Vaccination using high-density microprojection array patch

Abstract

To provide a method of stimulating an immune response in a human.SOLUTION: A microprojection array patch comprises a base and a large number of continuous non-porous projections extending from the base made of synthetic polymer, where at least one projection comprises an uncoated support section which transitions into an end section which is dry-coated with vaccine. Vaccine doses which are applied as a coating to a microprojection array patch are administered to people.SELECTED DRAWING: Figure 2

Description

Detailed Description of the Invention

[0001] [Background technology] The present invention relates to the use of microprojection array patches (MAPs) for the delivery of vaccines, in particular polymeric high density microprojection array patches (HD-MAPs) for the delivery of vaccines to patients, which deliver smaller vaccine doses (dose-sparing) than vaccine doses delivered by intramuscular injection, while providing equivalent or superior immunogenicity.

[0002] [Prior art] Reference herein to prior publication (or information derived therefrom) or known matter is not, and should not be, regarded as an admission, endorsement or any suggestion that the prior publication (or information derived therefrom) or known matter forms part of the common general knowledge in the field to which this specification pertains.

[0003] Most vaccinations are still administered intramuscularly using a needle and syringe invented in 1853. More efficient vaccine delivery methods are needed to make reduced-dose, inexpensive, heat-resistant, self-administered, and painless vaccinations available, especially in resource-poor countries. Patches used to deliver vaccines and drugs to the skin have been developed, such as vaccine-dry coated silicone microprojection skin patches (Fernando et al. 2018). Skin is an ideal site for vaccine delivery due to its high concentration of antigen-presenting cells (Fernando 2010). Studies using silicone patches have observed that a single 15 μg dose produced an immune response similar to that achieved with traditional intramuscular vaccination using a needle and syringe.

[0004] There are various microprojection / microneedle arrays and methods for administering vaccines via these arrays. One consideration when developing novel vaccine delivery technologies is the ability to globally manufacture the millions of doses required for vaccination. Cost is a consideration, and array administration must be less expensive or comparable to needle-and-syringe administration. While dissolving needle microarrays have been investigated (Rouphael 2017, Hirobe 2015), it is questionable whether these dissolving needle patches can be mass-produced under sterile conditions in the required quantities and at a cost comparable to needles and syringes. Another drawback of dissolving microneedles is that each clinical vaccination takes a minimum of 20 minutes to 6 hours to dissolve in the skin, depending on the needle design. Such a long dwell time would significantly delay mass vaccination. In contrast, patches made with sequentially inactivated synthetic polymers can be mass-produced inexpensively. The vaccine is dry-coated onto the polymer using a thin layer of vaccine that rapidly dissolves in the skin within 2 minutes of application. Automated coating methods have been developed to coat vaccines directly onto microprojection arrays, achieving mass production of vaccine-coated patches.

[0005] When vaccines are delivered to skin-resident APCs using high-density microprojection arrays, enhanced immunogenicity and reduced doses have been observed in several preclinical models, such as mice, compared with intramuscular injection using a conventional needle and syringe. Dose reduction in humans using vaccine dry-coated microprojection array skin patches has not been demonstrated. Studies have shown that up to a fivefold dose reduction can be achieved in humans by delivering liquid influenza vaccines to the skin through hollow microneedle arrays compared with intramuscular injection (Van Damme Vaccine 2009 27 p454). Dose reduction would make more vaccine doses available in situations such as pandemics, where high demand limits antigen supply. Furthermore, reducing the required antigen dose could make expensive vaccines, such as anti-cervical cancer vaccines, more affordable for resource-poor countries.

[0006] There is a need for a vaccination device and method that allows the vaccine to be stable and administer a reduced dose with the same efficacy as a needle and syringe.Furthermore, there is a need for a vaccination device that can be mass-produced at low cost under GMP conditions, which avoids reformulation and increases immunogenicity expression.In addition to the dose-saving and ease of mass production of MAP at very reasonable costs, MAP has further advantages.Vaccine dry coating MAP is generally more heat-resistant than the liquid vaccine required for injection using a needle and syringe.

[0007] Summary of the Invention In one broad form, embodiments of the present invention seek to provide a method of stimulating an immune response in a human comprising administering to the human a vaccine dose coated on a microprojection array patch (MAP).

[0008] In one embodiment, the MAP comprises a base and a number of continuous, imperforate projections extending from the base made from a synthetic polymer, wherein at least one projection comprises an uncoated support section that transitions to an end section that is dry-coated with the vaccine.

[0009] In one embodiment, the length of the projections is about 200 to 300 μm, the width at the base is about 100 to about 120 μm, and the density of the projections is about 1000 to about 5000 projections / cm 2 and the weight of the MAP is 0.1 to 0.6 g.

[0010] In one embodiment, the MAP is made from a synthetic polymer.

[0011] In one embodiment, the synthetic polymer is a liquid crystal polymer.

[0012] In one embodiment, administration of the composition to a human provides protective immunity against infection resulting from exposure of the human to an antigenic source.

[0013] In one embodiment, the human is 49 to 64 years old.

[0014] In one embodiment, the human is at least 65 years of age.

[0015] In one embodiment, the dosage is at least one selected from the group consisting of 0.5 μg, 1 μg, 2 μg, 2.5 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 10 μg, 15 μg, 20 μg, 25 μg, and 30 μg dosages.

[0016] In one embodiment, the dosage is at least one selected from the group consisting of 2.5 μg, 5 μg, 10 μg, and 15 μg dosages.

[0017] In one embodiment, the vaccine dose comprises one or more influenza antigens.

[0018] In one embodiment, the influenza antigen is a hemagglutinin influenza antigen.

[0019] In one embodiment, the influenza antigen is an influenza A antigen.

[0020] In one embodiment, the influenza antigen is an influenza B antigen.

[0021] In one embodiment, the influenza antigen is influenza C antigen.

[0022] In one embodiment, the method further comprises administering at least one subsequent dose of the vaccine to a human.

[0023] In one broad form, embodiments of the present invention seek to provide a method of stimulating an immune response in a human population, comprising administering to the human population a vaccine dose that is dry-coated onto a microprojection array patch (MAP) and inserted into the skin of the humans in the human population, the method comprising: wherein the seroconversion rate in a human population is at least 85%, measured at least 8 days after administration of the vaccine.

[0024] In one broad form, embodiments of the present invention seek to provide a method of stimulating an immune response in a human population, comprising administering to the human population a vaccine dose that is dry-coated onto a microprojection array patch (MAP) and inserted into the skin of the humans in the human population, the method comprising: wherein the seroconversion rate in a human population is at least 95%, measured at least 8 days after administration of the vaccine.

[0025] In one embodiment, the vaccine dose comprises one or more influenza antigens.

[0026] In one embodiment, the influenza antigen is a hemagglutinin influenza antigen.

[0027] In one embodiment, the influenza antigen is an influenza A antigen.

[0028] In one embodiment, the influenza antigen is an influenza B antigen.

[0029] In one embodiment, the influenza antigen is influenza C antigen.

[0030] In one embodiment, the dose comprises 2.5 to 15 μg of hemagglutinin influenza antigen.

[0031] In one broad form, embodiments of the present invention seek to provide a method of stimulating an immune response in a human population, comprising administering to the human population a vaccine dose that is dry-coated onto a microprojection array patch (MAP) and inserted into the skin of the humans in the human population, the method comprising: wherein the geometric mean antibody titer (GMT) in a human population, measured at least 8 days after administration of the vaccine, is at least 6-fold greater than the GMT following intramuscular injection of the same dose of vaccine.

[0032] In one embodiment, the GMT in a human population measured at least 8 days after administration of the vaccine is about 6 to about 10 times greater than the GMT following intramuscular injection of the same dose of vaccine.

[0033] In one broad form, embodiments of the present invention seek to provide a device for stimulating an immune response in a human, comprising a vaccine dose coated onto a microprojection array patch (MAP).

[0034] In one embodiment, the MAP comprises a base and a number of continuous imperforate projections extending from the base made from a synthetic polymer, at least one projection comprising an uncoated support section that transitions to an end section that is dry-coated with the vaccine.

[0035] In one embodiment, the projections have a length of about 200 to 300 μm, a width at the base of about 100 to about 120 μm, and a density of about 1000 to about 5000 projections / cm 2 and the weight of the MAP is 0.1 to 0.6 g.

[0036] In one embodiment, the MAP is made from a synthetic polymer.

[0037] In one embodiment, the synthetic polymer is a liquid crystal polymer.

[0038] In one embodiment, administration of the composition to a human provides protective immunity against infection resulting from exposure of the human to an antigenic source.

[0039] In one embodiment, the human is 49 to 64 years old.

[0040] In one embodiment, the human is at least 65 years of age.

[0041] In one embodiment, the dosage is at least one selected from the group consisting of 0.5 μg, 1 μg, 2 μg, 2.5 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 10 μg, 15 μg, 20 μg, 25 μg, and 30 μg dosages.

[0042] In one embodiment, the dosage is at least one selected from the group consisting of 2.5 μg, 5 μg, 10 μg, and 15 μg dosages.

[0043] In one embodiment, the vaccine dose comprises one or more influenza antigens.

[0044] In one embodiment, the influenza antigen is a hemagglutinin influenza antigen.

[0045] In one embodiment, the influenza antigen is an influenza A antigen.

[0046] In one embodiment, the influenza antigen is an influenza B antigen.

[0047] In one embodiment, the influenza antigen is influenza C antigen.

[0048] In one broad form, embodiments of the present invention seek to provide an apparatus for stimulating an immune response in a human population, the apparatus comprising a vaccine dose dry coated onto a microprojection array patch (MAP) configured for insertion into the skin of humans in the human population, the apparatus comprising: The seroconversion rate in a human population is at least 85%, measured at least 8 days after administration of the vaccine.

