Ryan Sanni contributed research on adjuvants and vaccines at Windsor University School of Medicine. Adjuvants help enhance the immune response to vaccines, allowing for lower doses of antigen or fewer immunizations. Common adjuvants include aluminum salts, emulsions, and toll-like receptor agonists. Ongoing research is exploring new delivery systems and immune potentiators to induce stronger and longer-lasting immunity through cellular and humoral responses.

1. Contributions made by Ryan Sanni
of Windsor University School of
Medicine
THE ROLE OF
ADJUVANTS IN
VACCINATION; CURRENT
STATUS AND
APPROACHES

2. Recent News

3. VBI Vaccines (VBIV) - eVLP

4. FDA Accepts VBI eVLP to treat GBM
•Announced August 15,2016
•IND (investigational new drug application)
enables VBI to initiate multi-center clinical
study evaluating eVLP in patients with
recurrent GBM in 2nd half of this year
•GBM tutors are susceptible to CMV with over
90% of GBM tumors expressing CMV antigens

5. Immune system works fine,
IDO pathway, tumors keep
growing

6. •Combined with GM-CSF and eVLP gB

7. • 300-500 nm in size
• Must be linked
physically
• B7 is
transmembrane
• (you need to attach
it)

8. CMV Vaccine Positive Phase 1 Study
•Announced July 27,2017
•Population: ~125 CMV-Negative Healthy Adults
•Design: Staggered Enrollment with Vaccinations at 0,
2, and 6 Months
•Primary Endpoint: Safety and Tolerability

9. CMV
•Approximately 5,000 U.S. infants development
permanent problems due to CMV. Some are
very severe including deafness, blindness,
sensory loss, and blindness
•40,000 cases of congenital HCMV each year in
U.S.
•Solid Organ Transplant Receipts CMV is the
most common viral infection

10. CMV cont’d
•In the U.S. the direct economic costs of CMV
infection exceeds $2.0 Billion annually
•Demand for CMV vaccines could exceed
7.6Million doses by 2030 with pricing at $140 a
dose, the prophylactic CMV vaccine market
could exceed $1B annually

11. Envelope eVLP
•VLP produced in cells after expression of
murine leukemia virus viral matrix protein gag
express full length CMV gb (gB-eVLP)
•CMV gB fused with transmembrane and
cytoplasmic domains from VSV G protein (gB G
eVLPs)

12. Research and Technology
•Glycoprotein B (gB) is a major target
•Two Phase II trials with adjuvant soluble
recombinant gB vaccine from seronegative
recipients from CMV positive solid organ donors
was tested
•Vaccine induced immunity waned quickly with the
greatest benefit seen in the first 12-15 months
shortly after the 6 month vaccination schedule.

13. Mammalian VLP
•Membrane expression of gB may enable induction of
neutralizing antibodies
•eVLPs produced in mammalian cells can be beneficial
due to their membrane fluidity afforded by the lipid
bilayer and mammalian glycosylation

14. Positive Results
•eVLPS expressing gB with an altered
conformation due to expression of vesicular
stomatits virus (VSV) G protein in
transmembrane and cytoplasmic domains
induced potent neutralizing antibody response
with a much greater propensity for epithelial
cells

15. Positive Results
•Antibody titers were not due to
immunogenicity of the gB-G eVLP but instead
due to the conformation structurally unique
that induces more potent neutralizing
antibodies
•Termed gB-G eVLP

16. Structural Studies
•HSV1 gB
•EBV gB
•VSV- G Protein
•Capable of undergoing large conformational
changes to enable viral fusion.
•Recombinant gB assumes a post fusion
conformation

18. Research
•Multiple adjuvants can improve gB induced
neutralizing antibody responses against
epithelial cells , this is an area of current
investigation
•Tropism via antigen structure - pentameric
•Particulate noninfections bodies are producing
during cell culture and they are being
investigated for humoral cellular responses

19. Tropism
•Study show that the structure of the antigen in a
pentameric complex is critical for epithelial cell
tropism of the vaccine
•Vaccines that target binding sites on epithelial cells
virus entry are still being investigated
•The gB protein has a universal role in viral fusion and
the conformational presentation of the gB antigen
might improve antibody response

20. Fusion Proteins
•Cytoplasmic domain is critical for cell fusion – EBV
gB
•VSV-G trans membrane and cytoplasmic domains
rather than just native CMV domains promotes
altered conformation of CMV gB on the surface of
eVLPs
•True because of syncytium formation, which
result in presentation of domains that elicit
antibodies with enhancing neutralizing effects
such as targeting epithelial cells.

21. Syncthia Studies
•Neutralizing antibodies blocking virus entry
into cells suggesting that the domain of gB
that functions for cell entry overlaps the
domain associated with synctium formation.

