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WO2020051566A1 - Vaccines with enhanced immune response and methods for their preparation - Google Patents

Vaccines with enhanced immune response and methods for their preparation Download PDF

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Publication number
WO2020051566A1
WO2020051566A1 PCT/US2019/050116 US2019050116W WO2020051566A1 WO 2020051566 A1 WO2020051566 A1 WO 2020051566A1 US 2019050116 W US2019050116 W US 2019050116W WO 2020051566 A1 WO2020051566 A1 WO 2020051566A1
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Prior art keywords
vaccine
capnp
antigen
formulation
peptide
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PCT/US2019/050116
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French (fr)
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Ramila Philip
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Immunotope, Inc.
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Publication of WO2020051566A1 publication Critical patent/WO2020051566A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/12Viral antigens
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • A61P31/14Antivirals for RNA viruses
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/555Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511Organic adjuvants
    • A61K2039/55566Emulsions, e.g. Freund's adjuvant, MF59
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/57Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/57Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011Details
    • C12N2770/24011Flaviviridae
    • C12N2770/24111Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • TITLE Vaccines with Enhanced Immune Response and Methods for their
  • the present invention relates to generating an effective T cell immunity in the context of prophylactic or therapeutic vaccination.
  • These vaccines comprise of any epitopic peptides specific for any virus or cancer with a built-in adjuvant, N-acetyl glucosamine incorporated into a calcium phosphate nanoparticle delivery system.
  • Current vaccines are generally designed to generate protective antibody response without a strong CTL response. In many cases, a CTL response alone or a combination of CTL response with antibody response is preferred.
  • a set of Dengue virus CTL epitopes as test samples to study immune response in presence of adjuvants in a nanoparticle delivery system.
  • Dengue virus is a member of the Flaviviridae family and is characterized by a single stranded RNA genome enclosed within a spherical enveloped virion.
  • Four distinct serotypes of DV circulate globally with most endemic countries reporting circulation of all four serotypes.
  • the incidence of dengue has grown dramatically around the world in recent decades; the numbers of dengue cases are underreported and in many cases misclassified due to asymptomatic presentation.
  • a recent estimate indicates there are roughly 3 90 million dengue infections per year, of which approximately 96 million manifest clinically. Other estimates indicate approximately 3.9 billion people in 128 countries are at risk of infection with dengue viruses.
  • CYD-TDV Dengvaxia
  • CYD-TVD induces neutralizing antibodies against all four DV serotypes, and induction of high-titer neutralizing antibodies can provide temporary cross protection to serotypes, lasting two years on average.
  • the efficacy of CYD- TVD for confirmed dengue cases was lower in seronegative individuals than in seropositive individuals.
  • the rate of hospitalization of seronegative individuals was considerably higher; especially among children younger than 9 years old. This observation was attributed to CYD-TDV inducing non-protective dengue antibodies that enhance infection. There still remains a significant need to develop efficacious immunotherapies for dengue virus infections.
  • T cell based vaccines are an attractive alternative strategy in that they can be used as 'stand alone' vaccines or be paired with current anti-viral treatments and/or the CYD-TDV vaccine.
  • CDS+ cytotoxic T lymphocyte cells (CTLs) are a major contributor of protection against dengue virus infection.
  • CDS+ T cells have been detected in patients after natural infection and in attempts at vaccination with some level of cross-reactivity between strains. Studies in children indicated that CDS+ T cell mediated secretion of IFN-y and TNF-a was more robust in asymptomatic or subclinical infections compared to symptomatic or severe disease. Additionally, CDS+ T cells play a major role in viral clearance and offer a robust cross protection against DV serotypes. These T cells are able to be activated via selected HLA-A2+ and HLA-A24+ in both healthy, seronegative individuals and in seropositive individuals who have been previously infected with DV.
  • Implementation of the approach to produce broad, cross-protective immunity involves the identification of conserved CDS+ T cell epitopes that can be induced in most members of the population and that can maintain the epitope-specific CDS+ T cells in a highly active state capable of controlling the infection.
  • Activation of T cells depends on complex interactions between the innate and adaptive immune systems. Enhancing innate immune responses is thought to drive more robust adaptive immunity and a primary method to enhance these innate responses in vaccination is through the use of adjuvants.
  • adjuvants Currently, only a few adjuvants have been approved for use in humans in the ETS, including alum and a few lipid based emulsions.
  • a variety of vaccine delivery systems are available but many have issues with stability which directly impacts effectiveness of a vaccine.
  • An ideal vaccine delivery system must induce a stronger, broader, and more persistent cellular and humoral immunity, as well as an improved immunological memory.
  • a vaccine must also improve immune responses in people with reduced or suppressed immunity, broaden the immune response to allow recognition of pathogenic strain variants, and be cost effective. Reducing the amount of antigen required to elicit an effective immune response or allowing for a reduction in the number of vaccine doses (dose sparing) is one way to contain costs.
  • Many vaccine delivery systems including liposomal formulations, virus-like particle (VLP) vaccines, DNA vaccines, viral vector-based vaccines, and synthetic gold nanoparticles, are currently being investigated in an effort to achieve long-term protection against a broad range of viral subtypes.
  • CaPNPs Calcium phosphate nanoparticles
  • the HLA-A2+ DV peptides selected for this work were previously identified through a comprehensive analysis of naturally presented epitopes on infected cells using an immunoproteomic approach. These novel HLA-A2+ DY-specific peptides are derived from conserved regions of the DV protein. Previous demonstrations showed that DV epitopes derived from conserved regions are capable of inducing cross-reactive T cell responses, a benefit when designing a vaccine to protect against multiple strains.
  • the vaccine is formulated by adsorbing the identified antigenic peptides to pre-formulated CaPNP. With the ability of these particles to induce CDS+ T cell responses, in both in vitro and in vivo experiments, the present invention discloses the potential development of a T cell vaccine against DV infection.
  • FIG. 1 Individual and multi-peptide CaPNP vaccine formulation stimulates CD8+ T cell activation in vivo. CTL response generated by individual vs multiple peptides formulated in CaPNP solutions in immunized HLA-A2 transgenic mice.
  • A Timeline for mice immunization and sample analysis.
  • FIG. 1A Low and high concentration of DV multi-peptide CaPNP vaccine formulation stimulate CD8+ T cell activation in vivo.
  • HLA-A2 transgenic mice were immunized as described in Fig. 1A with CTL response assessed in the following groups: Groupl- unimmunized as control; Group 2 - 1 Opg peptide/150m1 per mouse with montanide;
  • peptide/mouse with montanide Group 3 - 1 Opg peptide/150m1 CaPNP /mouse; Group 4 - 1 Opg peptide/150m1 CaPNP with lXGlcNAc; Group 5 - 1 Opg peptide/150m1 CaPNP with 3XGlcNAc.
  • FIG. 4 DV multi-peptide CaPNP vaccine formulation stimulate CD8+ T cell activation in vitro : CTLs were generated using peripheral blood from a healthy donor PBMCs were activated with: 1) pooled peptides, 2) multi peptide CaPNP with GlcNAc, or 3) DV peptide CaPNP in vitro.
  • A HLA-A2 specific The non-adherent PBMCs containing the epitope specific CTLs were harvested, washed, and cultured with individual peptide pulsed (2pg/ml) or dengue infected HepG2 cells overnight in an IFN-g ELISpot assay.
  • B Identical cultures were set up as in (A) but in 96 well round bottom plates.