[0049] In one broad form, embodiments of the present invention seek to provide an apparatus for stimulating an immune response in a human population, the apparatus comprising a vaccine dose dry coated onto a microprojection array patch (MAP) configured for insertion into the skin of humans in the human population, the apparatus comprising: The seroconversion rate in a human population is at least 95%, measured at least 8 days after administration of the vaccine.

[0050] In one embodiment, the vaccine dose comprises one or more influenza antigens.

[0051] In one embodiment, the influenza antigen is a hemagglutinin influenza antigen.

[0052] In one embodiment, the influenza antigen is an influenza A antigen.

[0053] In one embodiment, the influenza antigen is an influenza B antigen.

[0054] In one embodiment, the influenza antigen is influenza C antigen.

[0055] In one embodiment, the dose comprises 2.5 to 15 μg of hemagglutinin influenza antigen.

[0056] In one broad form, embodiments of the present invention seek to provide an apparatus for stimulating an immune response in a human population, the apparatus comprising a vaccine dose dry coated onto a microprojection array patch (MAP) configured for insertion into the skin of humans in the human population, the apparatus comprising: A device having a GMT in a human population measured at least 8 days after administration of the vaccine that is at least 6 times greater than the GMT of an intramuscular injection of the same dose of vaccine.

[0057] In one embodiment, the GMT in a human population measured at least 8 days after administration of the vaccine is about 6 to about 10 times greater than the GMT following intramuscular injection of the same dose of vaccine.

[0058] It will be understood that the broad aspects and features of the invention can be used simultaneously and / or independently, and reference to separate broad aspects is not intended to be limiting. Furthermore, it will be understood that method features can be implemented using a system or apparatus, and system or apparatus features can be implemented using a method.

[0059] BRIEF DESCRIPTION OF THE DRAWINGS Various examples and embodiments of the present invention will now be described with reference to the accompanying drawings.

[0060] FIG. 1A is a photograph of a polymeric microprojection array patch.

[0061] FIG. 1B is a photograph of a polymer array of microprojections coated with vaccine using an inkjet coating method.

[0062] FIG. 1C is a photograph of the microprojection array applicator.

[0063] FIG. 1D is a photograph of the application of the microprojection array to the forearm using the applicator.

[0064] FIG. 2 is a scanning electron micrograph of a vaccine-coated microprojection array.

[0065] 3A and 3B are flow charts of the design for Studies A and B described in this Example.

[0066] FIG. 4A is a plot of μg hemagglutinin against time for the 5 μg dose vaccine.

[0067] FIG. 4B is a plot of μg hemagglutinin versus time for the 15 μg dose vaccine.

[0068] Figure 5 is a plot of hemagglutinin inhibition titers for several vaccine formulations, where the symbol NP is microprojection array intradermal administration and IM is intramuscular injection. Figure 6 is a plot of hemagglutinin inhibition titers on day 1 versus day 22 for several vaccine formulations in Study A, where the designation NP is microprojection array intradermal administration and IM is intramuscular injection. FIG. 7 is a plot of hemagglutinin inhibition titers versus time in Study A.

[0069] FIG. 8 is a plot of microneutralization titers on day 22 in Study A.

[0070] Figure 9 is a plot of hemagglutination inhibition (HAI) titers for subjects in Part B on Study Day 1 (pre-vaccination), 4, 8, 22, and 61. Subjects in Part B were vaccinated with: 15, 10, 5, or 2.5 μg HA / dose of A / Singapore / GP1908 / 2015 H1N1 delivered by HD-MAP applied to the volar forearm (MAP-FA-15, MAP-FA-10, MAP-FA-5, MAP-FA-2.5); uncoated HD-MAP (MAP-FA-0); 15 μg HA / dose of A / Singapore / GP1908 / 2015 H1N1 delivered by HD-MAP applied to the upper arm (MAP-UA-15); or intramuscular injection as a component of the Afluria® tetravalent vaccine (IM-QIV-15). Symbols represent geometric mean titers (GMTs) and error bars indicate 95% confidence intervals.

[0071] Figure 10 plots microneutralization titers for subjects in Part B on days 1 (pre-vaccination) and 22 after vaccination with 15, 10, 5, or 2.5 μg HA / dose of A / Singapore / GP1908 / 2015 H1N1 delivered by HD-MAP applied to the volar forearm (MAP-FA-15, MAP-FA-10, MAP-FA-5, MAP-FA-2.5); uncoated HD-MAP (MAP-FA-0); 15 μg HA / dose of A / Singapore / GP1908 / 2015 H1N1 delivered by HD-MAP applied to the upper arm (MAP-UA-15); or intramuscular injection as a component of the Afluria® tetravalent vaccine (IM-QIV-15). Bars represent GMT, symbols represent titers for each subject, and error bars indicate 95% confidence intervals.

[0072] Figure 11A is a plot of midpoint ELISA titers, and Figure 11B is a plot of the fold change in midpoint titers on day 22 versus day 1 for HA-specific FcR-binding antibodies. Antibodies specific to A / Singapore / GP1908 / 2015 monovalent purified harvest involving dimeric soluble recombinant FcγRIII were measured by ELISA. Symbols represent individual responses on day 1 before and day 22 after immunization, with horizontal lines indicating median responses (A); bar graphs with error bars represent median values ​​with interquartile ranges (B).

[0073] Figure 12 shows plots of influenza-specific IgA titers in saliva samples. Subjects were vaccinated with either 15 μg of A / Singapore / GP1908 / 2015 H1N1 delivered to the volar forearm (MAP-FA-15) or upper arm (MAP-UA-15) by HD-MAP, or intramuscular injection as a component of the Afluria® tetravalent vaccine (IM-QIV-15) or uncoated HD-MAP (MAP-FA-0). Measurements were taken at four time points: prevaccination (day 1), 4, 8, and 22. Absorbance values ​​per group for each time point were averaged and compared to day 1. The fold change compared to prevaccination (day 1) was then plotted. Symbols represent the mean across all subjects per group, and error bars indicate 95% confidence intervals.

[0074] Figures 13A-13F are plots of memory cell (MBC) frequencies before and after vaccination. The frequency of HA-specific MBCs was assessed in cryopreserved PMBC samples by flow cytometry. Samples were gated for live CD19+, IgD- B cells, and specificity was determined based on binding to the A / Michigan / 2015 probe alone or in combination with the A / New Caledonia / 1999 or stabilized H1N1 stem probes. Figures 13A and 13B are for A / Michigan / 2015 H1N1; Figures 13C and 13D are for A / New Caledonia / 1999; Figures 13E and 13F are for H1 stem. Results are presented as the frequency of probe-binding cells on days 1 and 22 in Figures 13A, 13C, and 13E. Symbols represent individual responses for day 1 pre-immunization and day 22 post-immunization, and horizontal lines indicate median responses. Meanwhile, the fold change on day 22 compared to baseline is shown in Figures 13B, 13D and 13F, where the bar graphs represent the median fold change and the error bars represent the median with interquartile range.

[0075] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In one broad aspect, embodiments of the present invention relate to the use of microprojection arrays for the delivery of vaccines, particularly polymeric high-density microprojection arrays for the delivery of vaccines to patients that deliver vaccine doses that are lower (dose-sparing) than vaccine doses delivered by intramuscular injection, while providing comparable or superior immunogenicity. The devices and methods of the present invention also provide for vaccine heat resistance, ease of use, acceptability, and avoidance of vaccine reformulation.

[0076] Influenza causes significant morbidity and mortality in adults aged 65 years and older. Strategies to improve vaccine coverage, immunogenicity, and efficacy in this age group are needed. Currently, IIV in this population requires chemical adjuvants such as MF59 or large doses of antigen (e.g., 60 μg HA per strain per dose) to achieve a sufficient immune response. The enhanced immunogenicity observed with MAP delivery indicates that HD-MAP offers an alternative approach to increasing vaccine doses.

[0077] Furthermore, the exceptional heat resistance of the vaccine in the MAP formulation compared to standard formulations eliminates cold chain dependency and reduces vaccine waste due to cold chain deviations. A more stable vaccine also eliminates the need for excessive patch use to compensate for potency lost during vaccine shelf life. Use of the device and method of the present invention may increase the number of vaccine doses that can be produced from a basic vaccine manufacturing facility during a single season or pandemic, as the amount of antigen required per dose is reduced. Global capacity for seasonal influenza products declined between 2013 and 2015 due to the switch from TIV to QIV formulations, and pandemic vaccine products rely on the implementation of dose-sparing strategies. Dose-sparing and rapid onset of protective immunity will also be valuable attributes for many vaccines of global health importance whose use is limited by long-term supply constraints and cost, such as inactivated poliovirus or yellow fever vaccines. These vaccines are often needed in low-resource settings, where other key attributes of the present invention, such as heat resistance, ease of use, acceptability, and avoidance of reformulation, would also be beneficial.

[0078] The devices and methods of the present invention include a microprojection array patch (MAP). The patch has a width W and a minor axis B, with the projections separated by a spacing. The projections can be provided in an array defined by regular repetition of the microprojections along a square or rectangular array. However, other arrangements of projections, such as a circular array of projections, compatible with rotary spray coating, can also be used. To further improve or enhance targeting precision, the substrate can be designed so that the features to be coated are arranged radially from a center point of rotation, or on concentric circles or a continuous spiral. The substrate can be designed so that the spacing of the features in each arc corresponds to the number of motor steps (an integer) for a given radius. Each projection has a tip for penetrating the tissue of a biological subject, and the projections typically have a tapered profile from base to tip (Figures 1A-1D).

[0079] The microprojection array can be divided into regions such that different vaccine antigens or other substances such as excipients can be coated in each region. For example, the microprojection array can be divided into halves or four equal sections where different vaccine antigens or other substances such as excipients can be applied. These regions can have equal or unequal numbers of microprojections. In another embodiment, some microprojections can be uncoated.

