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					A High-Volume, Cost-Effective Microparticle-Based
           Vaccine Production Process

                    William Warren, Ph.D.
                  CEO, VaxDesign Corporation
                    2721 Discovery Drive
                      Orlando, FL 32826
                      Tel: 407.249.3677
                   wwarren@vaxdesign.com
                     www.vaxdesign.com




        Work funded by DARPA/DSO in the Rapid Vaccine
       Assessment & Pathogen Countermeasures Programs
         What Is the Bottleneck
in Getting More Therapies to the Market?




                            Reduce the cost & time
                            associated with scale-up
                            manufacturing

                            Reduce R&D and lost
                            opportunity costs
          Immuno-Engineering Technologies
Artificial Immune System                             Immunoparticle-
                                                        vaccines




                           Universal innate immune
                             signaling pathways
                      Previous Mistakes
        Beginning (Immunologists Ruled)
  We asked everyone what they wanted, what it should
                                                             Everything
 include, how it should operate, what it should produce   (Mimic Human Nature)
            and how it should be analyzed.


           Now (Engineers to the Rescue)
 Now we know what works, how things are prepared,             Elegant/
 how things are tested, and how they should operate.          Simplicity



Future – Add in Complexity – Immuno-Engineering
  Start with what works and add in complexity as the      Advanced Models
      immunology dictates (test then implement)
What we need for rapid vaccine assessment
           Tissue Engineering To Create
            Immunological Constructs




collagen



            http://books.nap.edu/html/science_based/Anthony%20Ratcliffe.pdf
       Vaccination Site: From Virtual To Actual
Input: Diapedesis of monocytes



                                 Output: Transendothelial
                                     migration of DCs




                                                            DC morphology

         DC
Lymphoid Tissue Equivalent:
   From Virtual To Actual

    DCs, T and B cells in
     a collagen matrix



                                                                Ag-specific naïve and
                                                                  recall responses

                                                      30                                     27.9 28.4




                        % Divided / activated cells
                                                      25   2D (No carrier)
                                                           3D (Carrier)



                            (CFSElowCD25+)
                                                      20
                                                      15
                                                                                     R=6.2
                                                      10                             7.44
                                                                             R=4.4
                                                      5                      1.98
                                                           0.45 1.2
                                                      0
                                                           DC - no Ag        DC - KLH        DC - C.a.
  Humoral Responses: An in vitro Germinal Center

                      in vitro self-assembled GC



  B and T cells



Dividing B cells




                                                                  Different immune cell types


     In previous murine in vitro GCs at VCU, Ig class switching, somatic hypermutation,
        selection of high affinity B cells, and affinity maturation were demonstrated
AIS Integration in a Well-Based Format

                                     Well Lid



                                 Vaccination Site
                                Membrane Bucket



                                     LTE with
                                Dialysis Membrane


                                 Media Well
          Immuno-Engineering Technologies
Artificial Immune System                             Immunoparticle-
                                                        vaccines




                           Universal innate immune
                             signaling pathways
          The Problem: Typical Adjuvants Simply Activate Toll Like
            Receptors and Do Not Provide Universal Protection

                                             Hep C virus
                                               protein     vaccinia




O’Neill, L. Current Opinion in Immunology 18:3-9, 2006.
   Universal Intracellular Signalling Pathways to Thwart Disease


The intracellular pathway is present in every cell
and regulates the NF-kB pathway more robustly
than the tissue restricted Toll Pathway.

We are now isolating new regulators (novel
genes/proteins, small molecules and other
compounds) of these pathways.

These activators can be used to potently
modulate the innate immune response

In the advent of virus infection, such activators
may function as universal anti-viral and anti-
bacterial therapeutics.
  Innatesome Intracellular Pathway Plays a Significant
        Protective Role Against Many Infections
                                                                        VSV




           Barber et al. Nature 432, 401-405 (2004)




Influenza & VSV replication is significantly augmented (>100 fold) in the FADD−/− MEFs
Continued…




             Barber et al., Nature 432, 401-405 (2004)
          Immuno-Engineering Technologies
Artificial Immune System                             Immunoparticle-
                                                        vaccines




                           Universal innate immune
                             signaling pathways
Tapping Into Potent Innate Signaling Pathways: Type 1 Interferons

