Why Mucosal Immunization?
• Mucosal surface is major portal of entry for pathogens
• Mucosa contains highest concentration of lymphocytes
Mucosal Immunology
6 x 1010 antibody-forming cells in mucosa-associated
and lymphoid tissue (MALT)
Vaccine Development versus
2.5 x 10 10 lymphocytes in lymphoid organs
• Mucosal immunization
- mucosal immunity
Mary Petzke, Ph.D. - systemic immunity
- prevents infection
Harvard Medical School
Systemic immunization
Children’s Hospital
- no mucosal immunity
- resolves infection before disease develops
Bordetella pertussis Bordetella pertussis
Parenteral Vaccination
• gram-negative coccobacillus Humans
• etiological agent of whooping cough Immunity diminishes in early adulthood
- serious childhood disease Only IgG is detected in nasal secretions
Mice
• colonizes respiratory mucosa
Only IgG is detected in respiratory tract
- systemic effects produced by toxins
- severe paroxysmal cough = convulsions, cyanosis,
Natural Infection/ Mucosal Vaccination
Humans
neurological damage, death
Natural infection = longterm immunity
• whole-cell vaccine introduced in 1950s
High titers of specific IgG and IgA in nasal secretions
- DPT = diphtheria toxoid, whole-cell pertussis,
Mice
tetanus toxoid
Mucosal vaccination generates specific IgG and IgA in
• no serological correlate of protection respiratory tract
Mucosal Immune System Mucosa-Associated Lymphoid Tissue (MALT)
• an integrated network of tissues, lymphoid and Gastrointestinal tract
constitutive cells and effector molecules which (gut-associated lymphoid tissue
= GALT) Mammary glands
protect the host from infection of the mucous
membrane surfaces
• separate from the peripheral (systemic) immune
system
• characterized by MALT
- production of secretory IgA (3 g/day)
- Th1 and Th2-type CD4+ responses
- CD8+ CTL responses
Respiratory tract Reproductive tract
(bronchus- and nasal-associated lymphoid tissues
= BALT and NALT)
Brush border and glycocalyx of intestinal
Mucosa: Diffuse lymphoid tissue enterocytes acts as a barrier
Brush border CD4+ T cell Glycocalyx
Epithelium B cell Microvilli brush border
Lamina propria
(connective tissue,
blood vessels, lymph)
Muscularis
mucosae
Submucosa Enterocyte
(collagen, glands,
large blood vessels)
Organized Lymphoid Tissue: Follicle
Follicle-associated epithelium M (Membraneous) Cells
(FAE)
M cell enterocyte •“Portals of entry” to the mucosal immune system
Villus
epithelium Dome Lumen
Microorganisms
Intraepithelial pocket
Corona
Villus (B cells)
lamina propria
M B T
Enterocyte
cell
Mφ
Germinal center
Interfollicular area
(T cells)
Dendritic cell
Muscularis mucosae
M cells are a major component of follicle-
M cell apical surface lacks brush border and glycocalyx
associated epithelium
Dome Enterocyte M cell
glycocalyx
brush border membraneous folds
Villus
(partial) M cells
Intraepithelial pocket
Crypt
Lymphocyte
Anti-vimentin staining for M cells in rabbit intestinal FAE
Antigen
Mucosal Immune Response Lamina propria
Effector
• Inductive phase functions
(sIgA
- priming of lymphocytes = antigen presentation production)
- migration of lymphocytes from inductive sites in mucosa
(eg., Peyer’s patches)
regional lymph nodes
Vein
Lymphatic
blood circulation
High endothelial venules
of inductive site Lymphocyte activation
mucosal effector sites (eg., lamina propria) Homing receptors completed BLOOD
(mannose 6-phosphate Spleen,
& other sugars) lymph nodes
High endothelial venules
of distal mucosal sites
Common Mucosal Immune System (CMIS) Mucosal Immune Response
• Immunological induction at one mucosal site often • Effector phase
results in immune responses at distal mucosal sites - production of antibodies
- Lymphocyte migration via the high endothelial venules _ predominantly secretory IgA (sIgA)
_ site-specific “homing”receptors on mucosal _ all isotypes expressed in lamina propria
lymphoid cells
- CD8+ T cell-mediated immunity (intraepithelial pocket)
- CD4+ T cell cytokine production (lamina propria)
∴Immunization of one mucosal inductive site may
induce mucosal immune responses in all mucosal
effector tissues
Distribution of IgA-, IgM-, IgG-, and IgD- Production of secretory IgA
producing B cells in human secretory tissues
Secretory IgA Secretory
componen
t
IgA IgA
Proteolytic cleavage
Enterocyte
Polymeric
Immunoglobulin
J-chain Receptor (pIg)
Dimeric IgA
B cell
Neutralization of antigens by mucosal IgA Strategies for Mucosal Immunization
LUMEN
• Mucosal adjuvants/delivery systems
- cholera toxin, lymphotoxin
- targeted delivery to M cells
M cell
- biodegradable microparticles
Enterocyte • Attenuated bacterial and viral vectors
- Salmonella
- adenovirus
• Site of antigen administration
B cell
B cell
- Utilization of Common Mucosal Immune System
LUMENAL INTRACELLULAR Neutralization in
neutralization neutralization LAMINA PROPRIA
Mucosal Adjuvants: Cholera toxin and lymphotoxin
Cholera toxin (CT) and E. coli lymphotoxin (LT) ADVANTAGES:
• circumvent the need for prolonged administration
• members of A-B toxin family of antigen, and for large doses of antigen, which
• highly immunogenic may induce oral tolerance
• when co-administered with antigen by mucosal • extremely effective mucosal adjuvants
route: PROBLEMS:
- induce strong systemic and mucosal T H2-type cytokine ! highly toxigenic: not acceptable for human use
responses to antigen - 5 ug CT = mild diarrhea
• IL-4, IL-5 - 25 ug CT = full 20-L cholera purge
IFN-γ, IL-2
• mechanism of adjuvanticity undefined
• serum IgG1 and mucosal sIgA
- enhanced intestinal permeability?