[0080] The projection density of the microprojection array is 1,000 to 7,500 / cm 2 , or 1500~7500 / cm 2 , 1500~5000 / cm 2 , 1500~2500 / cm 2 , 2000~7500 / cm 2 , 2000~5000 / cm 2 , 2000~4000 / cm 2 , 2000~3000 / cm 2 , 2500~7500 / cm 2 , 2500~5000 / cm 2 , 2500~4000 / cm 2 , 2500~3000 / cm 2 or 3000-7500 / cm2 , or 3000-5000 / cm 2 , 3000~4000 / cm 2 , 4000~7500 / cm 2 , 4000~5000 / cm 2 or 5000-7500 / cm 2 The applicators of the present invention are often utilized to project high-density microprojection arrays into the skin. Such high-density arrays are microprojection arrays of sufficient size and density that manually applied forces are insufficient to overcome the elasticity of the skin. The projections are typically separated by 10 μm to 200 μm, 30 μm to 150 μm, 50 μm to 120 μm, and more typically 70 μm to 100 μm, resulting in a patch with 10 to 1000 projections / mm. 2 , more typically 1000-3000 projections / mm 2 It has.

[0081] The protrusion lengths are 100μm~700μm, 100μm~600μm, 100μm~500μm, 100μm~400μm, 100μm~300μm, 100μm~250μm, 100μm~200μm, 150μm~700μm, 150μm~600μm, 150μm~500μm, 150μm~400μm, 150μm~300μm, 150μm~250μm, 150μm~200μm, 200μm~700μm, 200μm~ It can be 600 μm, 200 μm to 500 μm, 200 μm to 400 μm, 200 μm to 300 μm, 200 μm to 250 μm, 225 μm to 700 μm, 225 μm to 600 μm, 225 μm to 500 μm, 225 μm to 400 μm, 225 μm to 300 μm, 225 μm to 250 μm, 250 μm to 700 μm, 250 μm to 600 μm, 250 μm to 500 μm, 250 μm to 400 μm or 250 μm to 300 μm.

[0082] The projections may have one or more step shoulders (discontinuities). When discontinuities are provided, they are typically positioned so that at least one discontinuity reaches the dermis, where penetration of the projection stops and the tip extends into the dermal layer. Discontinuities are typically located 50-200 μm, 50-190 μm, 50-180 μm, 50-170 μm, 50-160 μm, 50-150 μm, 50-140 μm, 50-130 μm, 50-120 μm, 50-110 μm, 50-100 μm, 50-90 μm, 50-80 μm, 60-200 μm, 60-190 μm, 60-180 μm, 60-170 μm, etc. from the end of the tip. , 60~160μm, 60~150μm, 60~140μm, 60~130μm, 60~120μm, 60~110μm, 60~100μm, 60~90μm, 60~80μm, 70~2 00μm, 70~190μm, 70~180μm, 70~170μm, 70~160μm, 70~150μm, 70~140μm, 70~130μm, 70~120μm, 70~110μm , 70~100μm, 70~90μm, 70~80μm, 80~200μm, 80~190μm, 80~180μm, 80~170μm, 80~160μm, 80~150μm, 80~1 40μm, 80~130μm, 80~120μm, 80~110μm, 80~100μm, 80~90μm, 90~200μm, 90~190μm, 90~180μm, 90~170μm, Discontinuous microprojections can be placed at 90-160μm, 90-150μm, 90-140μm, 90-130μm, 90-120μm, 90-110μm, 90-100μm, 100-200μm, 100-190μm, 100-180μm, 100-170μm, 100-160μm, 100-150μm, 100-140μm, 100-130μm, 100-120μm, and 100-110μm. Discontinuous microprojections can provide greater drug / vaccine / excipient loading on the microprojections.

[0083] The microprojection array can be made of any suitable material, including, but not limited to, metal, silicon, polymer, and plastic. Polymer and plastic are preferred materials. Preferred materials include, but are not limited to, liquid crystal polymer. The overall mass of some embodiments of the microprojection array is about 0.3 gm. The microprojection array may have an overall weak convex patch to improve mechanical coupling with the skin and mitigate the effects of high-speed rippling application, i.e., a "high-speed / low-mass" system. The mass of the microprojection array may be less than 1 g, or less than 0.9 g, or less than 0.8 g, or less than 0.7 g, or less than 0.6 g, or less than 0.5 g, or less than 0.6 g, or less than 0.5 g, less than 0.4 g, less than 0.3 g, less than 0.2 g, less than 0.1 g, or less than 0.05 g. The microprojection array may be about 0.05 g to about 2 g, or about 0.05 g to about 1.5 g, or about 0.05 g to about 1.0 g, or about 0.05 g to about 0.9 g, or about 0.05 g to about 0.8 g, or about 0.05 g to about 0.7 g, or about 0.05 to about 0.6 g, or about 0.05 g to about 0.5 g, or about 0.05 g to about 0.4 g, or about 0.05 to about 0.3 g, or about 0. It can be about 0.05 g to about 0.2 g, or about 0.05 g to about 0.1 g, about 0.1 g to about 1.0 g, or about 0.1 g to about 0.9 g, or about 0.1 g to about 0.8 g, or about 0.1 g to about 0.7 g, or about 0.1 g to about 0.6 g, or about 0.1 g to about 0.5 g, or about 0.1 g to about 0.4 g, or about 0.1 g to about 0.3 g, or about 0.1 g to about 0.2 g. In one embodiment of the applicator / microprojection system, the array has a mass of about 0.3 g and is fired by the applicator at a velocity of about 20-26 m / s.

[0084] In some embodiments, more than one coating may be applied to the same protrusion, for example, different coatings may be applied to one or more layers, either simultaneously or at different times if the layers dissolve sequentially, to provide the same or different materials for delivery to tissue within a subject.

[0085] The amount of antigen used in the devices and methods of the present invention includes the amount necessary to provide an immune response. Dosages can be 0.1 μg, 0.5 μg, 1 μg, 2 μg, 2.5 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 15 μg, 20 μg, 25 μg, and 30 μg doses. The dosage is about 1 μg to about 100 μg, about 1 μg to about 75 μg, about 1 μg to about 50 μg, about 1 μg to about 25 μg, about 1 μg to about 15 μg, about 1 μg to about 10 μg, about 1 μg to about 5 μg, about 2.5 μg to about 100 μg, about 2.5 μg g ~ approx. 75 μg, approx. 2.5 μg ~ approx. 50 μg, approx. 2.5 μg ~ approx. 25 μg, approx. 2. μg ~ approx. 15 μg, approx. 2.5 μg ~ approx. The ranges may vary from about 5 μg to about 25 μg, about 5 μg to about 15 μg, about 5 μg to about 10 μg, about 10 μg to about 100 μg, about 10 μg to about 75 μg, about 10 μg to about 50 μg, about 10 μg to about 25 μg, about 10 μg to about 15 μg, about 15 μg to about 100 μg, about 15 μg to about 75 μg, about 15 μg to about 50 μg, about 15 μg to about 25 μg, about 20 μg to about 100 μg, about 20 μg to 75 μg, about 20 μg to about 50 μg, and about 20 μg to about 25 μg. Each dose may contain multiple antigens or multiple substances.

[0086] In a preferred embodiment, the microprojections of the microprojection array are coated by a sterile printhead-type device that rapidly delivers small droplets that dry quickly on the microprojections. In a preferred embodiment, a coating such as a vaccine formulation dries quickly on top of the microprojections, increasing the amount of vaccine that can be delivered (FIG. 2). The sterile printhead device can deliver multiple droplets to the microprojections either sequentially or in an alternative manner.

[0087] The devices and methods of the present invention provide comparable or superior antibody titers using MAP vaccine delivery to the skin compared to intramuscular injection using a conventional needle and syringe when using a smaller dose of vaccine. Thus, the devices and methods of the present invention reduce the dose by multiple times using MAP vaccine delivery to the skin compared to intramuscular injection using a conventional needle and syringe. The devices and methods of the present invention reduce the vaccine dose by 1.1-100-fold, 1.1-50-fold, 1.1-25-fold, 1.1-20-fold, 1.1-15-fold, 1.1-10-fold, 1.1-5-fold, 1.5-100-fold, 1.5-50-fold, 1.5-25-fold, 1.5-20-fold, 1.5-15-fold, 1.5-10-fold, 1.5-5-fold, 2-100-fold, 2-5 ... Reduced by 25x, 2x to 20x, 2x to 15x, 2x to 10x, 2x to 5x, 3x to 100x, 3x to 50x, 3x to 25x, 3x to 20x, 3x to 15x, 3x to 10x, 3x to 5x, 4x to 100x, 4x to 50x, 4x to 25x, 4x to 20x, 4x to 15x, 4x to 10x, 4x to 5x, 5x to 100x, 5x to 50x, 5x to 25x, 5x to 20x, 5x to 15x, or 5x to 10x.

[0088] Another advantage of MAP vaccine delivery over needles and syringes is that the vaccine is dry-coated on MAP, making it more heat-resistant and eliminating hazardous waste. Influenza vaccines were stable for at least 12 months when stored dry-coated on HD-MAP at up to 40°C. This is particularly useful in resource-poor countries where maintaining a cold chain is difficult. Another advantage is that MAP microprojections are invisible to the naked eye, making them invaluable when vaccinating people and children who are afraid of needles. The higher density of microprojections in HD-MAP may induce higher danger signals in the skin during vaccination, leading to physical adjuvantage and enhancing immunogenicity.

[0089] The devices and methods of the present invention provide higher geometric mean antibody titers (GMTs) using MAP vaccine delivery to the skin compared to intramuscular injection with a conventional needle and syringe, measured at the same time. The devices and methods of the present invention increase GMT by 2x to 500x, 2x to 100x, 2x to 50x, 2x to 25x, 2x to 20x, 2x to 15x, 2x to 10x, 2x to 5x, 5x to 500x, 5x to 100x, 5x to 50x, 5x to 25x, 5x to 20x, 5x to 15x, 5x to 10x, 10x to 500x, 10x to 100x, 10x to 50x, 10x to 25x, 10x to 20x, 10x to 15x, 20x to 500x, 20x to 100x, 20x to 50x, 20x to 25x, 50x to 500x, or 50x to 100x compared to intramuscular injection.

