFF) STEP 6. To remove the solvent and concentrate the particles to the desired concentration, use tangential flow filtration (Spectrum Labs). 160 mL of polymer NPs are concentrated to 2 mL by TFF, and the solvent is washed with 24 mL of 10 mM Tris buffer, pH 7.4 (12 washes).

[0176] STEP 7. Measure the size, PDI and ZP of the resulting particles. If the results are within the desired range, the particles are ready.

[0177] NOTE: If particles are larger than expected (because the desired particle range is very narrow), the particles can be bath sonicated for 90 seconds (1 cycle) using the Bioruptor.

[0178] analysis technology Measurement of particle size, particle size distribution and zeta potential using Malvern ZS The particle size of polymer nanoparticles was determined by dynamic light scattering (DLS) using a Malvern nano ZS (Malvern Instruments, Worcestershire, UK). Samples were diluted 10-fold with 10 mM Tris buffer, pH 7.4, and filtered through a 0.22 μm filter to achieve optimal particle concentration with an attenuator number (attenuator number) of 7–9. Triplicate measurements were performed at 25 °C. A 1 mL sample was filled into a square disposable plastic cuvette and analyzed using a 4 mW He-Ne 633 nm laser. For zeta potential, samples were diluted in the same manner as for size, and a cell capillary electrophoresis cuvette was used. Data collection and analysis were performed using Malvern Dispersion Technology Software (DTS) v.7.12 (Malvern Instruments, Worcestershire, UK).

[0179] Defining Critical Product Parameters (KPP) for Liquid Products To reliably define the physicochemical properties of the product, the HSPC:Chol:DSPE-PEG2000:DOX formulation was characterized using the following methods.

[0180] [Table 2]

[0181] Example 5 Preparation of cationic polymer nanoparticles with a particle size of 50 nm

[0182] [Table 3]

[0183] Nanoparticle formulations Preparation of lipid and polymer stocks and buffers STEP 1. Dissolve each lipid and polymer in a mixture of chloroform:methanol (9:1 v / v). The resulting stock concentrations are as follows:

[0184] - PLGA 50:50: 20 mg / mL in chloroform:methanol (9:1 v / v)

[0185] - DDA: 20 mg / mL in chloroform:methanol (9:1 v / v) STEP 2. Mix both stocks and pour into a round-bottom flask.

[0186] Weight ratio 1:1 Final concentration: 20mg / mL Final volume: 2 mL STEP 3. Prepare Tris buffer to 10 mM and pH 7.4.

[0187] NOTE: pH is adjusted with 0.1 M HCl.

[0188] STEP 4. Filter the buffer solution using a 0.22 μm filter to remove impurities and contaminants.

[0189] Fabrication of cationic polymer formulations using lipid films and ultrasonic baths STEP 5. Evaporate the organic solvent using a rotary evaporator at 200 rpm for 15 minutes.

[0190] STEP 6. Rehydrate the film with 2 mL of filtered Tris buffer 10 mM (pH 7.4) until completely dissolved.

[0191] STEP 7 Transfer the cationic polymer particles to a 15 mL Falcon tube and place it in a Bioruptor® Plus sonicator (Diagenode, Liege Science Park, 3 Rue bois Saint-Jean, 4102 Ougree, Belgium).

[0192] This system utilizes ultrasound waves generated by a magnet placed below the water bath to indirectly transmit ultrasonic energy to the sample.

[0193] STEP 8. Perform one 90-second cycle (x 10 times) with a 30-second delay between cycles.

[0194] analysis technology STEP 9. Measurement of particle size, particle size distribution, and zeta potential using Malvern ZS The particle size of polymer nanoparticles was determined by dynamic light scattering (DLS) using a Malvern nano ZS (Malvern Instruments, Worcestershire, UK). Samples were diluted (10-fold) with 10 mM Tris buffer, pH 7.4, and filtered through a 0.22 μm filter to achieve optimal particle concentration and optimal attenuator number (attenuator number 6–7). Triplicate measurements were performed at 25 °C. A square disposable plastic cuvette was filled with 1 mL of diluted sample and placed in an instrument using a 4 mW He-Ne 633 nm laser. For zeta potential, samples were diluted in the same manner as for size, and a cell capillary electrophoresis cuvette was used. Data collection and analysis were performed using Malvern Dispersion Technology Software (DTS) v.7.12 (Malvern Instruments, Worcestershire, UK).