• induce prolonged mucosal memory to antigen - depletion of CD8+ intraepithelial T-cells?
- upregulate antigen presentation?
Mechanism of cholera toxin and lymphotoxin action Possible alternatives to holotoxin:
cholera toxin B subunit (CTB)
Intestinal epithelial cell
Trypsin ADP
ribosylation Intestinal epithelial cell
digestion
B B Adenylate
A1 A1 NAD Gs cyclase
A2 A1 B B
(OFF)
B A2 B ADPR Adenylate
cyclase
B B B (OFF)
ADPR B
Gs Adenylate
cAMP cyclase
GM1 H2O Sodium, chloride cAMP cAMP (ON) CTB
ganglioside loss transport cAMP cAMP
• Cholera toxin B subunit nontoxic
• In mice, loss of adjuvant activity compared to holotoxin
Diarrhea • Adjuvant activity in humans unknown
Dehydration
Possible alternatives to holotoxin:
Site-directed mutagenesis of A subunit M Cell Targeting
Amino acid change in Intestinal epithelial cell
NAD-binding site
Trypsin NAD Lumen
digestion binding
CT B
A1
B
A2 A1 NAD Gs
Adenylate
cyclase
Antigen
Intraepithelial pocket
A1 (OFF)
B A2 B
B
Single amino acid change
M B T
Enterocyte
in trypsin-sensitive region cell
ADP Mφ
Trypsin ribosylation
LT B
A1
B
A2
digestion
A1 A2 A1
?
NAD Gs
Adenylate
cyclase
(OFF)
B A2 B
B
Dendritic cell
NO toxicity in animal models
Currently being evaluated in two phase-I safety trials in humans
Macromolecules and microorganisms which bind
exclusively or preferentially to M-cells Reovirus binds selectively to M cells
• Macromolecules Enterocyte M cell
- IgA --secretory, monoclonal or Ag-complexed
Virus
• Viruses
- reovirus
- poliovirus
- HIV ? Virus
• Bacteria
- Vibrio cholerae
- Salmonella typhi and S. typhimurium Lymphocyte
- Shigella species
- Yersinia pseudotuberculosis
- pathogenic E. coli strains
Do M cells have specific receptors for Are M-cell surfaces more accessible than enterocyte
microorganisms and macromolecules? surfaces?
• Macromolecules Receptor Frey, Neutra et al. (1996)
- IgA --secretory, monoclonal or Ag-complexed ? • GM1 ganglioside
• Viruses - receptor for cholera toxin subunit B (CTB)
- reovirus ? - found on membranes of diverse cell types, including enterocytes
- poliovirus ? - 2.5 nm above surface of membrane = cholera toxin must come
- HIV ? ? into close contact with membrane
• Bacteria Binding to:
- Vibrio cholerae ? Conjugate Size enterocytes M-cells
- Salmonella typhi and S. typhimurium ? CTB-fluorescein 6.4 nm + +
- Shigella species ? CTB-colloidal gold 28 nm - +
- Yersinia pseudotuberculosis ? CTB-latex particles 1000 nm - -
- pathogenic E. coli strains ?
