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Influenza Update

Dimitar Sajkov, MD, MMSc, DSc, PhD, FCCP, FRACP

BMJ Medical Milestones


Poll results: Top 5

Votes Proportion (%)

Sanitation
(clean water and sewage disposal)

1795 1642 1574 1337 1000

15.8 14.5 13.9 11.8 8.8

Antibiotics Anaesthesia Vaccines

Discovery of DNA structure

Influenza (Flu)

Highly contagious and potentially deadly disease Spread through coughing, sneezing, hands Caused by a virus and is not the same as a common cold It can cause serious and debilitating complications such as pneumonia, especially in the elderly and others in the 'at risk' group, such as patients with COPD

Issues to be addressed
Epidemiology - H1N1 pandemics Influenza vaccine correlates of protection Role of adjuvants Alternative manufacturing approaches Clinical trial outcomes Lessons learned

Influenza Burden

Outbreaks yearly, usually in winter months 5-20% of population get the flu Annually in Australia (non Pandemic):
1,500

deaths 80,000 GP visits and 15,000 hospitalisations

Annually in US:
200,000

hospitalizations 36,000 deaths

Influenza

Illness more severe for very young, elderly, or those with pre-existing health conditions People with a chronic disease, such as COPD, have a 40x increased risk of death from influenza A combination of heart and lung disease increases this risk 800x 1.4 million Australians aged under 65 are in the at risk group

Normal Tracheal Mucosa Influenza and The Respiratory Epithelium

7 Days Post-Infection

3 Days Post-Infection

Seasonal Flu vs Pandemic Flu


Seasonal

Pandemic

Occurs every year Occurs during winter (usually Dec-Mar) Most recover in 1-2 weeks without Rx Very young, very old, ill most at risk of serious illness

Occurs infrequently (3 per century) Occurs any time of year Some may not recover, even with Rx People of all ages may be at risk

Pandemic Influenza
Past

Pandemics:

2009 Swine Flu (H1N1) 1968 Hong Kong Flu (H3N2) 1957 Asian Flu (H2N2) 1918 Spanish Flu (H1N1)

1918-1919 Influenza pandemic


Worst of past century Estimated 2040% of world population ill 40-50 million people died 600,000 deaths in US High mortality in young adults

Infectious Disease Deaths 1900s

Loveland, 1917 & 1918


Deaths by age, influenza/pneumonia (Sept - Dec)
18 16 14 12

Deaths

10 8 6 4 2 0

0-4

5-14

15-24

25-34

35-44

45-54

55-64

65-74

over 75

Age

1917 (Sept-Dec)

1918 (Sept-Dec)

Why did young people die?


Over-reaction by the immune system called cytokine storm Those with the strongest immune systems affected Older people and youngest often die of bacterial pneumonia complicating flu - treatable now with antibiotics Even in 2011, no good treatment for cytokine storm

How Ready Are We?

What happened
As

with previous pandemics spread was only limited by the speed of travel
2.35 million passengers flew to 1018 cities in 164 countries from Mexico during March/April

April 25 2009:
WHO

declared a public emergency of international concern The Australian Health Management Plan for Pandemic Influenza was activated

Initial Epidemiology
Novel Swine-Influenza A virus (genes from both Nth

American and Eurasian swine virus lineages) Widespread community transmission in Mexico, Canada, United States, Japan, Panama, (&Vic) Household attack rate 25-30% High Hospitalization rates..especially young Estimated mortality 0.4% in Mexico Average age of deaths: 31 years 46% had no other illnesses Pregnancy associated with severe disease

Global cases

Global totals

WHO Phases of Pandemic Alert

Australian influenza data 2007- 09

Laboratory confirmed cases of pandemic (H1N1) 2009 in Australia to 11 September 2009 by jurisdiction

A 15-fold increase in ICU load compared to viral pneumonitis in previous winters

2009 H1N1 Pandemic Swine Flu


37,584 cases, 191 deaths in Australia 4,912 hospitalized cases 9 - 31% needed ICU Younger age distribution Indigenous, pregnant women
Seroprevalence varies by age 20% in <5 y/o, 40% in school age children, 30% in elderly

ICU series: 10% of ICU beds [peak utilisation]

722 admissions in Australia & NZ [H1N1\09 lab.confirmed] 15 times ICU admissions for usual Flu 93% <65 years [85% btn 25-64 yrs] 9% pregnant; 29% BMI>35; Asthma/COPD common 65% mechanical ventilation Mortality 14%
ECMO 18 times usual usage of ECMO n=68 [10 pregnancy /3 children] Median age 34 years obesity, asthma and diabetes mellitus common 21% mortality