[0195] Defining Critical Product Parameters (KPP) for Liquid Products

[0196] [Table 4]

[0197] Example 6 Antigen-specific antitumor immunity in mice

[0198] Materials and Methods Mice were vaccinated on days 0 and 7 with PBS, OVA peptide (10 μg) alone, or 50 nm PS particles (1 mg) and OVA peptide (10 μg), followed by 3.5 × 10 5 were challenged sc with B16-OVA tumor cells.

[0199] Results and Conclusions As shown in Figures 6A and 6B, potent antigen-specific antitumor immunity was driven by 50 nm PS nanoparticles. Figure 6A shows no tumor growth 22 days after induction, and Figure 6B shows 100% survival 20 days after induction.

[0200] Example 7 Particle size does not affect antibody size or neutralizing capacity, but does affect isotype and class switching

[0201] The success of traditional prophylactic vaccines relies on the induction of sustained antibody responses, most of which are directed against T-cell-dependent epitopes. As a result, vaccine design and adjuvant discovery have used antibody titers and neutralizing activity as the primary correlates of vaccine efficacy and adjuvant potency.

[0202] Therefore, we evaluated how particle size affects the magnitude and quality of antibody responses. The polymer polystyrene (PS) particles are biocompatible particles with unique adjuvant properties that allow for greater control of particle size compared to other biodegradable polymers, including PLGA. Therefore, we selected PS particles with diameters ranging from 50 nm to 100 μm as a model particle adjuvant.

[0203] PS particles were mixed with OVA at antigen / particle ratios optimized for intramuscular (im), intraperitoneal (ip), or subcutaneous (sc) administration. Mice were vaccinated with vehicle, OVA alone, or PS+OVA on days 0 and 14, and OVA-specific IgG responses in serum were assessed on day 21. All particle sizes, from 50 nm to 30 μm, elicited strong OVA-specific IgG responses, but larger 100 μm particles given ip were slightly less efficient. PS particles induced responses comparable in magnitude to the OVA-specific IgG titers induced by the gold-standard adjuvant, Alum, both im (Figure 7A) and ip (Figure 7B). We previously demonstrated that PLGA particles in the 400 nm range induced antigen-specific antibodies when given sc. To determine whether PS particles could elicit similar responses, mice were vaccinated sc with PS+OVA according to the above scheme, and additional nanodomain sizes (200 nm and 430 nm) were also tested. Again, all particle sizes, including all in the nanometer range, induced robust OVA-specific IgG after sc vaccination (Figure 7C). Finally, to establish whether the particles could induce protective humoral responses against clinically relevant antigens, mice were vaccinated sc with recombinant Staphylococcal clumping factor A (ClfA) admixed with the particles. ClfA is a surface protein and virulence factor of Staphylococcus aureus that binds to fibrinogen and fibrin, promoting bacterial adhesion to thrombi in vivo and to plasma protein-coated biomaterials ex vivo. Bacterial adhesion could be blocked by neutralizing antibodies against ClfA, allowing us to determine the functionality of antibodies induced after particle immunization by measuring the ability of sera from vaccinated mice to inhibit S. aureus adhesion to immobilized fibrinogen in vitro. Particles of all size ranges induced ClfA IgG (Figure 7D) and showed comparable neutralizing activity (Figure 7E).

[0204] Finally, we evaluated the quality of humoral responses by analyzing antibody isotypes. All particle sizes tested induced comparable IgG1 titers both im (Figure 8A) and sc (Figure 8D), and most particle sizes induced IgG2b when administered sc with ClfA (Figure 8E). Notably, only 50 nm particles induced class switching to IgG2c after im vaccination with OVA (Figure 8C) or sc vaccination with ClfA (Figure 8F). Collectively, these data indicate that while the overall magnitude of humoral responses and antibody functionality are independent of particle size, particle size determines the quality of antibody responses by providing the unique ability to control class switching and selectively induce antigen-specific IgG2c with 50 nm particles. IgG2c responses require IFN. Therefore, the selective enhancement of IgG2c supports our finding that the particles are effective in promoting Th1 responses.