M cell Targeting: Microparticle delivery systems Poly(lactide-co-glycolide) (PLGA) microparticles
• Encapsulation of antigen(s) within biodegradable
particles • PLGA = primary candidate for vaccine development
- antigens are protected from degradation in the gut - safety established by prior use of PLGA polymers in
following oral administration humans (resorbable sutures, bone plates, drug delivery
- facilitate uptake into M cells vehicles)
• Particles known to be transported through M cells: - biodegradable
Latex Poly(butyl-cyanoacrylate) - adjuvant activity comparable to Complete Freund’s
Carbon Poly(lactide-co-glycolide) Adjuvant (CFA)
Liposomes Poly(styrene) - controlled release of antigen due to polymer degradation
• Size of particles determines response • eliminates need for booster doses
in mice, particles >5 um remain in mucosa 1 dose = elevated antibody titers 1 year later
= mucosal antibody response
particles <5 um are transported to lymph nodes and spleen
= both systemic and mucosal antibody responses
Poly(lactide-co-glycolide) microsphere-
Structure of PLGA microparticles encapsulated influenza vaccine
Poly(lactide-co-glycolide)
Antigen
Microcapsule Microsphere
Adjuvanticity of PLGA microspheres
• Bordetella pertussis filamentous hemagglutinin (FHA)
- virulence factor--mediates attachment of bacterium to epithelium • Major disadvantage of microparticles and
- administered to mice intranasally in either PBS or encapsulated in
poly(lactide-co-glycolide) (PLGA) microspheres
traditional mucosal adjuvants:
- 2 doses, 4 weeks apart
- serum and bronchoalveolar lavage fluid (BAL) assayed for antibody
response 4 weeks after second vaccination Do not elicit CTL response
Mean endpoint titer
Immunization group Serum IgG BAL IgA ∴ Not effective against viruses and invasive
PBS control --- --- bacteria
10 ug FHA in PBS 70,000 (1/5) 25 (1/5)
10 ug PLGA-encapsulated FHA 330,000 (5/5) 300 (5/5)
1 ug FHA in PBS --- ---
1 ug PLGA-encapsulated FHA 90,000 (5/5) 100 (5/5)
Attenuated bacterial vaccines: Salmonella Salmonella typi Ty21a
• Salmonella • biosynthetic mutant
S. typhi: human pathogen - multiple deletions in the galE gene
S. typhimurium: mouse pathogen - defective in the enzyme uridine diphospho-(UDP)-
- colonizes intestine galactose-4-epimerase
- invades and destroys M cells • avirulent in humans
- migrates via macrophages to spleen and liver - 5 x 1010 organisms given orally = no adverse reactions
- causes bacteremia and death • immunogenic in humans
• Immune response - protective efficacies in field trials range from 43-96%
- elicits humoral, secretory and CMI responses - 100% of North American volunteers had detectable CMI
- live Salmonella are very effective inducers of mucosal response following oral ingestion of 10 9 live organisms
immune responses; killed Salmonella are poor
immunogens
S. typhi TY21a as a vector for vaccine antigens Salmonella typhi hybrid vaccines
• Salmonella typhi-Shigella hybrid vaccine
S. typhi TY21a expressing
Bacterium A Bacterium A antigen
- S. typhi TY21a expressing Shigella group D somatic
S. typhi TY21a
antigen
- safe and immunogenic in North American volunteers
- inconsistent protective efficacy = unstable association of
Antigen
the group D antigen with bacterial surface
Gene
• Salmonella typhi-Vibrio cholerae hybrid vaccine
- S. typhi TY21a expressing the O antigen polysaccharide
Plasmid of V. cholerae LPS
- safe and immunogenic in volunteers using doses of up to
6 x 106 viable organisms
- no significant degree of protection (25%) conferred,
although diarrhea volume was reduced
∴Although promising, no unequivocal results yet.
Recombinant viral vaccines: adenovirus Recombinant Adenovirus Vaccines
• Adenovirus
• Mucosal viral vaccines - high cloning capacity: can accomodate 8.3 kb of foreign
- stimulate both mucosal and systemic humoral and CMI DNA
responses - can be made replication deficient
- generate long-lasting immunity (Sabin oral polio vaccine) Mammalian cell
• Adenovirus Adenovirus
- double-stranded DNA virus Foreign gene
- structure and biology well characterized Gene expression
Infection
- infects and replicates in mucosal tissues: GI tract, upper Viral
DNA
respiratory tract, eye and urinary bladder
- some serotypes cause mild respiratory disease in humans
- safety established Oral and intranasal vaccination of rhesus macaques with
- delivery of antigens to oral or respiratory mucosa adenovirus expressing SIV envelope protein
= cellular and humoral systemic responses, mucosal response
= reduced viral load after vaginal challenge
Route of antigen delivery Nasal and oral vaccination of humans
• Compartmentalization occurs within the Common Maximal Fold Increase In:
Mucosal Immune System Vaccination Nasal Vaginal
-some sites better at inducing responses at distal sites Secretions Secretions
-not all distal sites respond equally IgA IgG IgA IgG
∴ Deliver vaccine at site which will induce response in
desired location Nasal 9.3 56 5.7 30
• Nasal and oral vaccination of humans to generate Oral --- 6.2 4.9 20
antibody responses in vaginal tract • Conclusions:
- 2 doses of cholera toxin subunit B (CTB) - Oral vaccination does not generate an IgA response in
- female volunteers aged 19-36 years nasal secretions
- secretions collected at 1,2,3,6 weeks and at 6 months - Both oral and nasal vaccination generate IgA and IgG
after second dose responses in vaginal secretions
∴ Not all mucosal sites respond equally to induction
at a distal site!
Strategies for Mucosal Immunization
• Mucosal adjuvants/delivery systems
- cholera toxin, lymphotoxin
- targeted delivery to M cells
- biodegradable microparticles
• Attenuated bacterial and viral vectors
- Salmonella
- adenovirus
• Site of antigen administration
- Utilization of Common Mucosal Immune System