  • spleen cells were harvested and mixed with APCs pulsed with either individual peptides (NIQ- Seq ID No.4, TIT- Seq ID No.3, VTL- Seq ID No.2, KLA- Seq ID No.5, AML- Seq ID No.6, LLC- Seq ID No.7) or infected with DV2 virus as targets in the ELISpot assay.
  • Data represent
  • Figure 6 Stability and efficacy of optimized DV multi-peptide CaPNP formulation with GlcNAc.
  • A Particle size of formulations measured over a time of three months (day 0, 30, 60 and 90) under different storage conditions (4°C or RT).
  • B In vitro CTL response by different formulations generated in PBMCs. Four groups were tested as: 1) pooled peptide with montanide, 2) peptide CaPNP with GlcNAc solutions stored at 4°C, 3) lyophilized peptide CaPNP with GlcNAc stored at 4°C and 4) lyophilized peptide CaPNP with GlcNAc stored at RT.
  • CTL responses were generated by pulsing PBMCs with no stimulated as control, pooled peptides, DV2 infected cells. IFN-g expression was measured by ELISpot assay.
  • C In vivo CTL response by different formulations generated in immunized HLA-A2 transgenic mice. Three groups were tested as: 1) pooled peptide with montanide, 2) peptide CaPNP with GlcNAc solutions stored at 4°C, 3) lyophilized peptide CaPNP with GlcNAc stored at 4°C. Mice were immunized as described in Fig. 1A.
  • Dengue virus infects an estimated 300 million people each year and even more are at risk of becoming infected as the virus continues to spread into new areas. Despite the increase in viral prevalence, few anti-viral medications or vaccines are approved for treating or preventing infection. Conventional vaccines normally target either DV virons or the viral structural proteins to provide the protection from all four dengue serotypes. All the candidate vaccines under development or in clinical trials are based on humoral immunity. Several concerns related to dengue pathogenesis caused by cross-reactive T cells during DV infection, antibody-dependent enhancement or insufficient response in younger age group have raised challenges to the development of a robust, broad and multi- functional dengue vaccine. Viral clearance is largely mediated by robust CD&+ T cell responses.
  • nanoparticles are ideal antigen delivery systems to target cells or organs for therapeutic purposes.
  • a number of previous studies show that they can be conjugated with drugs or biomolecules such as peptides or antibodies. Comparing to microparticles, they are at the range of nanometer which enhance the efficiency of cellular uptake by 15-250 fold.
  • Knuschke et al. demonstrated a calcium phosphate nanoparticle delivery system ( ⁇ 200nm) was taken up by dendritic cells in vivo and induced a potent T cell response.
  • gold nanoparticle (Au-NP) based vaccines have been studied extensively due to smaller particle size ( ⁇ l00nm), customizable shape, various binding mechanisms (covalent/non-covalent), stable etc.
  • the average particle size of the dengue vaccine formulation, described herein, is below lOOnm, which was small enough to enter lymphatic network and generate more potent immune responses. More importantly, it was biodegradable due to the component of calcium phosphate and had a good safety profile.
  • Intradermal and subcutaneous injection of CaPNP in HLA-A2 transgenic mice at levels of low and high dose on study days 1, 8 and 15 did not result in any adverse effects, treatment- related clinical signs of toxicity, or effects on body weight or food consumption (data not shown).
  • the CaPNP formulated vaccines disclosed herein were proved to be stable and active during long term storage either at 4°C or at RT. It is not uncommon that nanoparticles show a poor long-term stability due to physical and chemical instability.
  • CaPNPs have been shown to be stable for many years at room temperature, either as particulate suspension or as lyophilized or spray-dried powder.
  • the present invention employs lyophilization (freeze-drying) to improve the CaPNP-DV vaccine stability.
  • particle size of lyophilized CaPNP-DV vaccine (4°C or RT) showed increased functional efficacy when compared to the same vaccine in suspension; however it was not statistically significant (Figure 6, P>0.05).
  • the inventor further tested the immunological performance between solution and lyophilized powder. Both in vitro and in vivo results showed a potent T cell response among testing groups, which provided the evidence that lyophilization could be an option for vaccine long term non-cold chain storage and suitable for transportation and use in parts where dengue is endemic.
  • One of the important components for nanoparticle based vaccines is the candidate epitopes and/or antigens which will be adsorbed to the particle surface, or incorporated in the core, or both.
  • Previous data demonstrated that dengue specific T cell responses can be induced in vivo in transgenic mice, in vitro from healthy seronegative or seropositive individuals who have previously been infected with DV.
  • the present vaccine formulation with HLA-A2 and A2/A24 epitopes has been characterized extensively.
  • CaPNP can be conjugated with one or multiple antigens either on the surface or inside the particle. Therefore, we formulated six individual peptides with CaPNP and demonstrated that the mean particle size of all testing formulations remained in the range of 80-l00nm, which was smaller than previously reported CaPNP formulations, producing better performance in terms of immune cells uptake and immune response.
  • the six DV peptides were from conserved regions of DV serotypes and induced a potent T cell responses against all four serotypes of D V infected cells, which could be critical in the development of a universally immunogenic vaccine.
  • the multi-epitope vaccine formulation with CaPNP improved the overall immunogenic performance when compared to free peptides or peptides mixed with montanide-5 l adjuvant.
  • the CaPNP-DV vaccine formulation is antigen sparing, in which lower vaccine dose induced higher T cell response when compared to the higher dose (Fig. 2), suggesting that CaPNP was acting both as antigen delivery system and partially as an adjuvant to facilitate the antigen uptake by antigen presenting cells, trigger DC activation and induce IFN-y production.
  • CaPNP can be considered as a preferred adjuvant/delivery system candidate due to the fact that they have small particle size in the nanometer range, present high loading capacity, are biodegradable/biocompatible, and enhance vaccine immunogenicity while they are inert to immune system by themselves.
  • the present invention integrates CaPNP with epitopes that are naturally presented by DV infected cells and characterized the biological function of this novel vaccine strategy.
  • Vaccine formulation is also very simple which involves pre- mixing the CaPNP and antigenic peptides at room temperature for periods of several hours. Furthermore, we optimized the formulation and added GlcNAc to enhance the immune cell uptake and activation of innate and adaptive immune responses.
  • the most promising universal dengue vaccine candidate must come from combining the antigens for both T and B cell immunity.
  • the vaccine In order to be active the vaccine must mimic viral sizes and permit cytosolic uptake to target antigen presenting cells.
  • Integrated nanoparticle- biomolecule hybrid systems constitute useful tools to mimic the behavior of biomolecules in cells, helping to explore the mechanisms of biological processes with a variety of potential applications.
  • the present invention describes the development of a synthetic universal vaccine based on shared multiple T cell epitopes incorporated in CaPNP.
  • HLA-A2 and A24 which are prevalent in 40% of the world population (HLA-A2)
  • HLA-A2 and A24 which are prevalent in 40% of the world population
  • CaPNP calcium phosphate nanoparticle
  • CaPNP adjuvant at 0.3% final concentration, but not less than 0.3% produced the best immune response with most vaccine antigens.
  • SIINFEKL SEQ ID No. 1
  • a test antigenic peptide SEQ ID No. 1
  • the following formulations were tested: (i) 0.8% CaPNP alone (0.8% placebo), (ii) 0.8% CaPNP + SEQ ID NO. 1, (iii) 0.3% CaPNP alone (0.3% Placebo), or (iv)0.3% CaP + SEQ ID NO.