[0205] Example 8 Size-dependent induction of antigen-specific IFN-γ drives IgG2c class switching

[0206] Antibody isotype switching is influenced by T lymphocyte-derived cytokines. In mice, IL-4 promotes switching to IgG1 and IgE, while IFN-γ stimulates IgG2c / IgG2a in C57 / BL6 or BALB / c mice, respectively. Previously, we demonstrated that 50-nm polystyrene particles combined with OVA or the tuberculosis candidate antigen H56 have a superior ability to induce Th1 responses compared with Alum. We hypothesized that the unique ability of these small nanoparticles to promote IgG2c class switching is related to their superior ability to promote IFN-γ secretion compared with larger particles. To test this hypothesis, cytokine secretion was assessed by ex vivo antigen restimulation of splenocytes obtained from mice vaccinated im with OVA and different PS sizes. Spleen cells from animals vaccinated with 50-nm PS particles were the only ones to significantly upregulate IFN-γ secretion after OVA compared with those obtained from mice vaccinated with antigen alone or in combination with other particle sizes (Figure 9A). IFN-γ knockout (Ifng) mice were also significantly upregulated after OVA compared with those obtained from mice vaccinated with antigen alone or in combination with other particle sizes. - / - ) Inoculation of mice with 50 nm PS + OVA failed to induce IgG2c, confirming that IFN-γ is essential for IgG2c switching, but IFng - / - In 50-nm and wild-type (WT) mice, IgG, IgG1, and IgG2b levels remained comparable (Figure 9B). These results confirm that 50-nm particles promote IFN-γ-driven antibody class switching to IgG2c.

[0207] Example 9 Particle size contributes to durable antigen-specific CD8 responses, independent of vaccination route. + and CD4 + Determines the induction of T cell responses and confers protection against melanoma

[0208] Although intrinsic particle properties such as size, shape, and hydrophobicity influence the induction of CTL and IFN-γ responses, other factors such as the immunization pathway also play an important role. These factors influence the targeted DC subsets and therefore the CD8+ / CD8+ antigens, primarily by influencing the rate of antigen and adjuvant transport to lymph nodes. + Small nanoparticles (>200 nm) are taken up locally at the injection site but are free to reach lymph nodes shortly after injection, potentially affecting the efficiency of T cell cross-priming. + DC and CD8 + It is possible to directly target resident DCs, including plasmacytoid DCs (pDCs), which specialize in T cell cross-presentation and priming. In contrast, particles smaller than 200 nm are more likely to be taken up by resident cells at the injection site and transported to lymph nodes. Small nano-sized particles can efficiently prime CTL responses, but the CD8+ / CD8+ response with particle formulations remains poorly targeted. + The relative contribution of particle-specific attributes, i.e., size and the impact of immunization strategy, to the induction of T cell responses has not been fully resolved. To address whether particle size can drive CTLs independently of vaccination route, we analyzed antigen-specific CD8+ T cells after immunization with OVA and a blend of particles of different diameters, administered either i.p. or im. using the same prime-boost regime as before. + T cell responses were assessed. Seven days after the booster, antigen-specific CD8 + T cell responses were quantified by flow cytometry using a tetramer of the H-2 Kb OVA epitope SIINFEKL. Notably, only the 50 nm particles elicited antigen-specific CD8 T cell responses in the spleens of animals vaccinated either i.p. or im. + The polymer particles induced a significant increase in CD8 T cells (Fig. 10A-B). + This suggests that the magnitude of administration is more important than the route of administration in priming T cell responses.