Outcomes
Mild

seasonal influenza
case fatality (very old + very young)

0.1%

Pandemic
>

influenza

2% case fatality (mostly young adults)

2009

H1N1 pandemic
case fatality

0.5%

Influenza Prevention
Yearly

influenza shot Avoid those who are ill Wash your hands Antivirals (in special circumstances)
If you are ill - dont come to work, cover coughs and sneezes

Influenza Prevention

Annual vaccination is the single most effective measure to prevent influenza, but
Only 42% of the at risk group <65 years are being vaccinated annually

Only 38% of health care workers (US), who are at a greater risk of contracting and spreading influenza, are being vaccinated annually
15% of influenza related deaths and two thirds of hospitalisations are in the under 65 at risk group 10% of all workplace absenteeism associated with illness is due to influenza

Best way of prophylaxis Reported 50 100% effective to reduce mortality and morbidity in seasonal epidemics

Age dependant Similarity of vaccine antigen to circulating strain

Commercial influenza Trivalent Inactivated Vaccine (TIV)


No adjuvants Low immunogenicity hence require multiple dose Good antibody and weak cellular response Less effective in young infants and elderly > 65 years Limited supply (production and transportation) urgent need for improved vaccination strategy!!

Influenza Virus types

Type

A: Infects humans and other animals More severe illness Causes regular epidemics; can cause pandemics B: Infectious only to humans Causes epidemics, but less severe illness

Type

Influenza A
Subtyped by surface proteins:

Hemagglutinin (H) 16 different types Helps virus enter cells Neuraminidase (N) 9 different types Helps virus leave cell to infect others

Influenza A

All known subtypes of Influenza A found in birds H5 and H7 cause severe outbreaks in birds Human disease usually due to H1, H2, H3 and N1 and N2.

Influenza

The flu virus constantly changes


When it does, vaccines will be less efficient Immune system may be unable to recognize new virus

No immunity in population for new virus potential for pandemic

Vaccine Development

Inactivated trivalent vaccine (killed vaccine) 2 A, 1 B Effectiveness of vaccine depends on match between circulating strains and those in vaccine
2010 - 2011 Influenza Season
B/Brisbane/60/2008 (Victoria lineage)

A/California/7/2009 (H1N1)-like

A/Perth/16/2009 (H3N2)-like

Influenza Vaccine Production


Flu vaccines first produced in 1940s Only few manufacturers 6-9 months to produce vaccine

Prior Year January

Surveillance on circulating strains

Selection of specific strains


Preparation and distribution of virus stock to manufacturers Seed pools inoculated into eggs

February

March

April
N Engl J Med 351;20 November 11, 2004

May June July August


Vaccine inactivated and purified

Harvest and concentration of fluids

Vaccine blended, content verified

September
October
N Engl J Med. 351;20 November 11, 2004

Packaging, labeling, delivery

Major Challenges to Influenza Vaccine Development


Accelerate development of cell culture-based (recombinant) vaccine technology Develop novel vaccine approaches Evaluate dose-sparing strategies, especially adjuvants Broaden the use of live, attenuated vaccine approaches

Recombinant vaccines: potential benefits


Faster vaccine production

Not vulnerable to loss of egg supply


Highly scalable GMP compliant

Better characterised and more consistent product


Preserves wild type HA sequence and avoids egg-adaptation Avoids contaminants (egg proteins, viral RNA, antibiotics,
formalin, preservatives, bird adventitious agents)
Avoids risk of egg allergy Reduced likelihood of adverse reactions

Vaccine Adjuvants

Reduce amount of antigen needed


Antibody Response

Promote earlier, stronger, more durable immune responses

Vaccine with adjuvant

Vaccine without adjuvant

May increase cross-protective immune response

Days

Candidate Human Adjuvants


Adjuvant Major component Developer Antibody induction CD8 respo nse Tolerability Potential Safety Issues

Alum

Aluminium salt Squalene

++

+++

Injection pain, granulomas, eosinopjilia, macrophagic myofasciitis

MF59
Montanide

Novartis

++

++

Injection pain, adjuvant arthritis Injection pain, adjuvant arthritis Injection pain, haemolysis Injection pain, haemolysis, autoantibodies, hepatitis Injection pain, adjuvant arthritis, fever

Oil-water emulsion
Saponins Saponins liposomes LPS derivative alum

Seppic
Antigenics CSL GSK (Corixa)