  • SIA the treatment groups either from peptide alone or peptide formulated in CaPNP demonstrated higher level of antibody staining which indicated the processing of peptide and presentation in association with MHC class I molecule. Moreover, there was no difference between 0.8% vs 0.3% CaPNP formulation.
  • MUG assay Figure SIB presented a similar result for the antigen processing activity. Both the free peptide and peptide formulation pulsed APC groups activated T cell measured by the expression of increasing gal levels. As there was no observed difference between the two formulation groups, we selected 0.3% CaPNP formulation for further investigation.
  • mice were immunized in 5 groups: unstimulated (PBS), pooled free peptides (lOpg each) emulsified with montanide-5l, pooled free peptides (SOpg each) emulsified with montanide-5l, CaPNP formulated peptides (IOpg each) and CaPNP formulated peptides (SOpg each).
  • PBS unstimulated
  • lOpg each pooled free peptides
  • SOpg each emulsified with montanide-5l
  • CaPNP formulated peptides IOpg each
  • CaPNP formulated peptides SOpg each
  • FIG 4 demonstrates significant T cell responses against DV infected cells as measured by IFN-y secretion in an ELISpot assay (Figure 4A), and by the secretion of granzyme B by MagPix (Figure 4B), and up- regulation of CD 107a by flow cytometry ( Figure 4C).
  • Figure 4A CaPNP formulated with or without GlcNAc stimulated robust peptide specific T cell response, which was more intense than peptide alone group.
  • the granzyme B ( Figure 4B) and CD 107a marker expression (Figure 4C) data showed a slight increase in the group which was treated with CaPNP formulation containing GlcNAc, which was consistent with the previous finding from in vivo study ( Figure 3).
  • HLA-A2 transgenic mice were immunized to study CaPNP formulation under various conditions. There were three immunization groups: 1) antigenic peptides emulsified with montanide-5l, 2) peptides formulated with CaPNP and stored as solution (4°C), lyophilized peptides formulated with CaPNP (4°C).
  • the T cell responses were analyzed using DV2 infected, peptide pulsed and various DV serotypes ⁇ Tl, T2, T3, T4) infected cells as targets. Results shown in Figure 6C demonstrated that CaPNP formulated peptide vaccine activated DV specific T cells in vivo, which was consistent with in vitro analysis (Figure 6B).
  • CaPNP formulation induced T cell activation against all of the 4 serotypes of DV infected cells.
  • Lyophilized CaPNP formulation activated T cells in vivo as measured by IFN-y response against various targets.
  • the conserved antigenic peptides in CaPNP vaccine formulation activate T cell response against all four serotypes of DV.
  • the lyophilized vaccine formulation is capable of generating T cell response against all the four DV serotypes as well.
  • the present invention embodies generally vaccine compositions, and methods of use thereof, for the prevention, treatment, and diagnosis of dengue virus infection. Accordingly, one group of embodiments in the present invention includes compositions for a vaccine that induces a broad, multi-functional T cell response with substantial cross- reactivity between all virus serotypes.
  • One particular aspect includes a multi-epitope DV specific vaccine formulation where DV epitopes are formulated in CaPNP-containing solutions to activate a CTL response.
  • compositions such as, but not limited to, an adjuvant (e.g., Freund’s complete or incomplete adjuvant) or administration with traditional prophylactic viral vaccine formulations (e.g., live attenuated viruses, inactivated viruses, recombinant proteins, chimeric viruses, DNA vaccines, and synthetic peptides).
  • an adjuvant e.g., Freund’s complete or incomplete adjuvant
  • traditional prophylactic viral vaccine formulations e.g., live attenuated viruses, inactivated viruses, recombinant proteins, chimeric viruses, DNA vaccines, and synthetic peptides.
  • kits that contain these compositions and optionally include instructions for treating (prophylactic or therapeutic), vaccinating or immunizing a subject against a DV infection, or treating (prophylactic or therapeutic) a subject having or at risk of having a Dengue virus infection or pathology.
  • prophylactic methods including methods of vaccinating and immunizing a subject against a DV infection (acute) such as, but not limited to, protecting a subject against a DV infection to decrease or reduce the probability of a DV infection or pathology in a subject or to decrease or reduce susceptibility of a subject to a DV infection or pathology or to inhibit or prevent a DV infection in a subject.
  • the vaccine compositions are utilized for purposes of preventing, suppressing or treating diseases causing the expression of the immunogenic peptides disclosed herein.
  • prevention relates to a process of prophylaxis in which an animal, especially a mammal, and most especially a human, is exposed to an immunogen of the present invention prior to the induction or onset of the disease process. This would be most appropriate where an individual is at high risk for DV infection based on the living or travel to the DV endemic areas.
  • the immunogen could be administered to the general population as is frequently done for any infectious diseases.
  • the term “suppression” is often used to describe a condition wherein the disease process has already begun but obvious symptoms of said condition have yet to be realized.
  • the cells of an individual may have been infected but no outside signs of the disease have yet been clinically recognized.
  • the term prophylaxis can be applied to encompass both prevention and suppression.
  • the term "treatment” is often utilized to mean the clinical application of agents to combat an already existing condition whose clinical presentation has already been realized in a patient. This would occur where an individual has already been diagnosed as having confirmed DV infection.
  • the suitable dosage of an immunogen of the present invention will depend upon the age, sex, health, and weight of the recipient, the kind of concurrent treatment, if any, the frequency of treatment, and the nature of the effect desired. However, the most preferred dosage can be tailored to the individual subject, as determined by the researcher or clinician.
  • the total dose required for any given treatment will commonly be determined with respect to a standard reference dose as set by a manufacturer, such as is commonly done with vaccines, such dose being administered either in a single treatment or in a series of doses, the success of which will depend on the production of a desired immunological result (i.e., successful production of a CTL-mediated response to the antigen, which response gives rise to the prevention and/or treatment desired).
  • the overall administration schedule must be considered in determining the success of a course of treatment and not whether a single dose, given in isolation, would or would not produce the desired immunologically therapeutic result or effect.
  • compositions containing the immunogens disclosed herein may, in addition, contain other vaccine pharmaceuticals.
  • the use of such compositions with multiple active ingredients is left to the discretion of the clinician.

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Abstract

Although numerous investigations are focused on developing vaccines that induce protective humoral responses, fewer studies are aimed at developing vaccines that induce broad and robust T cell responses. In order to design a potent T cell based vaccine, two critical components are needed: the identity of specific T cell epitopes and an optimized vaccine formulation capable of delivering the antigens and the adjuvants simultaneously. Previously, we have identified and characterized HLA-A2 and A24 binding conserved epitopes and have characterized the feasibility of epitope based vaccine for dengue infection. Using these T cell epitopes and N acetyl glucosamine incorporated into a calcium phosphate nanoparticle (CaPNP) delivery system as the model, we studied efficacy of such CTL vaccine. The present disclosure describes such a CTL vaccine utilizing such combination.. Evaluating the immunogenicity of CaPNP/Dengue peptides formulation in-vitro and in-vivo in HLA-transgenic mice demonstrates that the CaPNP vaccine is immunologically relevant to induce T cellresponses against all four serotypes of dengue virus infection. This formulation is simple, has a low investment manufacturing process, and is stable at room temperature in a lyophilized form, further reducing the manufacturing time and costs. Further, the formulation is a candidate for the development of effective multi-serotype specific dengue virus vaccine to be used as prophylactic vaccine to prevent dengue infection in non-infected healthy individuals, and has the potential to be used as a therapeutic vaccine in already infected individuals, to minimize the antibody mediated complications caused by secondary infections.