[0209] To examine whether particle composition influences this response, we investigated antigen-specific CD8 responses in mice given biocompatible OVA alone or in combination with 50 nm PS particles, or biodegradable PLGA particles with diameters of 50-60 nm, 100 nm, and 500 nm. + T cells were also compared. Importantly, only the 50 nm PLGA particles induced significant numbers of antigen-specific CD8 T cells after im vaccination, comparable to the CTL responses elicited by the 50 nm PS particles. + In contrast, the 100 nm and 500 nm particles were able to induce T CD8 T cell responses (Figure 10C-D). + The 50 nm PLGA particles failed to stimulate a Th1 response (Figure 10E-F). Similarly, only spleen cells from animals vaccinated with 50 nm PLGA particles induced antigen-specific IFN-γ production comparable to that from mice vaccinated with 50 nm PS particles when stimulated ex vivo with antigen (Figure 10G). In contrast, no IFN-γ was detected in spleen cells isolated from animals immunized with antigen and 100 nm or 500 nm PLGA (Figure 10H), indicating that polymer size, not polymer composition, determines the induction of Th1 responses.

[0210] The size is CD4 + To determine whether influenza A nucleocapsid protein peptide (SEQ ID NO. 2) QVYSLIRPNENPAHK(NP) affected T cell responses, mice were administered the influenza A nucleocapsid protein peptide (SEQ ID NO. 2) QVYSLIRPNENPAHK(NP) 311-325 ) alone or in combination with the particles NP, a known class II restricted epitope in C57BL / 6 mice. 311-325 By using NP 311-325 The availability of a suitable MHC-II tetramer allowed us to overcome the poor performance of the OVA IA(b) tetramer. Alum was used as a positive control, and as expected (McKee et al., International Immunology, 20, 659-669, 2008) (Non-Patent Document 8), it increased antigen-specific CD4 +The 50 nm PS particles induced the highest increase in Ag-specific CD4 T cells. + The 50 nm particles followed Alum in their ability to induce T cell responses, whereas none of the other sizes tested induced significant responses compared to antigen alone (Fig. 11A). When 50 nm particles were mixed with proteins in addition to peptides, and induced CD4 T cells via the IM pathway, + To confirm that T cell responses can be induced, mice were administered NPs containing OVA mixed with 50 nm particles. 311-325 Fusion antigen (NP) produced by chemical complexation of 311 The mice were vaccinated im with either OVA (-OVA) or OVA alone. Seven days after the booster, splenic NP was 311-325 specific CD4 + The number of T cells was 50nmPS+NP 311 The expression of SIINFEKL class I-restricted CD8 T cells was enhanced in mice receiving SIINFEKL-OVA (Figure 11B). As expected, these animals also expressed SIINFEKL class I-restricted CD8 T cells. + T cells were upregulated (Figure 11C). The breadth of antigen-specific T cell responses induced by 50 nm particles was assessed 100 days after the booster. Class I SIINFEKL-specific CD8 + T cells were more sensitive to NP than to antigen alone, as opposed to animals receiving only antigen. 311 -OVA + 50 nm particles vaccinated animals remained upregulated (Fig. 11D, upper left panel and pointillism). These cells were characterized by upregulation of the canonical memory marker CD69 and the absence of CD62L expression and CD103 (Fig. 11D, histogram). Previously, we reported that CD4 + We determined that T cells were the primary producers of antigen-specific IFN-γ. NP at 100 days post-booster 311 -Re-stimulation of splenocytes with OVA was significantly enhanced by 50 nm PS+NP compared to antigen alone. 311 -significantly enhanced IFN-γ secretion in OVA-immunized mice and functional Ag-specific CD4 + This suggested the presence of memory T cells (Fig. 11D, lower left panel).

[0211] To further assess the functionality of the response induced by nanoparticle vaccination, we used cancer cells genetically engineered to express the vaccine antigen OVA (B16-OVA) and assessed their ability to confer prophylactic protection against the B16-F10 melanoma model. This model demonstrates that protection is mediated by IFN-γ, antigen-specific CD8 + and CD4 + This vaccine was chosen because it is known to be highly dependent on T cells. 14 days after the im booster vaccination, B16-OVA cells were implanted sc, and tumor growth was monitored and recorded daily (Fig. 12A). Interim monitoring showed that mice vaccinated with 50 nm PS particles and OVA were completely protected against melanoma challenge, whereas all animals in the mock (PBS) group developed tumors and died by day 25 after implantation. Vaccination with OVA alone delayed tumor development compared to PBS, but 80% of animals still developed tumors within 2 weeks after implantation, and 75% succumbed to challenge by day 35 (Fig. 12B). Extension analysis 100 days after tumor implantation showed that vaccination with 50 nm PS + OVA conferred long-term protective immunity against B16 melanoma (Fig. 12C).