[0090] Detection of antibodies after vaccination occurs more rapidly when MAP vaccine delivery to the skin is used compared to intramuscular injection using a conventional needle and syringe.

[0091] The devices and methods of the present invention provide a protective immune response in a population against an infectious agent (eg, influenza) using relatively low doses of antigen.

[0092] Seroresponsiveness means at least a four-fold increase in HAI antibody titer with a minimum post-vaccination titer of 40. Seroprotection means achieving a minimum post-vaccination HAI titer of 40 among subjects with pre-vaccination titers of less than 40. The seroconversion rate of anti-HA antibody responses is defined as the proportion of subjects in each group with a protective post-vaccination titer of 1:40 or greater. Seroprotection is the percentage of subjects with an HAI titer of less than 1:10 pre-vaccination and 1:40 or greater post-vaccination. However, if the initial titer is 1:10 or greater, at least a four-fold increase in antibody levels after vaccination is required.

[0093] In another aspect of the present invention, there is provided a composition, method, or use claimed herein, wherein the immune response generated by administration of a composition of the present invention induces functional (HAI) antibodies in a majority of elderly recipients, with dosages depending on the method. In certain embodiments, the composition induces a neutralizing antibody response with a titer of greater than about 50 after 7, 14, or 28 days. In other embodiments, the composition induces a neutralizing antibody response with a titer of greater than about 100 after 7, 14, or 28 days. In other embodiments, the composition induces a neutralizing antibody response with a titer of greater than about 150 after 7, 14, or 28 days. In another embodiment, the composition induces a neutralizing antibody response with a titer of greater than about 200 after 7, 14, or 28 days.

[0094] Thus, in one aspect of the invention, there is provided a composition, method or use as claimed herein, wherein the immune response generated by administration of the composition in a population meets or exceeds one of the following criteria:

[0095] - Seroconversion rates greater than 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, or 99%.

[0096] - Seroprotection rates greater than 50%, 55%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, or 99%.

[0097] - A mean increase of 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, or more in neutralizing antibody titers after vaccination.

[0098] An "effective amount," when referring to the amount of a vaccine composition administered to a human, refers to an amount or dosage of the composition that is sufficient for therapeutic efficacy when administered to a subject (e.g., an amount sufficient to stimulate an immune response in a subject, an amount sufficient to provide protective immunity in a subject).

[0099] The vaccine composition can be administered alone or in a mixture with a conventional excipient (e.g., a pharmaceutically or physiologically acceptable organic or inorganic carrier substance) that does not adversely react with the vaccine composition. Substances that stabilize the vaccine composition can be used in the vaccine composition. While conventional vaccines may contain adjuvants to enhance immune responses, the formulations of the present invention are preferably used without adjuvants.

[0100] The dosage and frequency (single or multiple doses) administered to a subject can vary depending on a variety of factors, including, for example, the subject's health, weight, body mass index, and diet, or health-related problems, prior to exposure to an infection resulting from exposure to an antigen. Other therapeutic regimens or agents can be used in conjunction with the methods and compositions, proteins, or polypeptides of the invention.

[0101] The composition can be administered to a human in a single dose or multiple doses (e.g., at least two doses). If multiple doses are administered to a subject, the second or third dose can be administered several days (e.g., 1, 2, 3, 4, 5, 6, 7), weeks (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), months (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), or years (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) after the first dose. For example, the second dose of the composition can be administered about 7 days, about 14 days, or about 28 days after the administration of the first dose of the composition comprising the fusion protein.

[0102] Ranges may be expressed herein as from about one particular value and / or to about another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will further be understood that the endpoints of each range are significant both in relation to the other endpoint, and independently of the other endpoint.

[0103] [Example] [Example 1] =Subjects and Study Design= A total of 210 healthy subjects (age 18-50 years, BMI 18-30 kg / m) were recruited, with at least 30% male and at least 30% female. 2 ) were recruited for the study. Part A: 60 healthy subjects (four groups of 15); Part B: 150 healthy subjects (seven groups of 20 plus two groups of 5 in which skin punch biopsies were performed). The study design is shown in Figures 3A and 3B. In addition to these groups, an additional 10 subjects were recruited and underwent vaccination site biopsies in Part B of the study.

[0104] The examples described below were conducted in two parts. The study was a two-part, randomized, partially double-blind, placebo-controlled trial conducted at Nucleus Network Pty Ltd (Melbourne, VIC). The primary objective was to measure the safety and tolerability of an A / Singapore / GP1908 / 2015 H1N1 (A / Sing) monovalent vaccine delivered by HD-MAP compared with intramuscular injection of uncoated HD-MAP and a quadrivalent seasonal influenza vaccine (QIV) delivering approximately the same dose of A / Sing HA protein. Exploratory outcomes included assessing the immune response to application of HD-MAP to the forearm with four dose levels of A / Sing compared with intramuscular injection of A / Sing at the standard 15 μg HA / dose per strain, and evaluating further measures of immune response through follow-up testing and assessment of local skin reactions via punch biopsy at the HD-MAP application site.

[0105] In both studies, subjects received a single vaccination and had their blood collected for analysis before (day 1) and after vaccination (days 4, 8, 22, and 61). The first part (A) consisted of four groups of 15 subjects, each vaccinated with one of the following: 1) MAP control without antigen applied to the volar forearm (A-MAP-FA-0); 2) MAP A / Singapore / GP1908 / 2015 [H1N1], 15 μg hemagglutinin [HA] / dose applied to the forearm (A-MAP-FA-15); 3) 15 μg A / Singapore / GP1908 / 2015 H1N1 HA antigen injected intramuscularly (IM) into the deltoid muscle (A-IM-ASing15); 4) Afluria® Tetravalent 2017 / 18 (Seqirus™, USA) containing 15 μg A / Singapore / GP1908 / 2015 H1N1 HA antigen plus three influenza antigen strains injected intramuscularly (IM) into the deltoid muscle (A-IM-QIV-15). In the first part of the experiment, antibody responses on days 1 and 22 were measured by ELISA and hemagglutination inhibition assay (HAI).

[0106] After evaluation of the results of Part (A), the second part of the experiment (B) was conducted. The second part of the study consisted of seven groups of 20 subjects each. Five groups were vaccinated with A / Singapore / GP1908 / 2015 (H1N1) using MAP applied in decreasing doses (15, 10, 5, 2.5, and 0 μg HA) to the forearm (MAP-FA-15, MAP-FA-10, MAP-FA-5, MAP-FA-2.5, and MAPFA-0, respectively). An additional group was vaccinated with 15 μg HA in the upper arm over the deltoid using MAP (MAP-UA-15). The final group was vaccinated with 15 μg A / Singapore / GP1908 / 2015 (H1N1) administered intramuscularly (IM) into the deltoid. Patients were vaccinated with Afluria® Quad 2018 (Seqirus™, Australia) containing the H1N1 HA antigen (IM-QIV-15). Antibody responses were measured on day 1 (pre-vaccination), 4, 8, 22, and 61 post-vaccination using hemagglutination inhibition assay (HAI) and virus microneutralization assay (VMN) (days 1 and 22 only). Saliva samples were also collected on days 1, 4, 8, and 22 post-vaccination. Saliva samples were analyzed using ELISA to determine influenza antigen-specific secretory IgA.

[0107] Abbreviations: A / Sing = disrupted inactivated A / Singapore / GP1908 / 2015 (H1N1) viral hemagglutinin. IM 15ug tetravalent = Afluria® tetravalent containing 15µg of A / Sing HA antigen injected intramuscularly (IM) in the deltoid muscle. 2018 (Seqirus™, Australia) vaccine. SD = standard deviation; N = North, E = East, S = South, W = West.

[0108] [Example 2] =Microprojection Array Patch (MAP)= The MAP used in this study is 5,000 / cm 2 The HD-MAP was a 10 x 10 mm square containing approximately 3136 microprojections at a density of 100 μm. Each microprojection was approximately 250 μm long and 120 μm wide at the base, tapering to a sharp point of less than 25 μm (Figure 2). The vaccine was aseptically applied to the tips of the gamma-irradiated (≥25 kGy, Steritech, Australia) HD-MAPs using a "Direct-jet" process (Vaxxas Pty Ltd, Australia), which deposits individual droplets onto the tip of each projection.

[0109] HD-MAPs were manufactured to deliver two different doses of antigen-sing, 2.5 μg and 5.0 μg (referred to as 2.5 μg and 5 μg HD-MAPs), as well as uncoated (placebo) HD-MAP. After coating, the HD-MAPs were placed in aluminum MediCan containers (Amcor, UK), sealed with foil, and stored at 2–8°C with desiccant until use. Antigen-coated HD-MAPs were used within 6 months of manufacture.

[0110] MAP was applied to the skin using a handheld spring-loaded applicator that ejected the patch at a velocity of 20 m / s ± 2 m / s. The microprojections penetrated the epidermis and dermis to an average depth of around 125 μm.

[0111] Synthetic polymer MAPs were fabricated by injection molding. The polymer materials were selected based on the polymer's ability to form microprojections, polymer hardness to allow effective skin penetration, adequate polymer ductility, material compatibility with gamma irradiation sterilization, and the biocompatibility of the synthetic polymer when in contact with skin tissue.