Description

TITLE: Vaccines with Enhanced Immune Response and Methods for their
Preparation
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims benefit of and priority to U.S. Provisional Patent Application No. 62/728,171, filed on 07 September 2018, and where permissible is incorporated by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to generating an effective T cell immunity in the context of prophylactic or therapeutic vaccination. These vaccines comprise of any epitopic peptides specific for any virus or cancer with a built-in adjuvant, N-acetyl glucosamine incorporated into a calcium phosphate nanoparticle delivery system.
BACKGROUND OF THE INVENTION
Current vaccines are generally designed to generate protective antibody response without a strong CTL response. In many cases, a CTL response alone or a combination of CTL response with antibody response is preferred. In this invention, we used a set of Dengue virus CTL epitopes as test samples to study immune response in presence of adjuvants in a nanoparticle delivery system.
Dengue virus (DV) is a member of the Flaviviridae family and is characterized by a single stranded RNA genome enclosed within a spherical enveloped virion. Four distinct serotypes of DV circulate globally with most endemic countries reporting circulation of all four serotypes. The incidence of dengue has grown dramatically around the world in recent decades; the numbers of dengue cases are underreported and in many cases misclassified due to asymptomatic presentation. A recent estimate indicates there are roughly 3 90 million dengue infections per year, of which approximately 96 million manifest clinically. Other estimates indicate approximately 3.9 billion people in 128 countries are at risk of infection with dengue viruses. Recovery from infection by one serotype provides lifelong immunity against that particular strain, but cross-protective immunity to the other serotypes after recovery is only partial and temporary. The subsequent infections by other serotypes increase the risk of developing severe dengue or dengue hemorrhagic fever, resulting in antibody dependent enhancement (ADE) of infection. Currently, there are no specific anti-viral treatments for dengue virus infection. Clinical management is based on supportive therapy. The improvements in case management have reduced the case fatality rate of hospitalized dengue illness to less than 1 %. Other primary forms of combating the virus have been targeting the viral vector, though the data supporting a positive impact on the incidence of dengue illness is limited. In terms of prophylactic measures, one dengue live attenuated tetravalent vaccine is approved for use in several countries (CYD-TDV, or Dengvaxia). CYD-TVD induces neutralizing antibodies against all four DV serotypes, and induction of high-titer neutralizing antibodies can provide temporary cross protection to serotypes, lasting two years on average. Interestingly, the efficacy of CYD- TVD for confirmed dengue cases was lower in seronegative individuals than in seropositive individuals. Furthermore, the rate of hospitalization of seronegative individuals was considerably higher; especially among children younger than 9 years old. This observation was attributed to CYD-TDV inducing non-protective dengue antibodies that enhance infection. There still remains a significant need to develop efficacious immunotherapies for dengue virus infections.
There are a number of unanswered questions that are important in developing a successful dengue virus vaccine. Both humoral and cellular immunity are imperative to forming a safe and effective cross-protective Dengue virus vaccine, therefore it is necessary to use tools that identify both B-cell and T-cell epitopes within viral proteins that stimulate protective immune responses but not the immune amplification. T cell based vaccines are an attractive alternative strategy in that they can be used as 'stand alone' vaccines or be paired with current anti-viral treatments and/or the CYD-TDV vaccine. CDS+ cytotoxic T lymphocyte cells (CTLs) are a major contributor of protection against dengue virus infection. DV specific CDS+ T cells have been detected in patients after natural infection and in attempts at vaccination with some level of cross-reactivity between strains. Studies in children indicated that CDS+ T cell mediated secretion of IFN-y and TNF-a was more robust in asymptomatic or subclinical infections compared to symptomatic or severe disease. Additionally, CDS+ T cells play a major role in viral clearance and offer a robust cross protection against DV serotypes. These T cells are able to be activated via selected HLA-A2+ and HLA-A24+ in both healthy, seronegative individuals and in seropositive individuals who have been previously infected with DV. The weight of evidence also suggests that a useful Dengue virus vaccine will require THI and/or CDS+ T-cell responses to not only successfully protect against infection by each of the four serotypes, but also against ADE as well. Having a vaccine that activates a CTL response against the virus, potentially paired with an efficient humoral immune- stimulating component, could be used to induce an increased, life-long immunity against DV.
The rationale for prophylactic vaccination against Dengue virus begins with the knowledge that natural infection protects against exogenous re-infection with the homologous viral type. However, little is known about which DV antigens are immunologically relevant in eliciting an effective T cell response to the four DV serotypes. Several groups have attempted to identify T cell epitopes by either screening overlapping peptides from structural and nonstructural Dengue proteins, including preM, E, and N3, or by predicting MHC peptide binding motifs of Dengue proteins, NSI, N6A, While these studies have revealed a few potential candidate epitopes, a comprehensive analysis of naturally presented epitopes on the infected cells has never been undertaken or reported.
Implementation of the approach to produce broad, cross-protective immunity involves the identification of conserved CDS+ T cell epitopes that can be induced in most members of the population and that can maintain the epitope-specific CDS+ T cells in a highly active state capable of controlling the infection. Activation of T cells depends on complex interactions between the innate and adaptive immune systems. Enhancing innate immune responses is thought to drive more robust adaptive immunity and a primary method to enhance these innate responses in vaccination is through the use of adjuvants. Currently, only a few adjuvants have been approved for use in humans in the ETS, including alum and a few lipid based emulsions. In addition, a variety of vaccine delivery systems are available but many have issues with stability which directly impacts effectiveness of a vaccine. An ideal vaccine delivery system must induce a stronger, broader, and more persistent cellular and humoral immunity, as well as an improved immunological memory. A vaccine must also improve immune responses in people with reduced or suppressed immunity, broaden the immune response to allow recognition of pathogenic strain variants, and be cost effective. Reducing the amount of antigen required to elicit an effective immune response or allowing for a reduction in the number of vaccine doses (dose sparing) is one way to contain costs. Many vaccine delivery systems, including liposomal formulations, virus-like particle (VLP) vaccines, DNA vaccines, viral vector-based vaccines, and synthetic gold nanoparticles, are currently being investigated in an effort to achieve long-term protection against a broad range of viral subtypes.
Calcium phosphate nanoparticles (CaPNPs) have shown promise for use as both an adjuvant and a drug delivery vehicle. In preclinical safety and toxicity studies, CaPNP was shown to be safe for intramuscular, subcutaneous, intradermal, oral, or inhalation routes. In a Phase I study with IND approved in the US, CaPNP was shown non-toxic, non-inflammatory, and non-allergic in human. Therefore, there was great interest in investigating the use of CaPNPs for vaccine development. Building on our prior work of discovered antigenic peptides from DV, the beneficial properties of CaPNP in terms of stability; formulation capability, conjugation simplicity and ease, and T cell responses is disclosed. The HLA-A2+ DV peptides selected for this work were previously identified through a comprehensive analysis of naturally presented epitopes on infected cells using an immunoproteomic approach. These novel HLA-A2+ DY-specific peptides are derived from conserved regions of the DV protein. Previous demonstrations showed that DV epitopes derived from conserved regions are capable of inducing cross-reactive T cell responses, a benefit when designing a vaccine to protect against multiple strains. The vaccine is formulated by adsorbing the identified antigenic peptides to pre-formulated CaPNP. With the ability of these particles to induce CDS+ T cell responses, in both in vitro and in vivo experiments, the present invention discloses the potential development of a T cell vaccine against DV infection.