[0212] Overall, our results demonstrate that antigen-specific Th1 and CD8 + We identify 50 nm as the optimal size to induce T cell responses and promote long-term and durable CMI after vaccination, indicating that size critically determines the ability of polymer particles. Furthermore, biodegradable PLGA particles elicited similar responses, demonstrating that CMI by 50 nm particles is independent of immune pathway and polymer composition.

[0213] Example 10 IL-1 and IL-18 were associated with nanoparticle-induced IFN-γ and CD8 + Required for T cell responses Importantly, IL-1 family cytokines regulate T cell priming, proliferation, and survival by promoting the maturation of antigen-presenting cells or by acting directly on lymphocytes.

[0214] Vaccination of IL-1R1- and IL-18-deficient mice with 50 nm particles and OVA demonstrated that neither cytokine was required for the induction of OVA-specific IgG responses. Overall, the size and IgG isotype profiles were significantly higher than those of Il18. - / - The results were similar in both KO and WT mice, except for IgG2b production, which showed a tendency to increase in titers in OVA-resistant mice (Figure 13A). In contrast, OVA H-2Kb restricted CD8 + Analysis of T cells revealed antigen-specific CD8 + Both the percentage and total number of T cells were significantly higher in IL18 mice compared to wild-type (WT) animals. - / - The CTL response induced by 50 nm particles was significantly reduced in mice, demonstrating that IL-18 is required for the CTL response. + Despite reports suggesting that OVA can directly support T cell expansion and effector function (Ben-Sasson et al., Journal of experimental medicine, 210, 491-502, 2013a; Ben-Sasson et al., Cold Spring Harbour symposia on quantitative biology, 78, 117-124, 2013b), OVA-specific CD8 + T cell numbers were not affected by the impairment of IL-1 signaling (Fig. 13B-C). When compared between mice vaccinated with 50 nm + OVA, Il1r1- / - consistently produced significantly less IFN-γ than WT when stimulated with OVA, whereas Il18 - / - Mice showed an unexpected, consistent, and significant increase in IFN-γ secretion compared to WT (FIG. 13D).

[0215] Example 11 The non-canonical inflammasome sensor caspase-11 and the pyroptosis effector GSDMD are essential mediators of nanoparticle-induced cell-mediated immunity The protease caspase-1 (casp-1) regulates the secretion of leaderless proteins, such as IL-18 and IL-1β. Upstream, sensing of various infectious and non-infectious stimuli, including PAMPs, particulate matter, ion flux, mitochondrial dysfunction, and redox stress, initiates the assembly of inflammasomes that recruit procaspase-1, leading to autoproteolysis and activation. In addition to the caspase-1 canonical inflammasome, non-canonical inflammasomes using mouse caspase-11 (or human caspase-4 / 5) or caspase-8 can promote the secretion of leaderless proteins, including IL-1 and IL-18. Therefore, we aimed to determine whether caspase-1 or caspase-11 contribute to antigen-specific CMI induced by 50 nm polymer particles. To assess this, we pharmacologically inhibited casp-1 by locally injecting Ac-YVAD-fmk at the time of vaccination. In parallel, casp-11-deficient mice (Casp11 - / - ) was also vaccinated.

[0216] Pharmacological inhibition of casp-1 did not result in antigen-specific CD8 +There was no significant change in T cell induction (Fig. 14A, B). However, antigen restimulation of splenocytes from Ac-YVAD-fmk-treated animals tended to decrease IFN-γ secretion (Fig. 14C). Casp-1 inhibition did not alter the magnitude or quality of the antibody response (Fig. 15A). On the other hand, casp-11 deficiency significantly reduced particle-induced antigen-specific CD8 T cells in both the percentage and total number of SIINFEKL-specific CTLs in the spleen compared to particle-fed WT mice. + Casp-11 deletion significantly impaired T cell responses (Fig. 13D, E). Furthermore, IFN-γ secretion upon antigen stimulation was four-fold reduced in cells compared to wild-type spleen cells (Fig. 13F). As in the previous study, casp-11 deletion did not affect antibody responses (Fig. 15B).