+++
+++ +++ +++

+
+++ + +

+
++ ++

QS21
Iscomatrix MPL/alum

CpG
Advax

Bacterial DNA
Inulin

Coley
Vaxine

+
+++

++++
+++

++
++++

Injection pain, fever, lupus

None

Adjuvants

Alum (aluminium phosphate) currently the main vaccine adjuvant Good at stimulating antibodies Very poor at stimulating cell-mediated immunity Poorly effective at inducing enhanced immune responses against influenza haemaggultinin

Gamma-Inulin

Potent humoral and cellular immune adjuvant


Works via activation of the alternate complement pathway resulting in levels of activated C3 activates the innate immune system and results in an improved vaccine response Micro-particulate inulin (Advax) is particularly effective at boosting cellular immune responses without the normal toxicity exhibited by other cellular adjuvants such as Freunds complete adjuvant

Gamma-Inulin

Can be combined with a variety of other human approved adjuvant components, e.g. aluminium phosphate, to produce specialised adjuvants with strong cell-mediated (Th1) and humoral (Th2) activity
Successfully tested in multiple animal models in combination with many standard vaccine antigens including influenza haemagglutinin, and have also been successfully validated in human trials including in a vaccine based on the E7 protein of human papilloma virus, and in a hepatitis B prophylactic vaccine Safe and effective for use in humans

Inulin adjuvants when added to current unadjuvnated influenza vaccines, e.g. Fluvax, can:

enhance the kinetics and magnitude of anti-influenza humoral and cellular protective immune responses induce a more robust Th1-type response for more effective influenza virus clearance thereby reducing viral transmission time increase the longevity and durability of influenza vaccine induced protection

Advax

Extensively tested in animals in combination with commercial influenza vaccines with no major toxicity in any of the species tested Produced strong combined antibody and T-cell immunity to influenza, with responses up to 100x superior to the immune responses generated by the commercial influenza vaccine alone Dramatic antigen sparing effect - Allowed the normal immune response to be obtained to influenza vaccine at 1/10 the normal dose, that if confirmed in human trials could mean that in event of a pandemic that limited influenza vaccine supplies could be stretched 10 fold, and thereby potentially save many additional lives within the population

0 Day 14

st 1

immunisation immunisation

nd 2

Vaccine antigen Trivalent Inactivated Vaccine (TIV) (Kitasato Institute, Japan) (15 ng HA antigen/strain per dose i.m.) A/New Caledonia/20/99 IVR116 (H1N1) A/Hiroshima/52/2005IVR IVR116 (H3N2) B/Malaysia/2506/2004 (Influenza B)

2 4 Week Post-Immunisation 5 6

S1 S2

Serological analysis

R1 S3 Recall assay

Immunisation groups (1 mg/dose)


8
9 10 23 24

S4 R2 S5 R6 S1 2 S1 3 R7

Non adjuvanted (control) ADVAX-1 ADVAX-2 ADVAX-3

26

29

Serology assay
serum CFSE recall assay

In vitro cellular assay


Spleen cells

Antigen-specific lymphoproliferation
ELISA

supernatant

Antibody subclass
Hemagglutination Inhibition (HI) assay
I. II. III. IV. IgM IgG (total) IgG1 IgG2a

CD4+, CD8+ T cells ELISA

HA neutralizing antibody

Th1/Th2 Cytokine profile

IFN-, IL-10
Protection: 40 titre

The effect of MPI adjuvants on anti-influenza humoral responses


Hemagglutination Inhibition (HAI) assay Protective HA neutralising antibody Antibody ELISA Evolution of antibody subclass over time

To measure protective HA neutralising antibody levels after immunisation

Influenza virus

erythrocytes

hemagglutination

Antibody/antigen complexes

Anti HA antibodies

No hemagglutination

Non Adjuvanted
Slower response Lower magnitude (for influenza B consistently below protective level) Shorter lived Weakest IgG2a induction

MPI (Advax) adjuvanted


Quick response (immediate protection?) Enhanced antibody production with increased immunogenicity of TIV Consistently above protective level up to 26 weeks p.i Strong IgG2a : enhanced cellular response

5 weeks p.i

29 weeks p.i

% CD8+ Proliferation

% CD4+ Proliferation

45 ng TIV

45 ng TIV + 1 mg 45 ng TIV + 1 mg 45 ng TIV + 1 mg ADVAX-1 ADVAX-2 ADVAX-3

45 ng TIV

45 ng TIV + 1 mg 45 ng TIV + 1 mg 45 ng TIV + 1 mg ADVAX-1 ADVAX-2 ADVAX-3

Responses

Non Adjuvanted

MPI (Advax) adjuvanted


Quick and higher magnitude Enhanced longevity of protective level Strong IgG2a ( cellular response)