BRIEF SUMMARY OF THE INVENTION We tested multiple carriers and adjuvants to give enhanced immune response. A combination of epitopic peptides with calcium phosphate nano-particles of size less than lOOnm and N-acetyl glucosamine gives robust CTL response as evidenced by in vitro and in vivo experiments. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Individual and multi-peptide CaPNP vaccine formulation stimulates CD8+ T cell activation in vivo. CTL response generated by individual vs multiple peptides formulated in CaPNP solutions in immunized HLA-A2 transgenic mice. (A) Timeline for mice immunization and sample analysis. (B) Result of IFN-g ELISpot assay. Mice were grouped (n=3) as: unstimulated (vehicle control); pool of peptides mixed with montanide- 51; multi - peptide in CaPNP formulation with GlcNAc; individual peptide in CaPNP formulation with GlcNAc and then mixed together. After the second boost, spleens were harvested and spleen cells were isolated and mixed with targets pulsed with individual peptides (NIQ, TIT, VTL, KLA, AML, LLC) or infected with DV2 virus in the ELISpot assay. Data represent Mean±S.D. (n=3)
Figure 2. Low and high concentration of DV multi-peptide CaPNP vaccine formulation stimulate CD8+ T cell activation in vivo. HLA-A2 transgenic mice were immunized as described in Fig. 1A with CTL response assessed in the following groups: Groupl- unimmunized as control; Group 2 - 1 Opg peptide/150m1 per mouse with montanide;
Group 3-50pg peptide/l50pl per mouse with montanide; Group 4-l0pg peptide/l50 mΐ CaPNP with GlcNAc; Group 5- 5 Opg peptide/ 150 pi CaPNP with GlcNAc. Spleen cells were harvested post last immunization and mixed with targets and pulsed with no peptides (negative control), or pooled peptides (PP including NIQ, TIT, VTL, KLA, AML, LLC), or infected with DV2 virus in the ELISpot assay. Data represent Mean±S.D. (n=3)
* Represents P<0.05, ** PO.Ol, ***P<0.00l, **** P0.0001
Figure 3. Different GlcNAc concentrations in DV multi-peptide CaPNP vaccine formulation stimulate CD8+ T cell activation in vivo. Mice were immunized as previously described in Fig 1 A. CTL response were assessed in following groups in HLA-A2 transgenic mice: Group 1 - unimmunized as control; Group 2 - 1 Opg
peptide/mouse with montanide; Group 3 - 1 Opg peptide/150m1 CaPNP /mouse; Group 4 - 1 Opg peptide/150m1 CaPNP with lXGlcNAc; Group 5 - 1 Opg peptide/150m1 CaPNP with 3XGlcNAc. Spleen cells were harvested and mixed with targets pulsed with no peptides (negative control), or pooled peptides (NIQ, TIT, VTL, KLA, AML, LLC), or infected with DV2 virus in the ELISpot assay. Data represent Mean±S.D. (n=3)
* Represents P<0.05, ** R<0.01, ***P<0.00l, **** PO.OOOl
Figure 4. DV multi-peptide CaPNP vaccine formulation stimulate CD8+ T cell activation in vitro : CTLs were generated using peripheral blood from a healthy donor PBMCs were activated with: 1) pooled peptides, 2) multi peptide CaPNP with GlcNAc, or 3) DV peptide CaPNP in vitro. (A) HLA-A2 specific The non-adherent PBMCs containing the epitope specific CTLs were harvested, washed, and cultured with individual peptide pulsed (2pg/ml) or dengue infected HepG2 cells overnight in an IFN-g ELISpot assay. (B) Identical cultures were set up as in (A) but in 96 well round bottom plates. The next day, supernatant was harvested and used to detect cytolytic activity (Granzyme B) using Luminex magnetic bead technology (MagPix) (C) The cells were also analyzed for the marker of degranulation, CD 107a, by flow cytometry. Data represent Mean±S.D. (n=3)
Figure 5. Optimized HLA-A2/A24 DV multi-peptide CaPNP vaccine formulation stimulate CD8+ T cell activation in vivo. CTL response was generated with: Group 1- unimmunized (control), Group 2-pooled peptides with montanide-5l (1 Opg pep/150m1), Group 3-pooled peptide in CaPNP formulation with GlcNAc (1 Opg pep/150m1), and Group 4- pooled peptide in CaPNP formuation without GlcNAc (10 pg pep/150 m 1 ) in HLA-A2 transgenic mice. As previously describe in Fig. 1 A, spleen cells were harvested and mixed with APCs pulsed with either individual peptides (NIQ- Seq ID No.4, TIT- Seq ID No.3, VTL- Seq ID No.2, KLA- Seq ID No.5, AML- Seq ID No.6, LLC- Seq ID No.7) or infected with DV2 virus as targets in the ELISpot assay. Data represent
Mean±S.D. (n=3)
Figure 6. Stability and efficacy of optimized DV multi-peptide CaPNP formulation with GlcNAc. (A) Particle size of formulations measured over a time of three months (day 0, 30, 60 and 90) under different storage conditions (4°C or RT). (B) In vitro CTL response by different formulations generated in PBMCs. Four groups were tested as: 1) pooled peptide with montanide, 2) peptide CaPNP with GlcNAc solutions stored at 4°C, 3) lyophilized peptide CaPNP with GlcNAc stored at 4°C and 4) lyophilized peptide CaPNP with GlcNAc stored at RT. CTL responses were generated by pulsing PBMCs with no stimulated as control, pooled peptides, DV2 infected cells. IFN-g expression was measured by ELISpot assay. (C) In vivo CTL response by different formulations generated in immunized HLA-A2 transgenic mice. Three groups were tested as: 1) pooled peptide with montanide, 2) peptide CaPNP with GlcNAc solutions stored at 4°C, 3) lyophilized peptide CaPNP with GlcNAc stored at 4°C. Mice were immunized as described in Fig. 1A. CTL responses were generated by pulsing spleen cells with unstimulated as control, pooled peptides, DV2 infected cells, Thai isolated DV serotypes (Tl, T2, T3, T4). Data represent Mean±S.D. (n=3).
DETAILED DESCRIPTION OF THE INVENTION
Dengue virus infects an estimated 300 million people each year and even more are at risk of becoming infected as the virus continues to spread into new areas. Despite the increase in viral prevalence, few anti-viral medications or vaccines are approved for treating or preventing infection. Conventional vaccines normally target either DV virons or the viral structural proteins to provide the protection from all four dengue serotypes. All the candidate vaccines under development or in clinical trials are based on humoral immunity. Several concerns related to dengue pathogenesis caused by cross-reactive T cells during DV infection, antibody-dependent enhancement or insufficient response in younger age group have raised challenges to the development of a robust, broad and multi- functional dengue vaccine. Viral clearance is largely mediated by robust CD&+ T cell responses. Therefore, effective vaccines that induce a broad, multi-functional T cell response with substantial cross-reactivity between all virus serotypes can have major impacts on reducing infection rates and infection related complications. Another common challenge in vaccine design is to deliver small/macromolecules to the target site and create appropriate immune response. To address those challenges, we developed a synthetic calcium phosphate nanoparticle (CaPNP) based multi-epitope T cell vaccine for DV infection, which can be used as a standalone vaccine or combination treatment for disease prevention or therapy.