[0217] Activation of the canonical casp-1 inflammasome and the non-canonical caspase-11 inflammasome results in downstream cleavage of GSDMD. The N-domain of the cleaved GSDMD interacts with acidic phospholipids on the inner surface of the plasma membrane and oligomerizes to form a transmembrane pore. The GSDMD pore allows the release of IL-1β and IL-18 as well as pyroptotic cell death. However, depending on the activation context, cytokine release can occur during or independently of cell death when cells adopt a hyperactivated state.

[0218] We hypothesized that the particles act by directly targeting skeletal muscle cells, inducing local death that subsequently releases danger-associated molecular patterns that may act as endogenous adjuvants to enhance immune responses. In vitro data showed that 50 nm particles induced cell death via the release of LDH in differentiated mouse myotubes. In contrast, 1 micron particles did not (Figure 16).

[0219] We aimed to investigate whether GSDMD is required for particle-induced CMI and humoral responses in vivo. The inhibitor necrosulfonamide (NSA) binds to the C191 residue of GSDMD and inhibits p30-GSDMD oligomerization. NSA selectively blocks pyroptosis and IL-1β release in human and mouse cells without affecting TLR signaling, inflammasome formation, or gasdermin E-mediated cell death. NSA inhibits the necroptosis effector mixed lineage kinase domain-like (MLKL) protein in humans, but not in mouse cells. To assess the relevance of local and systemic GSDMD activation in particle-induced responses, mice were administered NSA intraperitoneally 1 h before vaccination or intramuscularly at the time of immunization. GSDMD inhibition did not affect antibody responses, regardless of the route of inhibitor administration (Figure 15C). On the other hand, ip administration of NSA significantly inhibited antigen-specific CD8 + induced a partial depletion of T cells, which was abolished by local administration of NSA (Fig. 13G). This indicates that local activation of GSDMD is required for priming of CTL responses by 50 nm particles. Furthermore, local administration of NSA downregulated the ex vivo IFN-γ response of restimulated splenocytes (Fig. 13I).

[0220] Overall, these data suggest that 50 nm polymer particles induce local activation of the noncovalent inflammasome sensors caspase-11 and GSDMD, promoting pyroptosis of cells at the injection site and secondary to caspase-1 activation and subsequent release of active IL-1 and IL-18, resulting in antigen-specific IFN-γ and CD8 upregulation. + These results suggest that they promote T cell responses, respectively.

[0221] Consideration Although the number of candidates is increasing, polymer particle adjuvants have not yet been included in clinically approved vaccines. Two of the most important aspects of rational adjuvant design are defining design principles that link particle properties with specific immune profiles and elucidating their mode of action. The use of polydisperse formulations with overlapping sizes, different antigen incorporation methods, coadjuvant incorporation, or diverse immunization strategies has led to conflicting results, hindering the identification of optimal particle sizes associated with specific types of immune responses.

[0222] To address the long-standing question regarding the role of particle size in adjuvant-driven immunity, we used biocompatible, highly monodisperse, endotoxin-free polystyrene particles mixed with protein antigens as a model particulate vaccine. These works revealed that particle size differentially regulates distinct components of the immune response, namely, humoral and cellular responses. Particle size did not significantly affect the magnitude of the humoral response or the neutralizing potency of antibodies (Figures 7 and 8). However, particle size critically determined the ability of solid polymer particles to promote cellular immunity against protein antigens. We demonstrated that polymer nanoparticles enhance CD8 activation. + We clearly identified 50±10 nm as the optimal size for the induction of T cell responses and IFN-γ, regardless of the route of administration or the antigen used (Figures 9 and 10). In addition, we demonstrated that particles given prophylactically with antigens expressed by cancer cells provided protection (Figure 12) and durable CD8 + It provided evidence of a T cell response (FIG. 11D), demonstrating efficacy in a melanoma model.