Humoral

Slow and lower magnitude Short lived and waned below protective level

Cellular

Consistently weak CD4+ and Robust induction of CD4+ and CD8+ CD8+ recall response over time recall responses Waned over time but remained superior to non-adjuvanted group Typical IFN- antiviral responses Enhanced IL-10 and IFN- production Bias towards Th1 responses

Cytokine

MPI adjuvants enhance the durability of influenza vaccine responses in murine model

Phase 1/2 adjuvanted influenza


150 subjects randomised into 3 groups of 50 Group 1 standard unadjuvanted 45ug HA vaccine Group 2 15ug HA plus Advax D 20mg Group 3 15ug HA plus Advax PD 20mg Blood taken at days 0, 7, 21, (120) Primary endpoint 1 Safety analysis Primary endpoint 2 - TGA vaccine release criteria at day 21 Secondary endpoint T-cell responses

TGA influenza vaccine release criteria


Modeled on WHO criteria Two categories 18-60 and >60 50 subjects per group To pass must meet at least one criteria for each of 3 serotypes
18-60 years
Seroconversion HI*4 or HI<1:10 to 1:40

>60 years
>30%

>40%

Mean GMT increase Seroprotection HI>1:40

>2.5 >70%

>2 >60%

Performance Advax influenza antigensparing vaccine against H1N1 serotype

50 subjects per group To pass must meet at least one criteria


Influenza H1N1 Pass criteria
>2.5

Fluvax 45ug unadjuvanted


2.78

Fluvax 15ug +Advax


3.27

Result

Mean GMT increase

Pass

Seroconversion HI*4 or HI<1:10 to 1:40 Seroprotection HI>1:40

>40% >70%

48% 98%

54% 100%

Pass Pass

Advax is not associated with any extra injection site pain


10 8 6 4 2 0 Fluvax Fluvax 1/3 +Advax pain score

Other potential benefits of incorporating Advax in influenza vaccines as seen in animal studies

Enhances flu vaccine responses in neonates Improves flu vaccine responses in the elderly Improves vaccine responses in diabetes, obesity or chronic disease Enhances influenza viral protection even further over current vaccine by inducing anti-influenza T cells as well as neutralising antibodies

Influenza vaccine trials at FMC


H1N1 Adjuvanted Recombinant Vaccine Trial Efficacy in high risk groups - ENINVAT
COPD Diabetes CVD Renal

mellitus

impairment over 60s Paediatric populations

ENINVAT
- Enhanced Influenza Vaccine Trial Randomized, Controlled Trial of an Enhanced Potency Seasonal Influenza Vaccine in Subjects with Chronic Diseases and Elderly

Objectives

Primary Endpoints:
Efficacy:

Seroconversion and seroprotection rates for each included serotype for adjuvanted versus unadjuvanted influenza vaccine Safety: Adverse event rates and tolerability of adjuvanted versus unadjuvanted influenza vaccine

Objectives

Secondary Endpoints:
Kinetics

and persistence of seroconversion, seroprotection and T-cell immunity for adjuvanted versus unadjuvanted influenza vaccine Degree of cross-protection against drifted influenza strains for adjuvanted versus unadjuvanted influenza vaccine Rates of influenza-mediated respiratory tract infections and hospital admissions for adjuvanted versus unadjuvanted influenza vaccine

Study
Site FMC Design - a single-centre Phase 2/3 RCT Subjects - >1000 patients and elderly subjects (200 per 4 at risk groups plus 200 healthy volunteers over 65 y.o.) recruited and randomised 1:2 to receive either the standard commercial influenza vaccine alone, or combined with 20 mg Advax

Progress
Recruited and randomised 1,400 subjects over 2008 - 2011 seasons Monitored by a safety committee No significant safety issues with Advax Preliminary efficacy results encouraging Results expected late 2011

Expected Findings

Inulin-adjuvanted influenza vaccine


Is

safe for vaccination of elderly patients Has superior immunogenicity Has significant antigen-sparing properties May show a trend towards reduced infective exacerbations of COPD

Pilot data will be collected to design a large multicentre RCT of Advax on morbidity and mortality

Traditional flu vaccine approach


Embryo detection Virus inoculation Battery egg production Egg incubation

Inactivated vaccine

Virus inactivation (formaldehyde B-propiolactone)