It is well documented that nanoparticles are ideal antigen delivery systems to target cells or organs for therapeutic purposes. A number of previous studies show that they can be conjugated with drugs or biomolecules such as peptides or antibodies. Comparing to microparticles, they are at the range of nanometer which enhance the efficiency of cellular uptake by 15-250 fold. Knuschke et al. demonstrated a calcium phosphate nanoparticle delivery system (<200nm) was taken up by dendritic cells in vivo and induced a potent T cell response. Recently, gold nanoparticle (Au-NP) based vaccines have been studied extensively due to smaller particle size (<l00nm), customizable shape, various binding mechanisms (covalent/non-covalent), stable etc. The average particle size of the dengue vaccine formulation, described herein, is below lOOnm, which was small enough to enter lymphatic network and generate more potent immune responses. More importantly, it was biodegradable due to the component of calcium phosphate and had a good safety profile. Intradermal and subcutaneous injection of CaPNP in HLA-A2 transgenic mice at levels of low and high dose on study days 1, 8 and 15 did not result in any adverse effects, treatment- related clinical signs of toxicity, or effects on body weight or food consumption (data not shown). Besides safety, the CaPNP formulated vaccines disclosed herein were proved to be stable and active during long term storage either at 4°C or at RT. It is not uncommon that nanoparticles show a poor long-term stability due to physical and chemical instability. CaPNPs have been shown to be stable for many years at room temperature, either as particulate suspension or as lyophilized or spray-dried powder. The present invention employs lyophilization (freeze-drying) to improve the CaPNP-DV vaccine stability. Over a three month observation period, particle size of lyophilized CaPNP-DV vaccine (4°C or RT) showed increased functional efficacy when compared to the same vaccine in suspension; however it was not statistically significant (Figure 6, P>0.05). The inventor further tested the immunological performance between solution and lyophilized powder. Both in vitro and in vivo results showed a potent T cell response among testing groups, which provided the evidence that lyophilization could be an option for vaccine long term non-cold chain storage and suitable for transportation and use in parts where dengue is endemic.
One of the important components for nanoparticle based vaccines is the candidate epitopes and/or antigens which will be adsorbed to the particle surface, or incorporated in the core, or both. We utilized an immunoproteomic approach to identify epitopes which are naturally processed and presented by DV infected cells. Those novel MHC class I restricted epitopes were derived from four DV serotypes and HLA A2/A24 positive, and they were able to induce CDS+ T cell activation both in vivo and in vitro. Previous data demonstrated that dengue specific T cell responses can be induced in vivo in transgenic mice, in vitro from healthy seronegative or seropositive individuals who have previously been infected with DV. Thus, the present vaccine formulation with HLA-A2 and A2/A24 epitopes has been characterized extensively. CaPNP can be conjugated with one or multiple antigens either on the surface or inside the particle. Therefore, we formulated six individual peptides with CaPNP and demonstrated that the mean particle size of all testing formulations remained in the range of 80-l00nm, which was smaller than previously reported CaPNP formulations, producing better performance in terms of immune cells uptake and immune response. The six DV peptides were from conserved regions of DV serotypes and induced a potent T cell responses against all four serotypes of D V infected cells, which could be critical in the development of a universally immunogenic vaccine. Accordingly, the multi-epitope vaccine formulation with CaPNP improved the overall immunogenic performance when compared to free peptides or peptides mixed with montanide-5 l adjuvant. The CaPNP-DV vaccine formulation is antigen sparing, in which lower vaccine dose induced higher T cell response when compared to the higher dose (Fig. 2), suggesting that CaPNP was acting both as antigen delivery system and partially as an adjuvant to facilitate the antigen uptake by antigen presenting cells, trigger DC activation and induce IFN-y production.
One of the major advantages in using adjuvant clinically is to reduce the dose of antigens, as it will increase the magnitude of an adaptive response to a vaccine. Moreover, CaPNP can be considered as a preferred adjuvant/delivery system candidate due to the fact that they have small particle size in the nanometer range, present high loading capacity, are biodegradable/biocompatible, and enhance vaccine immunogenicity while they are inert to immune system by themselves. The present invention integrates CaPNP with epitopes that are naturally presented by DV infected cells and characterized the biological function of this novel vaccine strategy. Unlike Au-NPs, which are constructed by reducing a gold salt in the presence of thiol functionalized synthetic neogly co-conjugates, the manufacturing of CaPNP is very simple. Vaccine formulation is also very simple which involves pre- mixing the CaPNP and antigenic peptides at room temperature for periods of several hours. Furthermore, we optimized the formulation and added GlcNAc to enhance the immune cell uptake and activation of innate and adaptive immune responses.
Ultimately, the most promising universal dengue vaccine candidate must come from combining the antigens for both T and B cell immunity. In order to be active the vaccine must mimic viral sizes and permit cytosolic uptake to target antigen presenting cells. Integrated nanoparticle- biomolecule hybrid systems constitute useful tools to mimic the behavior of biomolecules in cells, helping to explore the mechanisms of biological processes with a variety of potential applications. The present invention describes the development of a synthetic universal vaccine based on shared multiple T cell epitopes incorporated in CaPNP. Although the CaPNP-epitope formulation focused on two of the major HLA haplotypes, HLA-A2 and A24, which are prevalent in 40% of the world population (HLA-A2), this approach, if successful, could quickly be extended to other major HLA super type specific epitopes that can be identified and incorporated into follow up vaccine formulations. Most importantly, the success of this vaccine will lead to a novel universal vaccine technology platform that could transform the clinical success of prophylactic vaccine strategies for heterologous viruses and that could be applied to therapeutic vaccines for chronic viral infections. From the manufacturing and cost effectiveness perspective, a synthetic universal vaccine strategy would have a significant positive impact.
Selection of calcium phosphate nanoparticle (CaPNP) for vaccine formulation
CaPNP adjuvant at 0.3% final concentration, but not less than 0.3% produced the best immune response with most vaccine antigens. To determine the optimum CaPNP concentration suitable for peptide antigen delivery and functional T cell activation, we formulated a test antigenic peptide, SIINFEKL (SEQ ID No. 1), a well characterized model peptide from ovalbumin, with various concentrations of CaPNP and tested its efficacy of T cell activation. The following formulations were tested: (i) 0.8% CaPNP alone (0.8% placebo), (ii) 0.8% CaPNP + SEQ ID NO. 1, (iii) 0.3% CaPNP alone (0.3% Placebo), or (iv)0.3% CaP + SEQ ID NO. 1, including one unpulsed sample as control. In Angel antibody assays (Figure SIA), the antigen presenting cells (Lkb cells; 100,000 cells per well) were pulsed with SUN peptide alone (placebo group), SUN peptide formulated in 0.8% CaPNP (dry particle weight/volwne) (0.8% CaPNP placebo) or SUN peptide formulated in 0.3% CaPNP (0.3% CaPNP placebo). Each group was treated with three different concentrations of SUN peptide, 0 1 pg, 0.2pg and lpg, respectively. As shown in Fig. SIA, the treatment groups either from peptide alone or peptide formulated in CaPNP demonstrated higher level of antibody staining which indicated the processing of peptide and presentation in association with MHC class I molecule. Moreover, there was no difference between 0.8% vs 0.3% CaPNP formulation. In addition, we tested the capability of these formulations pulsed APCs to present antigen and activate antigen specific T cells. MUG assay (Figure SIB) presented a similar result for the antigen processing activity. Both the free peptide and peptide formulation pulsed APC groups activated T cell measured by the expression of increasing gal levels. As there was no observed difference between the two formulation groups, we selected 0.3% CaPNP formulation for further investigation.