[0112] [Example 3] =Vaccine= cGMP inactivated split influenza A / Singapore / GP1908 / 2015 (H1N1) (IVR-180A) virus vaccine was obtained from Seqirus Pty Ltd, Australia (monovalent pooled harvest (MPH)). To ensure that the hemagglutinin (HA) antigen retained potency during MAP coating and subsequent storage, the stabilizing excipient sulfobutyl ether beta-cyclodextrin (SBECD, Captisol®, Cydex Pharmaceuticals, Kansas, USA) was added to the vaccine solution used with MAP. 4.8 mg / ml of HA was mixed with a small volume of 40% w / v SBECD solution (in Baxter irrigation water) to form a 2% w / v SBECD solution with A / Singapore (coating solution). For intramuscular injection of monovalent A / Singapore, the antigen preparation was diluted with pH-tested sterile saline. Commercially available vaccines were used for intramuscular injection of the tetravalent vaccine. In the first part of the study, Afluria® Tetravalent Influenza Vaccine 2017-2018 (Northern Hemisphere) from Seqirus™, USA was used. For Part B, Afluria® Tetravalent Vaccine 2018 (Southern Hemisphere) was used. The Afluria® Tetravalent Northern Hemisphere 2017 / 18 vaccine nominally contained 15 μg of HA of each of the following disrupted inactivated virus types: A / Singapore / GP1908 / 2015 (H1N1), IVR-180A; A / Hong Kong / 4801 / 2014 (H3N2), NYMC X-263B; B / Phuket / 3073 / 2013 BVR-1B; and B / Brisbane / 46 / 2015. The Afluria® Tetravalent Southern Hemisphere 2017 / 18 vaccine nominally contained 15 μg of HA of each of the following disrupted inactivated virus types: A / Singapore / GP1908 / 2015 (H1N1), IVR-180A; A / Hong Kong / 4801 / 2014 (H3N2), NYMC X-263B; B / Phuket / 3073 / 2013 BVR-1B; and B / Brisbane / 46 / 2015. The Hemisphere 2018 vaccine nominally contained 15 μg of HA of each of the following virus types: A / Michigan / 45 / 2015(H1N1)pdm09-like virus (A / Singapore / GP1908 / 2015(IVR-180A)); A / Singapore / INFIMH-16-0019 / 2016(H3N2)-like virus (A / Singapore / INFIMH-16-0019 / 2016(IVR-186)); B / Phuket / 3073 / 2013-like virus (B / Phuket / 3073 / 2013(BVR-1B)); and B / Brisbane / 60 / 2008-like virus (B / Brisbane / 46 / 2015).

[0113] To produce the A / Singapore MAP, sterilized A / Singapore antigen was added to a filter-sterilized SBECD solution and coated onto the tips of MAP microprojections by direct jet coating. The A / Singapore antigen was received as a suspension of particles in phosphate-buffered saline. The final solid formulation of antigen on the MAP therefore consisted of HA protein, other proteins (from A / Singapore bulk), SBECD, and buffer salts. The coated MAP was then immediately sealed in foil in a product pack, removed from the sterile room, and stored at 2–8°C. To simplify product manufacturing and testing, three patches were applied to deliver the required dose. Subjects in the 15 μg group received 3 x 5 μg patches, subjects in the 10 μg group received 2 x 5 μg and 1 x placebo patches, subjects in the 5 μg group received 1 x 5 μg and 2 x placebo patches, subjects in the 2.5 μg group received 1 x 2.5 μg and 2 x placebo patches, and subjects in the 0 μg group received 3 x placebo patches. The order of application was randomized and blinded.

[0114] The MAP applicator device (CAPD) was a portable, spring-powered device designed to reliably and reproducibly apply MAP to the skin. The MAP applicator utilizes spring-generated mechanical force to accelerate MAP to high velocities of 20 m / s over short distances (less than 5 mm), sufficient for the dense array of microprojections to tear the skin. The CAPD uses magnets to attach, position, and hold the MAP. The CAPD is a disposable device used in conjunction with a skin conditioning ring. The skin conditioning ring contacts the skin around the area of ​​MAP administration. A downward force of approximately 30 Newtons is required to activate the skin ring, resulting in preconditioning of the skin for MAP administration.

[0115] A / Singapore / GP1908 / 2015 dry coated on MAP The HA antigen was shown to be stable (measured by enzyme-linked immunosorbent assay and maintained at temperatures up to 40°C for 12 months after coating onto MAP) (see Figure Y). Placebo MAP (MAP-placebo / FA group) was sterilized by gamma irradiation (≥25 kGy, Steritech, Australia). All MAPs were placed in aluminum MediCan containers (Amcor, UK), sealed with foil, and stored at 2–8°C until use (Figure 4).

[0116] [Example 4] =Immunogenicity assessment= Serum samples were collected on days 1 (preinoculation), 4, 8, 22, and 61 and tested by hemagglutination inhibition (HAI) and viral microneutralization (VMN) assays on days 1 and 22. HAI assays were performed as previously described (Fernando et al., 2018). Briefly, serum samples for HAI were treated with receptor-destroying enzyme (Denka Seiken Co Ltd, Japan) and adsorbed onto washed, packed turkey red blood cells (TRBCs) for 30 minutes at room temperature (RT). Before testing, TRBCs were diluted to 1% v / v in PBS. Two-fold serum dilutions starting at 1:10 were prepared, and 4 HA units / 25 μL of A / Singapore / GP1908 / 2015 virus (WHO Collaborating Centre, Australia) were added to each test well and incubated for 45 minutes at room temperature (RT). TRBCs were then added and incubated for an additional 30 minutes at room temperature. The HAI titer was calculated as the reciprocal of the highest serum dilution that completely inhibited virus-induced agglutination of TRBCs.

[0117] The VMN assay was performed as previously described (Fernando et al., 2018). Briefly, serum samples were heat inactivated at 56°C for 30 min. Two-fold serum dilutions starting from 1:100 were prepared, and 100 TCID 50 of A / Singapore / GP1908 / 2015 virus (WHO Collaborating Centre, Australia) was added to each test well. Prevention of infection of MDCK cells by A / Singapore / GP1908 / 2015 virus was tested using ELISA detection of influenza nucleoprotein.

[0118] Antibodies capable of mediating antibody-dependent cellular cytotoxicity (ADCC) were analyzed using an ELISA that detects the ability of immobilized A / Sing MPH-specific antibodies to crosslink soluble recombinant FcγRIIIA receptor dimers (22). Serum samples collected on days 1 and 22 from subjects in the MAP-FA-0, MAP-FA-15, MAP-UA-15, and IM-QIV-15 groups were tested. Briefly, 96-well Nunc Maxisorp plates (Thermofisher Scientific, USA) were coated with 50 ng of A / Singapore / GP1908 / 2015 HA in PBS for 16 hours at 4°C, washed with PBS + 0.05% Tween 20 (PBST), and then filled with SuperBlock (Thermofisher After blocking with PBS (Thermofisher Scientific), serially diluted serum samples (1:20 to 1:43,740) were added in duplicate. The plates were incubated at 37°C for 1 hour and then washed with PBST. FcγRIIIA Val158 ectodomain biotin dimer (0.1 μg / mL) was added, incubated at 37°C for 1 hour, and then washed with PBST. Antibody-FcγRIIIA complexes were detected using a 1:10,000 dilution of streptavidin-HRP (Thermofisher Scientific) and developed with 3,3',5,5'-tetramethylbenzidine substrate (Sigma-Aldrich, USA). The reaction was stopped with 0.16 M H2SO4, and absorbance was measured at 450 nm. The serum concentration giving the half-maximal signal (EC50) was determined using a curve fitting (four-parameter logarithmic regression) and GraphPad Prism (GraphPad Software, USA).

[0119] [Example 5] =IgA in saliva= Saliva samples were collected from subjects in the MAP-FA-0, MAP-FA-15, MAP-UA-15, and IM-QIV-15 groups on days 1, 4, 8, and 22. Subjects bitten a cotton swab from a Salivette® saliva collector (Sarstedt, France) for approximately 1 minute. After centrifugation, the supernatant (saliva) was stored at -80°C. Influenza-specific IgA was detected by ELISA. Specifically, saliva samples serially diluted in 4 mg / mL BSA in PBS (PBSA) were added to Nunc Maxisorp plates (Thermofisher Scientific, USA) pre-coated overnight with A / Singapore / GP1908 / 2015 HA MPH (60 μl / well at 2 μg / mL) and blocked with PBSA. The presence of A / Sing HA-specific IgA was detected using HRP-1-conjugated goat anti-human polyclonal IgA (PA1-74395, Thermofisher Scientific, USA) and ABTS 232 substrate (Sera-Care, USA). The reaction was stopped with 1% SDS, and the absorbance was measured at 405 nm.

[0120] [Example 6] =Memory B cells= Peripheral blood mononuclear cells (PBMCs) were collected and cryopreserved from subjects in the MAP-FA-0, MAP-FA-15, MAP-UA-15, and IM-QIV-15 groups on days 1 and 22 and stored in liquid nitrogen until use. Recombinant HA proteins for use as flow cytometry probes for A / Michigan / 45 / 2015, A / New Caledonia / 20 / 1999, and stabilized H1N1 stem domains were derived as previously described (23). HA-specific B cells were identified within cryopreserved human PBMCs by co-staining with HA probes conjugated to SA-PE, SA-APC, or SA-Ax488 (Thermofisher Scientific). Monoclonal antibodies for surface staining include: CD19-ECD (J3-119) (Beckman Coulter, USA), IgM-BUV395 (G20-127), CD21-BUV737 (B-ly4), IgD-Cy7PE (IA6-2), IgG-BV786 (G18-145) (BD 243 Biosciences, USA), CD14-BV510 (M5E2), CD3-BV510 (OKT3), CD8a-BV510 (RPA-T8), CD16-BV510 (3G8), CD10-BV510 (HI10a), CD27-BV605 (Biolegend, USA), and IgA-Vio450 (REA1014) 245 (Miltenyi, USA). Background B cells interacting with SA were excluded by staining with SA-BV510 (BD 246 Biosciences). Cell viability was assessed using Aqua Live / Dead amine-reactive dye (Thermofisher Scientific). Samples were collected using a BD Fortessa configured to detect 18 fluorescent dyes, and analysis was performed using FlowJo software version 9.5.2 (TreeStar, USA).