[0223] PLGA 50nm biodegradable particles also showed similar antigen-specific CD8 activity as PS particles. +The CMI-inducing ability of 50 nm particles extends beyond that of model polystyrene particles, as we have shown that 50 nm particles induce T cell responses and IFN-γ production. In contrast, 100 nm and 500 nm PLGA particles failed to induce CMI (Figure 10C–H). PLGA particles are one of the most promising adjuvant candidates, as many PLGA particles and other products have been approved for human use by the FDA and EMA. While PLGA particles have been successfully used as drug carriers, they have not yet reached the vaccine industry. In general, CMI induction remains poor. This has led to alternative coformulation strategies with TLR agonists and other coadjuvants, or complex antigen encapsulation or complexation methods, which have led to challenges in formulation stability, increased costs, and challenges in converting these into clinically relevant formulations. Prior to this invention, limitations in the PLGA particle formulation process limited their production to sizes between 80 nm and 250 μm. However, new platforms, such as the microfluidic technology used here to fabricate the 50 nm particles, allow for fine control of particle size, low polydispersity index particles, avoidance of hazardous solvents, and scalability for large-scale adjuvant production. Importantly, this, along with the identification of optimal particle sizes to promote CMI, may accelerate the development of PLGA adjuvants for anti-cancer drugs and anti-viral vaccines.

[0224] We identified the non-canonical inflammasome sensor caspase-11 and the pyroptosis effector gasdermin D as novel actors in the induction of CMI induced by 50 nm particles (Fig. 14). It is important to emphasize that the role of caspase-11 in the induction of Th1 responses has previously been reported for GLA-SE. The ability of GLA-SE to participate in the non-canonical inflammasome pathway is due to its glucopyranosyl lipid moiety, which can interact with caspase-11 in a manner similar to its natural ligand, LPS. However, the particles and antigen used in this study did not induce the secretion of IL-6, TNF-α, or IL-1β in unprimed dendritic cells, which are known to produce inflammatory cytokines, after sensing LPS concentrations as low as 10 pg / ml, demonstrating no evidence of LPS contamination. It is therefore plausible that 50 nm particles induce the release of specific DAMPs that can act directly on caspase-11.

[0225] The requirement of GSDMD for particle-induced CMI suggests that different endogenous casp-11 ligands are induced by particles or that the downstream consequences of casp-11 activation are cell-dependent. Indeed, when cultured in vitro, particles caused limited cell death in DCs but induced hemolytic cell death and casp-11 activation in skeletal muscle tubes.

[0226] Nanoparticle-induced CMI required both IL-1 and IL-18. This data supports our understanding that IL-1R1 signaling is essential for the regulation of CD4 + It is required for the secretion of IFN-γ, which has been shown to be T cell dependent, whereas IL-18 is primarily expressed by CD8 + These findings suggest that IFN-γ deficiency contributes to the T cell response (Figure 13A), suggesting a division of labor between IL-1 and IL-18 in regulating Th1 and CTL responses. This finding supports the conclusion that IFN-γ deficiency may contribute to the nanoparticle-induced CD8 +This is consistent with the fact that it had no effect on T cell numbers (Fig. 9B).

[0227] Although canonical and non-canonical inflammasomes can induce pyroptosis, the cleavage of pro-IL-1β and pro-IL-18 classically requires caspase-1 activity. Thus, blocking casp-1 enzyme activation and the resulting activation of IL-1 and IL-18 may be a key factor in preventing IL-1-induced IFN-γ production and IL-18-driven CD8+ production. + This leads to the abolition of both T cell responses and IL-18 activation. However, administration of Ac-YVAD-fmk only affected IFN-γ secretion, while CD8 responses remained intact. Interestingly, many reports suggest that in Salmonella infection models, caspase-11, not caspase-1, mediates the proteolytic activation of IL-18, whereas the caspase-4 inflammasome (one of the human orthologs of mouse casp-11) directly cleaves IL-18 during intestinal infection with Salmonella enterica. Therefore, we propose that caspase-11 directly mediates the activation of IL-18 triggered by 50 nm particles.