23. Licensed Hepatitis B Vaccine
• Sci-B-Vac
• 3rd generation Hep B vaccine demonstrated safety and effiay in over
300,000 patients Sci-B-Vac approved for use in Israel and in 14 other
countires

24. Advantages of 3rd generation
• S antigen, Pre-s1, and Pre-S2 surface antigens present in 3rd
generation vs just the S antigen in second generation (surface
antigen)

25. Idea in Increase Efficacy of Influenza Adjuvants
• GIFT4 is a fusion of GM-CSF and IL-4 or IL-5. Helps to
stimulates B cell proliferation and effector function.
Incorporate GIFT4 into GPI anchor on VLP. Benefits of
increasing levels of IgG and IgA and stronger humoral
response.

26. Efficacy Route
•Micro needle skin route efficacy is higher than
intramuscular VLP demonstrated with
delivered via Micro needle stimulated
Langerhans cells in the skin and trafficked
dendritic cells to to the draining lymph nodes
for effective induction of protective immunity.

27. Gardasil 9 – Merck & Co. (NYSE:MRK)
•9 Valent HPV VLP vaccine
•Prepared from VLP of L1 Protein of HPV types
6, 11, 16, 18, 31, 33, 45, 52, 58
•L1 Protein produced by recombinant
Saccharomyces cervisiae and assembled into
VLPs.

28. Increased Immunogens
•Purified VLPs adsorbed onto preformed
Aluminum containing adjuvant (AAHS –
Amorphous Aluminium
Hydroxphosphate Sulfate)
•Prevents more than 90% of HPV
associated cancers opposed to it’s
predecessor a quadrivalent (4 major
immunogen) vaccine that prevented 70%
of cancers.

31. What is an adjuvant?
• Adjuvant comes from the Latin adjuvare, which means to help or aid
• Adjuvants can be defined as substances that increase
immunogenicity of a vaccine formulation when added/mixed to it
• The adjuvant keeps the antigen in longer allowing the adaptive
immune system to react and prevents the innate from taking it way

32. History of Adjuvants
• 1796 Edward Jenner used
inactivated cowpox on patient with
small pox
• 100 years later Shibasaburo Kitasato
discovered antitoxin activity by
using animal sera immune to
diphtheria
• Gaston Ramon used breadcrumbs to
induce sterile abscesses to confirm
higher specific antibody response

33. History of Adjuvants cont’d
• Alexander Glenny discovered the adjuvant effect of aluminum salts.
In 1925
• Jules Freund developed a powerful adjuvant composed of a water-
in-mineral oil emulsion that also contained heat-killed mycobacteria
1930
• 1950 Johnson – LPS and lipid A

34. Adjuvant Roles
•Enhance immunogenicity of highly purified or
recombinant antigens
•Reduce the amount of antigen or the number of
immunizations needed for protective immunity
•Improve rate of seroconversion in newborns,
elderly, and immuno-compromoised
•Facilitate mucosal immunity and induce more
rapid and long lasting immune responses

36. Local vs Systemic Reactions

38. Adjuvant Regulatory Requirements
•Rigorous compared to Veterinary Vaccines
•Toxicology subjection is necessary prior to
commencement of phase I clinical trials

39. •Delivery systems can function as carriers to
which the antigens can be associated
•Create local proinflammatory response that
recruit innate immune cells to the site of
injection
Delivery Systems

40. Immune Potentiators
•Activate innate immune response through PRR or
directly via cytokines
•PRRs come in classes of receptors including TLRs
on macrophages, NOD (nucleotide-binding
oligomerzation domain), and RIG-1 (retinoic acid
inducible gene I) receptor.
•PAMPs trigger activation of innate cells that can
ultimately migrate to other tissues and produce
cytokines

41. Mineral Salts
•Alum – most widely used adjuvant since
1920’s
•Alum do not use classical TLRs and MyD88 or
TRIF to activate innate immunity.
•Sensed by NOD like receptors (NLRs) through
direct activation of NLRP3/NALP3
inflammasome complex or from release or
uric acid

42. Alum
•Causes cell death releases urate crystals among
DNA components into blood stream (rupture of
endo-lysosome and release of uric acid which acts
as a DAMP (Damage Associated Molecular
Pattern)
•Induces TH2 response associated with Asthma,
Allergies, and Eczema (IL4  IgE, IL5 IgA)
•Immune response can form against these DNA
proteins, antibodies against our own DNA
potential risk

43. Alum Cont’d
• Activates macrophages and increases MHC II expression antigen
presentation
• Cannot elicit cell mediated TH1 or CTL to control most intracellular
pathogens such as TB, Malaria, Leishmania,, Babesia, Leprosy, Aids

44. TH1 vs TH2 Response

45. Is Alum Safe
• 6 mcg/kg/day is the approved FDA safety range
• Average 2 mo infant. 5KG = 25mcg/day is the max
• HiB (Haemophilus Influenza B) = 225 mcg
• PCV (Pneumococcal) = 125mcg (Prevnar 13)
• You decide!