[0121] [Example 7] =T cell flow cytometry= Cytokine production by CD4+ and CD8+ T cells was assessed using a modified method described by Landry et al. (24). PBMCs were thawed, plated at 1.5 × 106 / well, and incubated for 6 hours. After washing, PBMCs were stimulated with A / Sing MPH (20 μg / ml) for 20 hours or with a pool of overlapping synthetic peptides (17 amino acids long, overlapping by 11 amino acids, 5 μg / ml) spanning the A / Sing HA sequence (Mimotopes Pty Ltd, Australia) for 6 hours. Medium alone and PMA / ionomycin were used as negative and positive controls, respectively. Golgi blockers (monensin and brefeldin A) were added 5 hours before the end of the incubation period. Cells were labeled with surface stains Live / Dead Aqua (for viability), anti-CD3 BV785, anti-CD4 FITC, and anti-CD8 APC / Cy7 (all from Biolegend), then fixed, permeabilized, and labeled with anti-IFN-γ Ax647, anti-TNF-α BV421, and anti-IL-2 PE (all from Biolegend). Samples were analyzed on a Becton Dickinson LSR Fortessa X20 within 24 hours of the final wash step. Approximately 500,000 events were acquired, and raw data were first analyzed using FlowJo (to obtain percent positive values ​​for each cytokine), followed by background-subtracted analysis using SPICE (http: / / exon.niaid.nih.gov / spice) software.

[0122] [Example 8] =Heat resistance= The A / Sing-coated HD-MAP was stored for 12 months at 2–8°C, 25°C ± 2°C / 60% ± 5% relative humidity (RH), and 40°C ± 2°C / 60% ± 5% RH. At the indicated time points, the coating was eluted from the HD-MAP in 1 mL of elution buffer (0.041% w / w hypromellose, 0.0295% w / w trehalose dihydrate) using water bath sonication at 20–28°C, and HA potency was determined by enzyme immunoassay (Bodle et al., 2013).

[0123] [Example 9] =Statistical analysis= Fold increases in HAI and MN titers were compared between groups using Student's t-test. Pearson's χ with continuity correction 2 The proportion of subjects who were seroprotected or seroconverted was compared between groups using the SAS test (SAS version 9.4, SAS Institute Inc., USA). Because this is an early-phase study, exploratory efficacy analyses were not performed on ADCC and memory B cell response assays. All groups were compared using the Kruskal-Wallis nonparametric test (without correction for multiple comparisons) and Dunn's multiple comparison post-test. On days 1 and 22, CD4 + Intragroup comparisons of cytokine production by cells were performed using the Wilcoxon rank sum test. 〔result〕 =Stability= To obtain stability data prior to clinical manufacturing, GLP stability studies were conducted to test 5 μg and 15 μg of HA A / Sing loaded onto HD-MAP. This loading range was selected to encompass the range of HA A / Sing loadings intended for use in this study. A / Sing antigen coated at 5 μg and 15 μg per HD-MAP was stable when stored at 2-8°C or 40°C for at least 12 months (Figure 4).

[0124] = Serological response = Figure 5 shows the geometric mean titers (GMTs) of HAI antibodies on days 1, 4, 8, 22, and 61 for subjects in Part A. These data demonstrate that 2.5 μg delivered to the skin by MAP induces HAI titers that are statistically similar to those induced by 15 μg of A / Singapore HA delivered either alone or as part of a tetravalent vaccine using a needle and syringe. Compared to the 15 μg dose delivered intramuscularly to humans, MAP reduces the antigen dose by sixfold. It is possible to reduce the dose below 2.5 μg and still maintain antibody titers comparable to intramuscular injection. While dose reduction has been demonstrated in mouse models with MAP, this is the first time it has been demonstrated in humans. At day 22, no statistically significant differences were observed between vaccinated groups. Figure 6 plots hemagglutinin inhibition titers on days 1 versus 22 for several vaccine formulations in Study A. Figure 7 is a plot of hemagglutinin inhibition titers versus time for Study A. Figure 8 is a plot of microneutralization titers at day 22 for Study A.

[0125] The HAI GMTs, seroprotection rates, seroconversion rates, and fold increase in GMT titers are shown in Table 1. Table 1 shows that for Study B, the hemagglutination inhibition response to vaccination exceeded pre-vaccination levels in terms of seroprotection rates, seroconversion rates, and fold increase in GMT.

[0126] [Table 1] JPEG2025157452000003.jpg109169JPEG2025157452000004.jpg73169

[0127] Geometric mean titers (GMTs) of HAI antibodies on days 1, 4, 8, 22, and 61 for subjects in Part B are shown in Figure 9. Subjects receiving uncoated HD-MAP did not experience increases in HAI titers. In subjects vaccinated with either HD-MAP or IM, titers did not rise above baseline on day 4, but on day 8, GMTs were highest in the MAP-FA-10 (GMT 437, 254-751 95% CI), MAP-UA-15 (GMT 243, 133-442 95% CI), and MAP-FA-15 (GMT 219, 112-427 95% CI) groups. The increase in GMT titers from baseline was significantly higher at day 8 (MAP-FA-10 p=0.0002, MAP-UA-15 p=0.0167, MAP-FA-15 p=0.0384) than in the IM-QIV-15 group (GMT 83, 42-161 95% CI). Titers continued to increase in all active treatment groups through day 22 and remained significantly higher than in the IM-QIV-15 group at day 22 for the MAP-FA-10 and MAP-UA-15 groups (p=0.0011 and p=0.0201, respectively) and at day 61 for the MAP-FA-10 and MAP-UA-15 groups (p=0.0062 and p=0.0277, respectively). The GMI in the MAP-FA-2.5 group (subjects receiving 1 / 6 the standard dose of HA) was not significantly different from that in the IM-QIV-15 group at any time point (day 4, p = 0.4034; day 8, p = 0.7449; day 22, p = 0.9312; day 61, p = 0.7297). Furthermore, at day 22, the HAI 405 GMT was similar in the MAP-FA-15 (GMT 320, 161-638 95% CI) and MAP-UA-15 (GMT 368, 198-683 95% CI) groups, indicating that the application site of HD-MAP does not affect the subsequent antibody response.

[0128] The fold increase in GMT on day 8 was significantly higher in the MAP-FA-10 and MAP-UA-15 groups (22.6 and 18.4, respectively) compared with the IM-QIV-15 group (1.0) (p=0.0069 and p=0.0095, respectively), indicating a more rapid antibody response compared with intramuscular injection. The fold change from baseline remained significantly higher in the MAP-UA-15 group on days 22 and 61 (p=0.0240 and p=0.0265, respectively). HAI titers observed in Part A subjects receiving vaccines delivered by HD-MAP were not significantly different from the corresponding groups in Study B at any time point (i.e., A-MAP-FA-15 compared with MAP-FA-15, all p-values ​​>0.4180), indicating consistency of antibody delivery and induction. The GMT induced by IM-QIV-15 in parts A and B was also not significantly different on days 4, 8, and 22, but was higher in study A on day 61 (p=0.0320).

[0129] MN assays were performed on serum samples obtained from all Part B subjects on Day 1 (pre-vaccination) and Day 22. Similar to HAI antibody results, titers increased from Day 1 to Day 22 in all vaccinated treatment groups (Figure 10). MN titers on Day 22 in the MAP-FA-10 and MAP-UA-15 groups were significantly higher than those in the IM-QIV-15 group (p=0.0005 and p=0.0096, respectively). Similar to the HAI results, MN GMTs on Day 22 were similar in the MAP-FA-2.5 (1 / 6 dose) (GMT 5,301, 2,509-11,196 95% CI) and IM-QIV-15 (GMT 3,880, 1,924-7,824 95% CI) groups (Figure 10).

[0130] HA-specific FcR-binding antibody titers capable of mediating ADCC were assayed on days 1 and 22. Midpoint titers significantly increased after vaccine delivery in the MAP-FA-15, MAP-FA-0, MAP-UA-15, and IM-QIV-15 groups (p<0.001, p<0.001, and p=0.002, respectively), but not in the MAP-FA-0 group (p>0.99) (Figure 3A). No significant differences were observed among the midpoint titers at day 22 in these three active groups (p>0.99 for all comparisons), nor were significant differences observed when results were expressed as fold changes from baseline, which accounts for the degree of within-group variability (Figure 11).

[0131] Salivary influenza-specific IgA was assayed by ELISA in samples collected on days 1, 4, 8, and 22 from subjects in the MAP-FA-0, MAP-FA-15, MAP-UA-15, and IM-QIV-15 groups. No significant increase in titers was observed compared to day 1 in any group. However, on day 8, the MAP-FA-15 and MAP-UA-15 groups showed a 1.92-fold and 1.57-fold increase above baseline, respectively, compared with no increase in the MAP-FA-0 group (1.01-fold), and a 1.22-fold increase in the IM-QIV-15 group at the same time point. IgA titers returned to near baseline levels by day 22 (Figure 12).

[0132] A flow cytometry-based assay using fluorescently labeled recombinant HA was used to assess the frequency and specificity of HA-specific B cells after immunization. The frequency of HA-Michigan probe-binding memory B cells (MBCs) (antigenically matched to A / Singapore / GP1908 / 2015) was elevated on day 22 after immunization with either QIV or active HD-MAP (MAP-FA-15 p<0.0001, MAP-UA-15 p<0.0001, and IM-QIV-15 p<0.0001), but not in the placebo group (MAP-FA-0 p<0.0001). However, the frequency of HA-Michigan-specific MBCs on day 22 was not significantly different among the three vaccine groups (p>0.99 for all comparisons). Binding to the A / New Caledonia / 99 probe was used to assess H1N1 cross-reactivity; only a small proportion of A / Michigan / 15-binding cells exhibited HA-recognition cross-reactivity. A significant increase in frequency was observed between days 1 and 22 in the MAP-FA-15 (p<0.0001) and MAP-UA-15 (p<0.0001) groups, and to a lesser extent in the IM-QIV-15 group (p=0.0522). Again, no difference in MBC frequency was observed in the vaccine groups at day 22 (p<0.99 for all comparisons). A similar pattern was observed for cross-reactive B cells binding to the HA-stalk domain probe. A significant expansion was observed from days 1 to 22 in the MAP-FA-15 (p=0.006) and IM_QIV-15 (p=0.468) groups. The expansion in the MAP-UA-15 group did not achieve significance, likely due to within-group variability (p=0.167). There was no difference in the frequency of Stark-reactive MBC between the active HD-MAP and IM groups on day 22 (p>0.99 for all comparisons). Figures 13A-F.