Multi-epitope DV specific vaccine formulation
Six previously characterized DV epitopes (Table 1) were chosen and formulated in 0.3% CaPNP solutions either individually or pooled together. GlcNAc (N-acetylglucosamine) was included as a surface modifying agent to increase surface-binding of antigens to CaPNP (37, 44), and act as adjuvant in targeting APC by binding to mannose receptor to facilitate antigen uptake and processing ( 45). IFN-y ELISpot assay results are shown in Figure 1. The data indicates that peptides formulated in CaPNP individually or collectively activate significant CTL response compared to unstimulated group (negative control) in all immunized HLA-A2 transgenic mice. Specifically, 6 peptide pool (Seq ID No. l; Seq ID No.2; Seq ID No 3; Seq ID No 4; Seq ID No 5; Seq ID No. 6; Seq ID No. 7) plus CaPNP formulation induced the highest CTL response in DV2 (Seq ID N03; Seq ID NO; 5; Seq ID NO. 6; Seq ID NO.7, and individual peptide target (NIQ- SEq ID N04, TIT- Seq ID NO. 3, AML-SEq ID NO.6) group, when compared to individual peptide plus CaPNP formulations pooled together or free peptides emulsified with montanide 51 (positive control). Hence, the multi-epitope formulation was selected for further in vitro and in vivo functional characterization.
Table 1 : Dengue Virus Specific MHC Class 1 associated peptides identified previously by immunoproteomics
Figure imgf000014_0001
To further characterize the multipeptide DV CaPNP formulation, we investigated varying concentration of the peptides for T cell responses in vivo using HLA-A2 transgenic mice. The mice were immunized in 5 groups: unstimulated (PBS), pooled free peptides (lOpg each) emulsified with montanide-5l, pooled free peptides (SOpg each) emulsified with montanide-5l, CaPNP formulated peptides (IOpg each) and CaPNP formulated peptides (SOpg each). The results depicted in figure 2 demonstrate a significant difference in response to DV2 infected targets (P<0.000l, one way ANOV A). Both peptides emulsified with montanide-5l and peptides formulated with CaPNP elicited higher T cell responses compared to unstimulated group. Also at lower peptide concentrations (IOpg each), CaPNP showed a higher CTL response than peptides (IOpg each) emulsified with montanide 51 group (P<0.0l). Comparing these 2 groups, the lower concentration of peptides plus CaPNP formulation group generated the highest T cell responses against DV2 infected targets, which was also significantly higher than the higher concentration ofCaPNP peptide group (P<0.05).
Subsequently, we compared the T cell responses generated by CaPNP formulated peptides with or without GlcNAc in HLA-A2 transgenic mice. The results from an IFN-y ELISpot data are shown in figure 3. Significant T cell responses (P<0.000l) against DV2 infected targets were observed in all treatment groups. The CaPNP formulated with GlcNAc (IX or 3X) showed a higher response compared to PBS injection group (P<0.0l, P<0.000l, respectively). Similarly, the addition of GlcNAc to the CaPNP plus peptide formulation demonstrated higher T cell response in comparison to formulation without GlcNAc, which is statistically significant (P<0.05, P<0.00l, respectively). However there was no significant difference between IX and 3X GlcNAc formulation group. Overall, there was robust peptide specific and DV specific T cell response in CaPNP formulation groups. Specifically, the response against DV infected targets were more pronounced than peptide loaded targets.
in vitro and in vivo functional analysis of multi-peptide DV CaPNP vaccine
formulation
Our previous studies(24, 25) demonstrated unformulated DV specific epitopes were able to activate CDS+ T cell responses in both seronegative, healthy and seropositive individuals who had previously been infected with DV, which might serve as candidate antigens for the development of effective multi-serotype DV vaccines. In order to assess whether the CaPNP formulated epitopes elicits similar anti-DV specific T cell responses, we formulated the DV specific epitopes into CaPNP particles and analyzed in vitro using PBL obtained from healthy individuals. The data in Figure 4, demonstrates significant T cell responses against DV infected cells as measured by IFN-y secretion in an ELISpot assay (Figure 4A), and by the secretion of granzyme B by MagPix (Figure 4B), and up- regulation of CD 107a by flow cytometry (Figure 4C). As shown in Figure 4A, CaPNP formulated with or without GlcNAc stimulated robust peptide specific T cell response, which was more intense than peptide alone group. However, the granzyme B (Figure 4B) and CD 107a marker expression (Figure 4C) data showed a slight increase in the group which was treated with CaPNP formulation containing GlcNAc, which was consistent with the previous finding from in vivo study (Figure 3).
Having confirmed that CaPNP vaccine formulation was capable of inducing CDS+ T cell activation in healthy PBMCs, we decided to investigate whether it could activate T cells in vivo. The HLA-A2 mice were immunized in four groups: PBS, peptide with montanide-5l, peptide formulated with CaPNP and GlcNAc, peptide formulated with CaPNP. Similarly, the activated T cell responses were measured by IFN-y secretion in an ELISpot assay (Figure 5). Similar to the in vitro studies, the CaPNP fonnulation with peptides elicited robust peptide specific and DV infection specific T cell responses in vivo. Additionally, fonnulation with GlcNAc showed slightly higher response than the fonnulation without GlcNAc group. The response against DV infected cells were more pronounced than the peptide loaded targets (Figure 5).
Stability and efficacy of multi-peptide DV CaPNP vaccine formulation
One of the ultimate goals of this study is to develop a T cell activating DV vaccine that is stable and does not require cold storage. To test if the CaPNP fonnulated peptides can be Iyophilized and stored as dry powder, we fonnulated the peptides and GlcNAc in CaPNP, lyophilized, and stored at 4 C or room temperature for periods of time, and periodically tested the stability of the particles and functional capability of the vaccine in in vitro and in vivo studies. We first tested the vaccine fonnulation integrity by evaluating the particle size at various conditions and time points. The CaPNP fonnulation stored as particulate suspension at 4 C, CaPNP fonnulation lyophilized and stored at 4 C and CaPNP lyophilized and stored at room temperature (RT). The data from particle size analysis at various time points (30, 60, and 90 days post-fonnulation) is shown in Figure 6A. The data clearly indicated that all three groups of fonnulations were stable for at least 90 days (P>0.05). There was no significant difference in particle size between 4°C or RT for lyophilized fonnulation (P>0.05). Although there was an increasing trend in particle size for lyophilized fonnulation stored at RT (93.4±26.0 nm at day 30, l25.65±45.04 nm at day 60, l03.l±5l.9 nm at day 90, mean±SD, n=3), the result did not show any statistical difference (P>0.05,). We further tested the functional stability for solution and lyophilized CaPNP fonnulation. In vitro T cell response was analyzed by an IFN-y ELISpot assay using PBL from healthy donors. Peptide specific T cells were generated by stimulating peripheral blood T cells with either pooled peptides, or CaPNP
formulation stored at 4 C, or CaPNP peptide formulation lyophilized and stored at 4C/RT. As shown in Figure 6B, all groups induced a higher expression of IFN-y in peptide pulsed cells. However, in DV2 pulsed cells, all groups except lyophilized formulation at 4 C showed a higher T cell response. Overall, the CaPNP formulation lyophilized and stored at either 4C or RT stimulated a strong DV specific T cell response in vitro. There
was no significant difference in responses generated by solution or lyophilized format under the tested storage conditions (P>0.05, n=3).