                       Multifunctional
                      immunoparticles
                                                                 Surface modified particles
                                                                                type I IFNs
                                                                    producealpha induction in DC2
                                                                     Interferon
   Cell engulfment
                                                             5
 activates pathways
 to produce INF a/b




                                         IFNa Expression
                                          Fold Increase in
                                                             4




                                           Fold Induction
                                                             3

                                                             2

                                                             1

                                                             0
                                                                     Protasan       Protasan-dsRNA

                                                             DC2 cells, which express little or no
                                                              TLR-3, appear to produce IFNa in
                                                                 response to poly(I:C)-loaded
                                                                 immunoparticles through an
                                                                intracellular signaling pathway
Immunoparticles Activate NFkB and TLR Pathways For Protective Immunity
The particles that are coated with poly(I:C) can directly activate the NFkB pathway as
                      well as activate it through TLRs 3, 8, and 9


                                                                     Protosan particles
                                                                     Protosan + poly(I:C)
                                                                     Protosan + Ag
                                                                     Protosan + Ag + poly(I:C)
                                                                     Protosan + Alexa Fluor 594




                                                           HEK293 SEAP cell line is a
                                                           NFkB reporter cell
                                                           line and does not contain
                                                           any Toll-like receptors
 Particles   Particles
with dsRNA    alone
   0     3   0      3    hrs dsRNA
                           IFIT
                           Map3k
                           Ccl5
                           IL15
                           IFIT3
                           GBP-2
                           Cxcl11
                                       Particles (PGLA) assembled
                           Mx1          with dsRNA as an adjuvant
                           Mx2             and potently activate
                           IFNa4     innate immune response genes
                           IFNa2
                           IFNa5         in human dendritic cells
                           IRF7        compared to particles alone
                           VHS                   (P-alone).




       DNA Array
                Poly(I:C) is Efficiently Adsorbed to
                        PLGA/PEI Particles
        PLGA/PEI particles obtained via
       electrospray; the poly(IC) sorption   Fluorescent PLGA/PEI/FITC particles;
               220 mg/mg particle             poly(I:C) sorption 70 mg/mg particle




• Enhancing innate immunity by activating two dsRNA signaling pathways
  to produce IFNa/b
APCs Efficiently Phagocytose PLGA Immunoparticles




   cytoplasm is stained with CFSE (green), the nucleus is stained with
                  DAPI (blue) while the particles are red
Poly (I:C)-Loaded Particles Activate Dendritic Cells
                              PLGA particles




                                               CD86


             % of Max




                                               HLA-DR




                          Red = no Treatment
                        Green = particles alone
                        Blue = particles/poly(I:C)
                                                                        Poly (I:C) & Ag-Loaded Particles Activate
                                                                        CD4+ and CD8+ Murine C57BL/6 T Cells
                                                                     600
                                                                                                                          Figure 7. OVA-loaded
                                                                                medium
                                                                                                                          chitosan and PLGA
Number of INFg spots/106 splenocytes


                                                                                Ova epitope-I
                                                                                                                          particles induce specific
                                                                     400                    CD8 response                            MHC cell
                                                                                                                          CD4+ and CD8+ Tclass         MHC class
                                   Number of spots/106 splenocytes




                                                                                                                                    I-restricted
                                                                                                                          responses in vivo.
                                                                                                                                                       II-restricted
                                                                                                                          C57BL/6 mice were
                                                                                                                                    257-264
                                                                                                                          injected i.p. with 50 mg     323-339
                                                                     200
                                                                                                                                    epitopes
                                                                                                                          OVA-equivalent of the
                                                                                                                                                       epitopes
                                                                                                                          indicated particles at
                                                                                                                          days 0 and 30. 6 days
                                                                       0                                                  later, splenocytes were
                                                                     600                                                  stimulated with the MHC
                                                                               medium                                     class I-restricted
                                                                               Ova epitope-II                             OVA257-264 (upper
                                                                                                                          panel) or MHC class II-
                                                                     400
                                                                                                                          restricted OVA323-339
                                                                                                                          (lower panel) peptides
                                                                                                                          for 18 hr in anti-IFNg Ab-
                                                                           CD4 response
                                                                     200                                                  coated plates. Media
                                                                                                                          alone served as a
                                                                                                                          negative control. Spots,
                                                                       0                                                  indicating specific IFNg
                                                                                               )                      )   production, were
                                                                                  VA        (IC         VA        (I:C
                                                                             n-O        VA
                                                                                          -p        A-O       VA
                                                                                                                -p        counted using an
                                                                           sa                     LG         O            automated ELISPOT
                                                                     Ch
                                                                        ito          n-O         P        A-
                                                                                   sa                  LG                 analyzer.
                                                                               it o                   P
                                                                            Ch
                   Poly (I:C) & Ag-Loaded Particles Trigger
                         OVA-specific Cytolytic Activity