[0133] T cell responses were assessed by analyzing the frequencies of influenza-specific CD4+ and CD8+ T cells producing IFN-γ, IL-2, and TNF-α in PBMCs collected from subjects in the MAP-FA-0, MAP-FA-15, MAP-UA-15, and IM-QIV-15 groups on days 1 and 22. PBMCs were stimulated with either the A / Sing MPH or 536 overlapping peptides spanning the A / Sing HA sequence.

[0134] An overall increase in the frequency of CD4+ cells producing IFN-γ, IL-2, or TNF-α after in vitro peptide stimulation was observed on day 22 compared with day 1 in the MAP-FA-15, MAP-UA-15, and IM-QIV-15 groups, but not in the MAP-FA-0 group (Figure 8A). Notably, the abundance of polyfunctional CD4+ T cells expressing all three cytokines (IFN-γ, TNF-α, and IL-2) significantly increased on day 22 compared with day 1 for the MAP-FA-15 (p = 0.0002), MAP-UA-15 (p = 0.0045), and IM-QIV-15 (p = 0.0110) groups. Furthermore, significant increases in CD4+ cells producing TNF-α and IL-2 were observed in the MAP-FA-15 group (p = 0.0008), MAP-UA-15 group (p = 0.0003), and IM-QIV group (p = 0.0012). Furthermore, the MAP-FA-15 group significantly increased CD4+ cells expressing TNF-α alone (p = 0.0053) or TNF-α together with IFN-γ (p = 0.0013) (Figure 8A). When comparing the MAP-FA-15, MAP-UA-15, and IM-QIV-15 groups with each other, no statistically significant differences were observed in the percentage of CD4+ T cells producing any combination of cytokines at day 22 (p > 0.0565 for all comparisons).

[0135] The overall frequency of cytokine-producing CD4+ cells before and after vaccination was greater after stimulation with A / Sing MPH compared with the overlapping peptide. This is likely due to the greater number of epitopes present in the MPH formulation. A / Sing MPH stimulation appeared to induce more CD4+ cells producing TNF-α alone compared with peptide stimulation. However, when comparing the MAP-FA-15, MAP-UA-15, and IM-QIV-15 groups with each other, no statistically significant differences were observed in the proportions of CD4+ T cells producing each cytokine combination at day 22 (p>0.0515 for all comparisons).

[0136] CD8+ T cell responses to the peptide pool and A / Sing MPH were also measured on days 1 and 22 and were weaker than CD4+ responses. The weak CD8+ T cell responses were not surprising given that the nature of the antigens used for restimulation (inactivated, split A / Sing MPH and 17-amino acid peptide) favored the stimulation and detection of CD4+ T cells.

[0137] This study demonstrated MAP dose sparing in clinical practice. The safety and reactogenicity profile of HD-MAP was very similar to that observed with silicone nanopatches using a similar H1N1 antigen, A / California / 7 / 2009 (3, 18), and the fact that erythema remained present 7 days after vaccination is also consistent with intradermal (ID) delivery of influenza vaccine. No differences in HAI or MNT responses were observed after HD-MAP application to the volar surface of the upper arm or forearm.

[0138] In terms of seroprotection and the proportion of seroconverted subjects, the HAI responses induced by HD-MAP delivery in this study were similar to those previously observed with intramuscular injection of inactivated influenza vaccine (IIV) using a needle and syringe. However, the more rapid antibody response seen with HD-MAP delivery, as indicated by higher HAI titers at the early 8-day time point, has not been observed with intradermal injection of IIV unless the intradermal injection site was pretreated with the topical adjuvant imiquimod, a TLR7 agonist (34). Obtaining higher titers by using HD-MAP to deliver seasonal or travel vaccines soon after vaccination would be beneficial to vaccine recipients and would be particularly valuable if shown to be applicable to vaccines against pandemic influenza strains and vaccines used in outbreak response.

[0139] To limit the impact of regional and global influenza epidemics, an influenza vaccine that provides broader protection and induces longer-lasting immunity than current seasonal vaccines is needed. Studies suggest that ADCC-mediating antibodies recognize epitopes that are more conserved than those bound by neutralizing antibodies and may contribute to protection against heterologous strains. The induction of antibodies capable of inducing ADCC followed a similar pattern to the responses to HAI and MN data, with slightly higher titers observed in groups vaccinated with HD-MAP compared with IM injection. The frequencies of B cells recognizing HA-stark and historical H1N1 HA probes were also increased to a similar extent after intramuscular injection of HD-MAP vaccine.

[0140] Several HD-MAP groups showed statistically higher responses at 8, 22, and 61 days post-vaccination compared with intramuscular injection, with 1 / 6 dose eliciting antibody levels equivalent to full-dose intramuscular injection.

[0141] The synthetic polymer MAP vaccine is safe and well tolerated by human subjects. MAP can deliver disrupted, inactivated A / Singapore / GP1908 / 2015 (H1N1) virus antigen (2.5 μg HA) to human skin and elicit HA-specific antibody responses equivalent to those generated by a conventional intramuscular vaccination with 15 μg HA. This six-fold dose reduction represents a potential reduction in vaccine costs, potentially making more vaccines available, especially when antigen availability is limited, such as in the case of a pandemic. Furthermore, if these dose reductions are possible for most vaccines, in addition to influenza vaccines, reducing the cost of expensive vaccines such as the anti-cervical cancer vaccine would help resource-poor countries more easily access these expensive vaccines.

[0142] MAP delivery at a low dose of 2.5 μg HA induced HAI and MNT titers similar to IM QIV (15 μg HA / dose). Seroconversion and seroprotection rates at day 8 were higher with MAP delivery. No differences were observed between MAP application to the forearm and upper arm. MAP has excellent high-temperature stability.

[0143] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" or "comprising" are understood to mean the inclusion of the stated integer, group of integers, or step, but not the exclusion of other integers or groups of integers. As used herein, unless otherwise stated, the word "about" means ±20%.

[0144] Those skilled in the art will recognize that numerous variations and modifications will be apparent, and all such variations and modifications apparent to those skilled in the art are to be considered within the spirit and scope of the invention as broadly described above. [Brief explanation of the drawings]