Virus harvesting

preservatives - ethyl mercury adjuvants - aluminium hydroxide/squalene oil

Recombinant flu vaccine approach


New flu virus sequenced
Haemagglutinin gene cloned Insect cell expression

GMP cell culture/protein purification

Adjuvant addition
Adjuvanted Recombinan t H1N1/2009 vaccine

Baculovirus expression system

Source: Protein Sciences

Source: Protein Sciences

Adjuvant Evolution
1st Generation Adjuvants (CFA, emulsions, alum, ISCOMS) Generate danger signals by inducing tissue damage associated with high reactogenicity/toxicity 2nd Generation Adjuvants (MPL, CpG) Trigger innate immune activation via receptor binding lower reactogenicity but potential autoimmunity 3rd Generation Adjuvant (Advax) Bypass innate immunity Directly induce T- and B-cell memory responses (adaptive immunity) Non-inflammatory High tolerability and absence safety issues in human studies

Advax adjuvant
Nano-particle made from delta inulin isoform Advax provides Enhanced antibody and T-cell responses Antigen-sparing Prolonged immune memory Restoration immune defects (neonates, elderly etc) TLR independent Avoids reactogenicity/toxicity of other adjuvants Avoids formulation complexity
TEM of Advax particles Advax chemical structure

Plant source

Advax enhances influenza vaccine immunogenicity


IgG Titer
2.5

IgG1 Titer

***
IgG1 Titer (OD)

0.8 0.6 0.4 0.2 0.0

***
IgG2a Titer (OD)

2.0 1.5 1.0 0.5 0.0

IgG2a Titer

*
HI GMT

150

HI Titer

**
100

IgG Titer (OD)

2.0 1.5 1.0 0.5 0.0

50

No Adjuvant

Advax

No Adjuvant

Advax

No Adjuvant

Advax

No Adjuvant

Advax

Advax enhances vaccine immunogenicity in the elderly


60

0.8

***
Total IgG (OD)
0.6 0.4

*** n.s

HI GM T

40

n.s

20

0.2 0.0

0 No Adjuvant CpG Advax

No Adjuvant CpG

Advax

Old Balb/c mice were immunized with influenza vaccine alone, CpG or Advax on day 0 and day 14 then serum was collected for HAI and ELISA assay

H1N1/2009 vaccine timelines



Late April 2009 A/H1N1/California/2009 sequence available Early May - teleconference Vaxine and Protein Sciences 11 June 2009, WHO declared a pandemic June 2009, Protein Sciences produced a GMP rHA for H1N1/2009, that was then formulated with Advax adjuvant to increase immunogenicity

July 2009 immunization 272 subjects with 2 doses of vaccine +/adjuvant

August 2009 preliminary trial results Represents one of the fastest vaccine developments in history

Recombinant H1N1/09 approach

Two doses 3 weeks apart of recombinant H1/2009 antigen at 3 doses (3, 11, 45ug) without or with Advax adjuvant Target: 6 groups of 50 subjects (300 total). Ages 18-70 Recruitment time frame 3 weeks. Budget $0

Trial endpoints: Sero-protection against H1N1/2009 (haemagglutinin inhibition assay 3 weeks after 1st and 2nd doses of vaccine) Vaccine safety by solicited and unsolicited adverse events and routine safety bloods Vaccine tolerability by visual analogue pain scores
Source: Prof. N. Petrovsky

H1N1/2009 trial result summary

<10% background immunity to H1N1/09 Recombinant Advax-adjuvanted vaccine safe and well tolerated Protection antigen - dose and age dependent HA 45ug with Advax induced protective titers in >80% of subjects <50 years No study subjects with flu symptoms in 6 months of follow-up despite this being time of peak H1N1 spread in Australia <1% subjects have shown unexpected rise in H1N1 titer post-vaccination (? subclinical infection)

Innovation in flu vaccine design



Genetically-engineered pure recombinant protein Plant-sugar derived immune-enhancer (Advax)

Doesnt involve production of flu virus


Egg independent

No problems of egg allergy Bird flu wont disrupt vaccine supply Highly pure protein vaccine (no antibiotics, formaldehyde,
viral RNA, ethyl mercury preservative)

Result: Faster, purer, safer, more scalable, pandemic


vaccine

Why new vaccine alternatives are needed

Lessons learned

The past future Pandemic planning = structure and flexibility Pandemic = widespread transmission virulence In Epidemiology, the denominator is important Poor diagnostics = huge handicap Small numbers can overwhelm critical care services Health care system buy in and assessing/responding to new information critical Important to maintain a balanced message Political and media imperatives significant Vaccination not straightforward

H1N1/2009 was only a practice run Pandemic vaccine plans need more investment Danger of complacency Egg-based manufacture too slow, cumbersome and inadequately scaleable Multiple product recalls due to inadequate potency, poor stability New technologies are needed to better counter future pandemics

Pandemic Crisis Management Plan