In addition to in vitro T cell activation assay, HLA-A2 transgenic mice were immunized to study CaPNP formulation under various conditions. There were three immunization groups: 1) antigenic peptides emulsified with montanide-5l, 2) peptides formulated with CaPNP and stored as solution (4°C), lyophilized peptides formulated with CaPNP (4°C). The T cell responses were analyzed using DV2 infected, peptide pulsed and various DV serotypes {Tl, T2, T3, T4) infected cells as targets. Results shown in Figure 6C demonstrated that CaPNP formulated peptide vaccine activated DV specific T cells in vivo, which was consistent with in vitro analysis (Figure 6B). CaPNP formulation induced T cell activation against all of the 4 serotypes of DV infected cells. Lyophilized CaPNP formulation activated T cells in vivo as measured by IFN-y response against various targets. Hence, the conserved antigenic peptides in CaPNP vaccine formulation activate T cell response against all four serotypes of DV. We concluded that the lyophilized vaccine formulation is capable of generating T cell response against all the four DV serotypes as well.
The present invention embodies generally vaccine compositions, and methods of use thereof, for the prevention, treatment, and diagnosis of dengue virus infection. Accordingly, one group of embodiments in the present invention includes compositions for a vaccine that induces a broad, multi-functional T cell response with substantial cross- reactivity between all virus serotypes. One particular aspect includes a multi-epitope DV specific vaccine formulation where DV epitopes are formulated in CaPNP-containing solutions to activate a CTL response. These embodiments incorporate useful pharmaceutical compositions such as, but not limited to, an adjuvant (e.g., Freund’s complete or incomplete adjuvant) or administration with traditional prophylactic viral vaccine formulations (e.g., live attenuated viruses, inactivated viruses, recombinant proteins, chimeric viruses, DNA vaccines, and synthetic peptides).
The invention embodies kits that contain these compositions and optionally include instructions for treating (prophylactic or therapeutic), vaccinating or immunizing a subject against a DV infection, or treating (prophylactic or therapeutic) a subject having or at risk of having a Dengue virus infection or pathology.
Further embodiments provide prophylactic methods including methods of vaccinating and immunizing a subject against a DV infection (acute) such as, but not limited to, protecting a subject against a DV infection to decrease or reduce the probability of a DV infection or pathology in a subject or to decrease or reduce susceptibility of a subject to a DV infection or pathology or to inhibit or prevent a DV infection in a subject.
The vaccine compositions are utilized for purposes of preventing, suppressing or treating diseases causing the expression of the immunogenic peptides disclosed herein. As used in accordance with the present invention, the term "prevention" relates to a process of prophylaxis in which an animal, especially a mammal, and most especially a human, is exposed to an immunogen of the present invention prior to the induction or onset of the disease process. This would be most appropriate where an individual is at high risk for DV infection based on the living or travel to the DV endemic areas.
Alternatively, the immunogen could be administered to the general population as is frequently done for any infectious diseases. Alternatively, the term "suppression" is often used to describe a condition wherein the disease process has already begun but obvious symptoms of said condition have yet to be realized. Thus, the cells of an individual may have been infected but no outside signs of the disease have yet been clinically recognized. In either case, the term prophylaxis can be applied to encompass both prevention and suppression. Conversely, the term "treatment" is often utilized to mean the clinical application of agents to combat an already existing condition whose clinical presentation has already been realized in a patient. This would occur where an individual has already been diagnosed as having confirmed DV infection.
It is understood that the suitable dosage of an immunogen of the present invention will depend upon the age, sex, health, and weight of the recipient, the kind of concurrent treatment, if any, the frequency of treatment, and the nature of the effect desired. However, the most preferred dosage can be tailored to the individual subject, as determined by the researcher or clinician. The total dose required for any given treatment will commonly be determined with respect to a standard reference dose as set by a manufacturer, such as is commonly done with vaccines, such dose being administered either in a single treatment or in a series of doses, the success of which will depend on the production of a desired immunological result (i.e., successful production of a CTL-mediated response to the antigen, which response gives rise to the prevention and/or treatment desired). Thus, the overall administration schedule must be considered in determining the success of a course of treatment and not whether a single dose, given in isolation, would or would not produce the desired immunologically therapeutic result or effect.
Regardless of the nature of the formulations given, additional vaccine compositions may also accompany the immunogens of the present invention. Thus, for purposes of preventing or treating DV infection (e.g., prophylactic or therapeutic vaccine), compositions containing the immunogens disclosed herein may, in addition, contain other vaccine pharmaceuticals. The use of such compositions with multiple active ingredients is left to the discretion of the clinician.
While examples are provided to illustrate the invention, it is to be understood that these methods and examples in no way limit the invention to the embodiments described herein and that other embodiments and uses will no doubt suggest themselves to those skilled in the art. All publications, patents, and patent applications cited herein are hereby incorporated by reference, as are the references cited therein. It is also to be understood that throughout this disclosure where the singular is used, the plural may be inferred and vice versa and use of either is not to be considered limiting.

Claims

Claims:
1. A method for preparing a vaccine composition comprising an antigen and a calcium phosphate carrier with a bacterial carbohydrate adjuvant.
2. The method according to claim 1, where the bacterial carbohydrate adjuvant is N acetyl Glucosamine.
3. The method according to claim 1, wherein the carrier is a calcium phosphate nano- particle less than approximately 100 nm.
4. The method according to claim 3, wherein the calcium phosphate nano-particle is less than 200 nm.
5. The method according to claim 4, wherein the calcium phosphate nano-particle is between 80nm to 100 nm.
6. The method according to claim 1, wherein N acetyl Glucosamine is in a concentration of 0.3%.
7. The method according to claim 1, wherein the adjuvant is an alum or a lipid based emulsion in a drug delivery vehicle having a target site providing a standalone vaccine for the treatment of disease prevention and therapy.
8. The method according to claim 1, wherein the antigen has a conformation other than its native conformation.
9. The method according to claim 1, wherein the antigen further elicits a T cell response that recognizes a native epitope.
11. The method according to claim 9, wherein the native epitope is a mammalian, bacterial or viral epitope.
12. The method according to claim 1, wherein the antigen is a non-native, recombinant or denatured protein, a recombinant or synthetic peptide, or a fragment thereof.
13. The method according to claim 1, wherein the antigen is viral, bacterial, protozoal or mammalian antigen.
14. The method according to claim 1, wherein the antigen is from a viral, bacterial, fungal, yeast or tumor source.
14. The method according to claim 1, wherein the treatment is an intradermal injection of CAPNP conjugated with one or multiple antigens for haplotypes HLA-A2 and A2/A24 epitopes.
13. The method according to claim 12, wherein the treatment is a subcutaneous injection.
14. The method according to claim 1, wherein the standalone vaccine is stable at 4°C or at RT as a suspension or lyophilized in a spray-dried powder.
15. The method according to claim 1, wherein the antigen is an antigen from a viral, bacterial, fungal, yeast or tumor source.
16. The method according to claim 1, wherein the antigen elicits an immune response related to viral infection or cancer.
17. The method according to claim 1, wherein the antigen elicits an immune response related to a biological condition other than viral infection or cancer.
PCT/US2019/050116 2018-09-07 2019-09-07 Vaccines with enhanced immune response and methods for their preparation WO2020051566A1 (en)

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