[0145] [Figure 1] Figure 1A is a photograph of a polymeric microprojection array patch. Figure 1B is a photograph of the microprojections of the polymeric array coated with vaccine using an inkjet coating method. Figure 1C is a photograph of a microprojection array applicator. Figure 1D is a photograph of the application of the microprojection array to the forearm using the applicator. [Figure 2] FIG. 2 is a scanning electron micrograph of a vaccine-coated microprojection array. [Figure 3A] 3A and 3B are flow charts of the design for Studies A and B described in this Example. [Figure 3B] 3A and 3B are flow charts of the design for Studies A and B described in this Example. [Figure 4A] FIG. 4A is a plot of μg hemagglutinin against time for the 5 μg dose vaccine. [Figure 4B] FIG. 4B is a plot of μg hemagglutinin versus time for the 15 μg dose vaccine. [Figure 5] Figure 5 is a plot of hemagglutinin inhibition titers for several vaccine formulations, where the symbol NP is microprojection array intradermal administration and IM is intramuscular injection. [Figure 6] Figure 6 is a plot of hemagglutinin inhibition titers on day 1 versus day 22 for several vaccine formulations in Study A, where the designation NP is microprojection array intradermal administration and IM is intramuscular injection. [Figure 7] FIG. 7 is a plot of hemagglutinin inhibition titers versus time in Study A. [Figure 8] FIG. 8 is a plot of microneutralization titers on day 22 in Study A. [Figure 9]Figure 9 is a plot of hemagglutination inhibition (HAI) titers for subjects in Part B on Study Day 1 (pre-vaccination), 4, 8, 22, and 61. Subjects in Part B were vaccinated with: 15, 10, 5, or 2.5 μg HA / dose of A / Singapore / GP1908 / 2015 H1N1 delivered by HD-MAP applied to the volar forearm (MAP-FA-15, MAP-FA-10, MAP-FA-5, MAP-FA-2.5); uncoated HD-MAP (MAP-FA-0); 15 μg HA / dose of A / Singapore / GP1908 / 2015 H1N1 delivered by HD-MAP applied to the upper arm (MAP-UA-15); or intramuscular injection as a component of the Afluria® tetravalent vaccine (IM-QIV-15). Symbols represent geometric mean titers (GMTs) and error bars indicate 95% confidence intervals. [Figure 10] Figure 10 plots microneutralization titers for subjects in Part B on days 1 (pre-vaccination) and 22 after vaccination with 15, 10, 5, or 2.5 μg HA / dose of A / Singapore / GP1908 / 2015 H1N1 delivered by HD-MAP applied to the volar forearm (MAP-FA-15, MAP-FA-10, MAP-FA-5, MAP-FA-2.5); uncoated HD-MAP (MAP-FA-0); 15 μg HA / dose of A / Singapore / GP1908 / 2015 H1N1 delivered by HD-MAP applied to the upper arm (MAP-UA-15); or intramuscular injection as a component of the Afluria® tetravalent vaccine (IM-QIV-15). Bars represent GMT, symbols represent titers for each subject, and error bars indicate 95% confidence intervals. [Figure 11A]Figure 11A is a plot of midpoint ELISA titers, and Figure 11B is a plot of the fold change in midpoint titers on day 22 versus day 1 for HA-specific FcR-binding antibodies. Antibodies specific to A / Singapore / GP1908 / 2015 monovalent purified harvest involving dimeric soluble recombinant FcγRIII were measured by ELISA. Symbols represent individual responses on day 1 before and day 22 after immunization, with horizontal lines indicating median responses (A); bar graphs with error bars represent median values ​​with interquartile ranges (B). [Figure 11B] Figure 11A is a plot of midpoint ELISA titers, and Figure 11B is a plot of the fold change in midpoint titers on day 22 versus day 1 for HA-specific FcR-binding antibodies. Antibodies specific to A / Singapore / GP1908 / 2015 monovalent purified harvest involving dimeric soluble recombinant FcγRIII were measured by ELISA. Symbols represent individual responses on day 1 before and day 22 after immunization, with horizontal lines indicating median responses (A); bar graphs with error bars represent median values ​​with interquartile ranges (B). [Figure 12] Figure 12 shows plots of influenza-specific IgA titers in saliva samples. Subjects were vaccinated with either 15 μg of A / Singapore / GP1908 / 2015 H1N1 delivered to the volar forearm (MAP-FA-15) or upper arm (MAP-UA-15) by HD-MAP, or intramuscular injection as a component of the Afluria® tetravalent vaccine (IM-QIV-15) or uncoated HD-MAP (MAP-FA-0). Measurements were taken at four time points: prevaccination (day 1), 4, 8, and 22. Absorbance values ​​per group for each time point were averaged and compared to day 1. The fold change compared to prevaccination (day 1) was then plotted. Symbols represent the mean across all subjects per group, and error bars indicate 95% confidence intervals. [Figure 13A]Figures 13A-13F are plots of memory cell (MBC) frequencies before and after vaccination. The frequency of HA-specific MBCs was assessed in cryopreserved PMBC samples by flow cytometry. Samples were gated for live CD19+, IgD- B cells, and specificity was determined based on binding to the A / Michigan / 2015 probe alone or in combination with the A / New Caledonia / 1999 or stabilized H1N1 stem probes. Figures 13A and 13B are for A / Michigan / 2015 H1N1; Figures 13C and 13D are for A / New Caledonia / 1999; Figures 13E and 13F are for H1 stem. Results are presented as the frequency of probe-binding cells on days 1 and 22 in Figures 13A, 13C, and 13E. Symbols represent individual responses for day 1 pre-immunization and day 22 post-immunization, and horizontal lines indicate median responses. Meanwhile, the fold change on day 22 compared to baseline is shown in Figures 13B, 13D and 13F, where the bar graphs represent the median fold change and the error bars represent the median with interquartile range. [Figure 13B] Figures 13A-13F are plots of memory cell (MBC) frequencies before and after vaccination. The frequency of HA-specific MBCs was assessed in cryopreserved PMBC samples by flow cytometry. Samples were gated for live CD19+, IgD- B cells, and specificity was determined based on binding to the A / Michigan / 2015 probe alone or in combination with the A / New Caledonia / 1999 or stabilized H1N1 stem probes. Figures 13A and 13B are for A / Michigan / 2015 H1N1; Figures 13C and 13D are for A / New Caledonia / 1999; Figures 13E and 13F are for H1 stem. Results are presented as the frequency of probe-binding cells on days 1 and 22 in Figures 13A, 13C, and 13E. Symbols represent individual responses for day 1 pre-immunization and day 22 post-immunization, and horizontal lines indicate median responses. Meanwhile, the fold change on day 22 compared to baseline is shown in Figures 13B, 13D and 13F, where the bar graphs represent the median fold change and the error bars represent the median with interquartile range. [Figure 13C]Figures 13A-13F are plots of memory cell (MBC) frequencies before and after vaccination. The frequency of HA-specific MBCs was assessed in cryopreserved PMBC samples by flow cytometry. Samples were gated for live CD19+, IgD- B cells, and specificity was determined based on binding to the A / Michigan / 2015 probe alone or in combination with the A / New Caledonia / 1999 or stabilized H1N1 stem probes. Figures 13A and 13B are for A / Michigan / 2015 H1N1; Figures 13C and 13D are for A / New Caledonia / 1999; Figures 13E and 13F are for H1 stem. Results are presented as the frequency of probe-binding cells on days 1 and 22 in Figures 13A, 13C, and 13E. Symbols represent individual responses for day 1 pre-immunization and day 22 post-immunization, and horizontal lines indicate median responses. Meanwhile, the fold change on day 22 compared to baseline is shown in Figures 13B, 13D and 13F, where the bar graphs represent the median fold change and the error bars represent the median with interquartile range. [Figure 13D] Figures 13A-13F are plots of memory cell (MBC) frequencies before and after vaccination. The frequency of HA-specific MBCs was assessed in cryopreserved PMBC samples by flow cytometry. Samples were gated for live CD19+, IgD- B cells, and specificity was determined based on binding to the A / Michigan / 2015 probe alone or in combination with the A / New Caledonia / 1999 or stabilized H1N1 stem probes. Figures 13A and 13B are for A / Michigan / 2015 H1N1; Figures 13C and 13D are for A / New Caledonia / 1999; Figures 13E and 13F are for H1 stem. Results are presented as the frequency of probe-binding cells on days 1 and 22 in Figures 13A, 13C, and 13E. Symbols represent individual responses for day 1 pre-immunization and day 22 post-immunization, and horizontal lines indicate median responses. Meanwhile, the fold change on day 22 compared to baseline is shown in Figures 13B, 13D and 13F, where the bar graphs represent the median fold change and the error bars represent the median with interquartile range. [Figure 13E]Figures 13A-13F are plots of memory cell (MBC) frequencies before and after vaccination. The frequency of HA-specific MBCs was assessed in cryopreserved PMBC samples by flow cytometry. Samples were gated for live CD19+, IgD- B cells, and specificity was determined based on binding to the A / Michigan / 2015 probe alone or in combination with the A / New Caledonia / 1999 or stabilized H1N1 stem probes. Figures 13A and 13B are for A / Michigan / 2015 H1N1; Figures 13C and 13D are for A / New Caledonia / 1999; Figures 13E and 13F are for H1 stem. Results are presented as the frequency of probe-binding cells on days 1 and 22 in Figures 13A, 13C, and 13E. Symbols represent individual responses for day 1 pre-immunization and day 22 post-immunization, and horizontal lines indicate median responses. Meanwhile, the fold change on day 22 compared to baseline is shown in Figures 13B, 13D and 13F, where the bar graphs represent the median fold change and the error bars represent the median with interquartile range. [Figure 13F] Figures 13A-13F are plots of memory cell (MBC) frequencies before and after vaccination. The frequency of HA-specific MBCs was assessed in cryopreserved PMBC samples by flow cytometry. Samples were gated for live CD19+, IgD- B cells, and specificity was determined based on binding to the A / Michigan / 2015 probe alone or in combination with the A / New Caledonia / 1999 or stabilized H1N1 stem probes. Figures 13A and 13B are for A / Michigan / 2015 H1N1; Figures 13C and 13D are for A / New Caledonia / 1999; Figures 13E and 13F are for H1 stem. Results are presented as the frequency of probe-binding cells on days 1 and 22 in Figures 13A, 13C, and 13E. Symbols represent individual responses for day 1 pre-immunization and day 22 post-immunization, and horizontal lines indicate median responses. Meanwhile, the fold change on day 22 compared to baseline is shown in Figures 13B, 13D and 13F, where the bar graphs represent the median fold change and the error bars represent the median with interquartile range.

Claims

1. An apparatus for stimulating an immune response in a human population, comprising: an applicator that fires a microprojection array; and a vaccine dose that is dry coated onto a microprojection array patch and inserted into the skin of humans in the human population, the microprojection array patch comprises a base and a plurality of continuous, imperforate projections extending from the base made from a synthetic polymer, at least one projection comprising an uncoated support section transitioning to an end section dry-coated with a composition comprising an influenza antigen; The following features - the vaccine dose is between 2.5 μg and 15 μg; - the microprojection array has (a) a projection length of 250 μm to 600 μm, (b) a projection density of 1500 to 5000 / cm 2 , and (c) a mass of the microprojection array of 0.1 g to 0.8 g; The microprojection array is projected by an applicator at a velocity of 20-26 m / s; An apparatus having:

2. The device described in claim 1, wherein the vaccine dose comprises one or more influenza antigens.

3. The device described in claim 2, wherein the influenza antigen is a hemagglutinin influenza antigen.

4. The device described in claim 3, wherein the influenza antigen is an influenza A antigen.

5. The device described in claim 3, wherein the influenza antigen is influenza B antigen.

6. The device described in claim 3, wherein the influenza antigen is influenza C antigen.

7. The device of claim 3, wherein the dose comprises 2.5 to 15 μg of hemagglutinin influenza antigen.

8. An apparatus for stimulating an immune response in a human population, comprising: an applicator that fires a microprojection array; and a vaccine dose that is dry coated onto a microprojection array patch and inserted into human skin in the human population, 1. A method of manufacturing a device, wherein the microprojection array patch comprises a base and a plurality of continuous, imperforate projections extending from the base made from a synthetic polymer, at least one projection comprising an uncoated support section transitioning to an end section dry-coated with a composition comprising an influenza antigen, the method comprising: The following features - the vaccine dose is between 2.5 μg and 15 μg; - the microprojection array has (a) a projection length of 250 μm to 600 μm, (b) a projection density of 1500 to 5000 / cm 2 , and (c) a mass of the microprojection array of 0.1 g to 0.8 g; The microprojection array is projected by an applicator at a velocity of 20-26 m / s; The composition dry coated onto the microprojections is stable for at least 12 months between 2 and 40°C as measured by elution of the vaccine from the microprojections and assessed for potency using an enzyme immunoassay; A method comprising:

9. The method of claim 8, wherein the vaccine dose comprises one or more influenza antigens.

10. The method of claim 9, wherein the influenza antigen is a hemagglutinin influenza antigen.

11. The method of claim 10, wherein the influenza antigen is an influenza A antigen.

12. The method described in claim 10, wherein the influenza antigen is an influenza B antigen.

13. The method of claim 10, wherein the influenza antigen is influenza C antigen.

14. The method of claim 10, wherein the dose comprises 2.5 to 15 μg of hemagglutinin influenza antigen.

Images