                                     Blank     OVA       OVA/poly(I:C)
                              40                           #1-EG7
                                    #1-EG7    #1-EG7
                                                           #2-EG7
           % Specific Lysis



                                    #2-EG7    #2-EG7
                              30                           #1-EL4
                                    #1-EL4    #1-EL4
                                    #2-EL4    #2-EL4       #2-EL4
                              20

                              10

                               0

                              -10



                                             E:T ratio


The lytic activity of splenic CD8+ T cells from immunized C57BL/6 mice was also
assessed directly ex vivo against tumor lines negative (EL4) or positive (EG7) for
                        OVA in a standard 51Cr-release assay
      Poly(I:C) & Antigen-Loaded Particles Trigger
                   Antibody Responses
                                              1.6
                                                    IgG1




                              O.D. (450 nm)
OVA-loaded carriers also                      1.2

trigger the production of                     0.8
IgG1, though poly(I:C) also
                                              0.4
induced the synthesis of
IgG2a. These results                           0

support our hypothesis that                    1
carriers loaded with dsRNA                          IgG2a



                              O.D. (450 nm)
                                              0.8
will promote a protective                     0.6
Th1-like response.                            0.4

                                              0.2

                                               0
                                                    Blank   OVA      OVA/
                                                            alone   Poly (I:C)
Immunoparticles to Elicit Broad Immunity
Against Influenza Virus That Is Ag-Specific
    Cross-protective Influenza Vaccines

• Multifaceted approach to generate strong protective immunity:
   – Innate defense ligands (adjuvant)
   – Humoral response (M2e)
   – Cellular response (NP)

• Activate novel intracellular innateosome and traditional
  extracellular pathways to generate potent IFNa/b responses

• Target immunity against conserved proteins of the virus
  (possibly heterologous protection)

• Rapid production – no eggs; recombinant bacterial proteins
  (NP) and/or synthetic peptides (M2e)
   Multifunctional Immunoparticles Against Influenza



      M2- and NP-            +
  loaded microparticles
                             Phagocytosis




       M2-loaded
      nanoparticles


                             Traffic to lymph nodes

Incorporating innate stimulatory signals and multiple influenza antigens into the
particles, a more robust and protective anti-influenza response should be realized
 Putting It All Together – Innate & Adaptive
Activating Immunoparticles Against Disease
       Sometimes You Have To Think
Differently – Turn the Problem Upside Down




       START
 Do We Really Need Ag-Specific Protection,
       Especially If Time Is Critical?


What If …you could temporarily boost one’s
            immune system?

Examples why one would care:

Pandemic

BWD
    Thanks to the VaxDesign Group
   William Brown, Ph.D., MBA, JD     Eric Mishkin, Ph.D.
   Tony Byers, Ph.D.                 David Moe, MS
   Don Drake, Ph.D.                  Janice Moser, Ph.D.
   Heather Fahlenkamp, Ph.D.         Luis Mosquera, MS
   Kristyn Feldman                   Mike Nguyen, MS
   Russell Higbee, Ph.D, DVM         Robert Parkhill, Ph.D.
   Anatoly Kachurin, Ph.D.           Guzman Sanchez-Schmitz, Ph.D.
   Olga Kachurina, Ph.D.             Brian Shanen
   Riyaz Mehta, MS                   Gary Sui, Ph.D.

Thanks to our University Collaborators
         Glen Barber, Ph.D., University of Miami
         John Tew, Ph.D., Virginia Commonwealth University
         Darrell Irvine, Ph.D., MIT
         Gwen Randolph, Ph.D., Mt Sinai School of Medicine.
         Kaveh Zamani, Ph.D.

      Thanks to the DARPA Team
                    Michael Callahan, MD
                    Brett Giroir, MD
                    Clay Holloway, Ph.D.
                    Kaveh Zamani, Ph.D.

 Work funded by DARPA/DSO in the Rapid Vaccine
Assessment & Pathogen Countermeasures Programs

				
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