46. Additional Features
• 0.3% of oral aluminium is abosrbed in GI tract
• 95% is bound to transferrin and albumin
• Aluminum can bind to BBB
• Bind to phosphate in nucleus and influence many genes

47. Emulsion Adjuvants
•Freund’s Adjuvants
•CFA (complete Freund’s) water-in-oil
emulsion that contains heat killed
mycobacteria
•Used to evaluate immunogenicity of antigens
in mice and induction of autoimmune disease
like uveitis and encephalomyelitis

48. Emulsion Adjuvants
•Mycobacteria direct T lymphocytes to acquire
TH1 pattern that mediates delayed
hypersensitvity.
•Strong long last local inflammation, leads to
ulcer

49. Incomplete Freund’s (IFA)
•Water in oil emulsion without mycobacteria
•1950’s use of IFA in human influenza led to
higher long lived antibody titers compared to
without.
•Immunization of one million individuals with
IFA 40,000 showed sterile abscesses.

50. Size and Why Size Matters
•130nm in size – MF59
•30-100nm size acts via depot effect, slow
release of antigen
•Microspheres less than 10nm activate
APC’s
•Size Matters!

51. MF59 cont’d
•Initially focused on elderly subjects but later
tested in young children and infants and was
successful.
•Approved for H1N1 pandemic for pregnant
woman.

52. MF59 cont’d
•2015 MF59 Fluad approved for
accelerated licensure by US FDA for
elderly over 65
•Prepared from virus propagated in
the allantoic cavity of embryonated
hens eggs

53. Fluad - Trivalent
•Each 0.5 mL dose contains at least 15 mcg
of pure hemagglutinin (HA) from each of
the following three influenza strains
recommended for the 2017/2018 influenza
season:
•FLUAD is prepared by combining the
three virus antigens with the MF59
adjuvant.

54. ASO3
•Oil-in-water adjvuant
•(squalene + alpha tocopherol).
•Stimulates immune system by NF-kb pathway,
subsequent transcription, cytokine production
•Must administer ASO3 with antigen at same site at
same time to avoid diminished response

55. •Children 6-35 months immunized with one dose of
ASO3 developed strong immune response that was
observed 6 months after vaccination

56. Why Fluad initally not licensed in Europe?
•Concern of narcolepsy following Pandemrix
(GSK) H1N1 swine flu vaccination in 2009 had
a role of negative opinion.
•Narcolepsy reports were only associated with
the use of vaccine adjuvant with ASO3
(squalene + alpha tocopherol).
•Narcolepsy was not reported in association
with squalene only adjuvant (MF59) – 3.5
million doses in children.

57. Pandemrix by GSK
•High levels of H1N1 nuclear protein stimulate
production of antibodies to the influenza virus
and the hypocretin receptor.
•Antibodies cross blood brain barrier due to an
unrelated infection of inflammation and in the
brain the antibodies latch onto hypocretin
receptors

58. Pandremix
•Destroy/suppresses brain cells critical to
regulating sleep wake cycle.
•Out of 4 million doses in children, a total
of 380 reports of Narcolepsy.

59. MPL
•TLR 4 Agonist
•Release of TNF, IL-2, IFN-Y generates TH1
response
•Monophosphoryl lipid A produced by
chemically detoxifying bacterial LPS
(Salmonella minnesota)

61. MPL Toxicity
•Too toxic for humans due to potent
endotoxin and complications of septic
shock
•1970 detoxication for LPS was created, by
hydrolytic process which LPS from
Salmonella minnesota converted into
acetylated di-glucosamines.

63. MPL Con’td
•LPS activates MYD88 high amount of TNFa
•Canada stage 4 melanoma (Melacine)
•Ongoing trials showing Leishmanisasis, malaria
and Herpes
•Used in complex adjuvant formulations with
alum,QS21,emulsions

65. Tensoactive Adjuvants

66. Saponins
• Integrate into cell through interaction with cholesterol resulting in
pores through which antigens enter
• Peptides from these antigens may be processed and presented via
MHC class I leading to CTL CD8 response
• Severe injection site pain is a limitation

67. Sources
• Thank YOU!
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68. THANK YOU

69. Acknowledgements
Thanks Sam and Chris for the help in preparing slides

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