[0228] In summary, we demonstrate that particle size modification induces long-term protective T CD8 + We demonstrate that this approach can be used as a strategy to enhance the ability of polymeric particulate adjuvants to induce Th1 responses. We are the first to demonstrate the involvement of the non-canonical inflammasome sensor, caspase-11, and the pyroptic effector, gasdermin D, in the mode of action of particulate adjuvants, highlighting the importance of non-canonical inflammasome activation for the induction of cellular immunity by nanoparticle vaccines. These results open new avenues for the rational design of anti-cancer and anti-viral vaccines.

Claims

1. 1. A polymeric nanoparticle having a diameter of 30 nm to 80 nm for use in inducing a CD8 and / or Th1 response against an immunogenic species of a subject, comprising: Polymer nanoparticles, wherein the polymer is poly(D,L-lactide-co-glycolide) (PLGA), polylactic acid (PLA), polyphosphazene, or chitosan. （１６）

2. The polymeric nanoparticles for use according to claim 1, wherein the response is induced without a co-adjuvant.

3. The polymeric nanoparticles for use according to any one of claims 1 to 7, wherein the polymeric nanoparticles have a diameter of from 30 nm to 65 nm.

4. 4. The polymeric nanoparticles for use according to claim 3, wherein the polymeric nanoparticles have a diameter of 40 nm to 60 nm.

5. 5. The polymeric nanoparticles for use according to any one of claims 1 to 4, wherein the polymeric nanoparticles are formulated as a composition, said composition comprising the nanoparticles and an immunogenic species, and wherein the nanoparticles and the immunogenic species are mixed in the composition.

6. 6. The polymeric nanoparticles for use according to claim 5, wherein the immunogenic species is adsorbed to the surface of the nanoparticles and / or is free in the composition.

7. The polymeric nanoparticles for use according to any one of claims 1 to 6, wherein the nanoparticles are endotoxin-free.

8. Polymeric nanoparticles for use according to any one of claims 1 to 7, wherein the nanoparticles are biocompatible.

9. Polymer nanoparticles for use according to any one of claims 1 to 8, wherein the nanoparticles are solid particles.

10. Polymeric nanoparticles for use according to any one of claims 1 to 9, wherein the nanoparticles are biodegradable.

11. The polymeric nanoparticles for use according to any one of claims 1 to 10, wherein the immunogenic species is an antigen.

12. A composition comprising a mixture of an adjuvant and an immunogenic species, wherein the adjuvant comprises biocompatible and / or biodegradable polymeric nanoparticles having a diameter of 30 nm to 80 nm; The composition, wherein the polymer is poly(D,L-lactide-co-glycolide) (PLGA), polylactic acid (PLA), polyphosphazene, or chitosan.

13. The composition of claim 12 , wherein the immunogenic species is adsorbed to the surface of the nanoparticles and / or is unbound in the composition.

14. A composition described in claim 12 or 13, comprising a plurality of nanoparticles, at least 80% of which have a diameter of 30 nm to 80 nm.

15. The composition of any one of claims 12 to 14, wherein the nanoparticles are endotoxin-free.

16. The composition of any one of claims 12 to 15, wherein the nanoparticles are solid.

17. The composition according to any one of claims 12 to 16, which is a vaccine composition.

18. A composition according to any one of claims 12 to 17 for use in a method of inducing a CD8 and / or a Th1 response in a subject.

19. 18. A composition according to any one of claims 12 to 17 for use in a method of treating or preventing a disease or condition in a subject.

20. 20. The composition for use according to claim 19, wherein the disease or condition is a bacterial infection or a viral infection or cancer or a chronic infection.

21. 21. The composition for use of claim 20, wherein the disease is selected from the group comprising SARS-CoV-2, influenza, hepatitis, shingles, dengue, Zika, infections caused by intracellular bacteria or bacteria that require effective activation of macrophages for intracellular killing, mycobacteria, intracellular parasites or infections caused by parasites in an intracellular developmental stage.

22. 18. The vaccine composition of claim 17 for use in vaccine therapy for preventing or treating a condition or disease in a subject.

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