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Mucosal vaccine for broad protection against divergent influenza subtypes

Chul-Joong Kim, DVM, PhD

College of Veterinary Medicine Chungnam National University

2014. 05. 06

1. Target antigens for universal vaccine

Viral protein Targeted site Proposed mechanism of protection

Hemagglutinin (HA2)

Stalk domain with fusion activity

(Non-neutralizing) antibodies, inhibition of the fusion, maturation of the HA, and antibody dependent cell mediated cytotoxicity

Matrix 2 ion channel (M2)

Ectodomain of M2 (M2e)

(Non-neutralizing) antibodies, alveolar macrophages, Fc receptor, antibody dependent natural killer cell activity, complement mediated lysis, antibody dependent cell mediated cytotoxicity, CD4 and CD8 T cell mediated protection

Nucleoprotein (NP)

T-cell epitopes Cell lysis by CD8+ cytotoxic T-lymphocytes (CTL), CD4+ T lymphocyte mediated cytolysis

Neuraminidase (NA)

Conserved sialidase active site

Non-neutralizing antibodies, inhibition of the virus release, virus spread

Matrix (M1) T-cell epitope Cell lysis by CD8+ CTL, CD4+ T lymphocyte mediated cytolysis

Universal influenza vaccines

2. Manipulation and delivery of universal antigens Peptides with adjuvants : require high dose of antigen and extensive safety approval in human Peptides fused to carrier proteins : M2e + HBc, M2e + flagellin etc. unnecessary antibody production Peptides expressed in viral vectors : preexisting immunity VLPs : Poor incorporation of antigens and economical issue DNA or phage nanoparticle : limitation of immunogenicity

3. Mucosal universal vaccine strategy

Multiple peptides (M2 without TM + HA2 ) and mucosal adjuvants (CTA1 and biopolymer nanoparticle)

Mucosal delivery system (LAB surface-displayed M2 + HA2 )with adjuvant (CTA 1)

Universal influenza vaccines

Mammary glands Gastrointestinal tract (gut-associated lymphoid tissue

= GALT)

Respiratory tract (bronchus- and nasal-associated lymphoid tissues

= BALT and NALT)

• production of secretory IgA (3g/day)

• Th1 and Th2-type CD4+ responses

• CD8+ CTL responses

MALT

Reproductive tract

Mucosa-Associated Lymphoid Tissue (MALT)

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Mucosal surface is portal of entry for most of pathogens

Mucosa contains highest concentration of lymphocytes

: 6x1010 antibody-forming cells

in mucosa-associated lymphoid tissue (MALT)

: 2.5x1010 lymphocytes in lymphoid organs

Mucosal immunization

: induce both mucosal immunity and systemic immunity

Parenteral immunization (no mucosal immunity)

: Peripheral immunity

Efficient delivery vehicles is necessary: safe, repeat administration, no cross rx

with Ab

Mucosal Immunization

Lactobacillus (LAB) surface display for

mucosal vaccine

Safe microorganism - widely used for the manufacturing of foodstuffs

without causing any known health problem

Intrinsic Immunogenicity – function of adjuvant

[Abundant presence of unmethylated CpG DNA, lipoteichoic acid, and

peptidoglycan component]

Induce the mucosal and systemic immunity [Lactobacillus casei, L.

plantanum]

Immobilized antigen expressed on surface- more potent

in activating dendritic cells to present antigen to CD4+ cells

(Lee et al. Nature Biotech 2000)

Pediococcus spp Enterococcus spp Leuconostoc spp Lactobacillus spp

Probiotic property – beneficial effects on the health of the host

[Nutrient effects, Inhibition of viral & bacterial infection,

Decrease of cholesterol level, anticancer effects etc]

Advantages of Lactic Acid Bacteria as Vaccine Vehicle

Synthesis of -PGA by PgsBCA complex

PgsBCA complex [PgsA, PgsB, PgsC]

: pgsBCA complex helps high molecular -PGA pass through the cell

wall as well as attach it on the surface.

PgsA is selected as a display motif.

: Transmembrane anchor protein on cell membrane

: a stabilizer of PgsBCA complex on cell membrane

Development of Anchor Protein for LAB Display System

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Target Protein

pgsA motif

Lactobacillus

Lactobacillus

Target Protein

pgsA motif

Ce

ll n

um

be

rs

Fluorescence intensity

FACScan analysis

FITC Microscopy

A Novel LAB Display System Surface display of consensus sM2 with or without CTA1 on L. casei

Surface display of consensus sM2 with or without CTA1 on L. casei Anti-M2 IgG & IgA titers following IN or oral immunization with recombinant L. casei

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Determination of cytokines (IFN-γ and IL-4) producing Splenocytes Protection efficacy against divergent Influenza subtypes (Intranasal Inoculation)

Protection efficacy against divergent Influenza subtypes (Oral Inoculation) Lungs virus titer at 3 and 5 dpi following infection with influenza subtypes

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Lungs histopathology and immunohistochemistry of vaccinated mice after infection Long-lasting protection effects against Influenza virus

Summary

The mucosal immune system is the first immunological barrier against the pathogens.

The well conserve matrix 2 (M2) proteins and hemagglutination stalk domain (HA2) could induce

cross protective immune responses (humoral & CMI) against divergent influenza virus infections.

CTA1 induced more efficient cross protection as fusion peptides with M2 and HA2.

-PGA/chitosan nanoparticles enhanced the immune responses and confer broad protection against

lethal influenza virus infection.

We also developed a unique mucosal delivery system using surface-displayed LAB.

Cellular and mucosal immunity induced by mucosal administrations (IN or Oral) of M2 and HA2

fusion peptides appear to protect against lethal challenge of divergent influenza A viruses.

The surface-displayed M2 and HA2 on LAB further demonstrated long-lasting immunity and

conferred protection, even at 6 months after the final vaccination.

Recombinant LAB expressing CTA1-M2-HA2 fusion peptide could induce systemic and local, as

well as, cell-mediated immune responses against divergent influenza virus subtypes without hurdle

of time-consuming and high expenses for preparation of influenza vaccine for both human and

animals.

Acknowlegement

Chunam National University Chungbuk National University Jong-Soo Lee, DVM PhD Young-Ki Choi, DVM, PhD Mohammed Y.E. Chowdhury, DVM, PhD Rui Li, DVM, MS Jae-hoon Kim, DVM, MS Tae-hwan Kim, DVM

International Vaccine Institute Man-Ki Song, PhD

Bioleaders Corp. Moon-Hee Sung, PhD Seung-Pyo Hong, PhD Jae-Chul Choi , MS