A/Prof Katie Flanagan Infectious Diseases Physician and Clinical Associate Professor

Director of Clifford Craig Vaccine Trial Centre, University of Tasmania, Australia

Adjunct Senior Lecturer, Dept of Immunology & Pathology, Monash University, Melbourne

Shifting paradigms in vaccinology: immune

modulation and sex differences explored

WAidid Meeting, Milan, Italy, February 2015

o Vaccination was the greatest public health intervention of the 20th

Century

o Nobody understands how many of the commonly used vaccines work

o Historically vaccines only analysed for induction of vaccine-specific antibodies

o Never considered that they might have broader non-specific / heterologous effects on the immune system

o Never considered that the sexes might differ in their immune response to vaccination

Studies conducted in Senegal, Guinea Bissau, Haiti showed:HTMV protects against measles infectionBUT

cf standard MV females and cf males (both gps)

1992 HTMV withdrawn by WHO

First widely accepted evidence of altered all-cause mortality after vaccination and sex differences in this effect

Later shown that it was the subsequent DTP dose that increased the mortality two-fold in females Aaby et al. Lancet 2003; 361: 2183

1989 E-Z HTMV recommended by WHO at 6 mo in high measles endemic settings

Senegal Data 1987-92

Guinea-Bissau 1986-90

MV Efficacy Against Death10 studies (size of square proportional to data quality)Aaby et al., BMJ 1995; 311: 481

Randomised TrialsExtra early MV @ 4.5 months 30% in death from 4.5mo to 3yrs (45% when measles cases excluded) Aaby et al. BMJ 2010; 341: c6495

Sudan – survival benefit of early 2-dose MV Aaby et al. Vaccine 2006; 24: 2764

None of the above could be explained by improved protection against measles

Introduction of DTP into rural Guinea Bissau (1984 – 1987)Aaby et al. Int J Epidemiol 2004; 33: 374

Randomised trials of delaying DTP deemed unethical

Recent analysis of all available studies (n=35) of DTP vaccination on child survival concluded that: DTP vaccinated children had higher mortality than unvaccinated children DTP vaccinated females have higher mortality than males in all studies Reducing time of exposure to DTP as most recent vaccine by giving BCG or MV

reduced child mortalityAaby et al. BMJ Open 2012; 2: e000707

N = 868

Female-Male Mortality Ratio.5 .6 .7 .8 .9 1 2 5 10

Combined

GUINEA-BISSAU

GUINEA-BISSAU

GUINEA-BISSAU

SENEGAL

GUINEA-BISSAU

GUINEA-BISSAU

SENEGAL

GUINEA-BISSAU

GUINEA-BISSAU

SENEGAL

SENEGAL

SENEGAL

GUINEA-BISSAU

DTP VaccinationDeleterious effects worse in females cf males

Female-Male Mortality Ratio.05 .1 .5 1 2

Combined

GB + SENEGAL

MALAWI

SENEGAL

GUINEA-BISSAU

SENEGAL

GUINEA-BISSAU

SENEGAL

SENEGAL

GUINEA-BISSAU

HAITI

SENEGAL

GUINEA-BISSAU

GUINEA-BISSAU

GUINEA-BISSAU

SUDAN

Measles Vaccination Beneficial in females cf males in many observational studies

Aaby et al. Personal Communication

Three randomised trials of BCG at birth in LBW children. Combined MMR = 0.54 (0.38-0.76)

46% Reduction in Death

Aaby et al. J Infect Dis 2011; 204: 245; Biering-Sørensen, Ped Inf Dis J 2012; 31: 306

BCG used to treat bladder cancer & melanoma

6 controlled trials involving 45,662 children in US and UK in 1940’s and 50s showed BCG reduced mortality by 25%Shann Arch Dis Childhood 2010; 95: 662

o Live vaccines beneficial (BCG, MV)

o Killed vaccines deleterious (DTwP)

o Very substantial effects

o Stronger in girls generally

o Strongest in the 6 months after vaccination

o Determined by the most recent vaccine given

o When live and killed vaccines are combined the effects cancel one another

Shann. Arch Dis Child 2010; 95: 662 Flanagan et al. Vaccine 2011; 29(13):2349-54Flanagan et al. Clin Infect Dis 2013; 57(2): 283-9 Benn et al. Trends Immunol 2013; 34: 431

Terms of Reference…to review the evidence concerning the possible non-specific effects of vaccines included in the routine infant immunization schedule.

…to determine if the current evidence is sufficient to lead to adjustments in policy recommendations or to warrant further scientific investigation….

Conclusions (May 2014)Epidemiological evidence supports protective heterologous effects of BCG and MV; insufficient evidence for deleterious effects of DTwP.Not enough evidence to lead to policy changeMore studies needed powered by sex including detailed immunological studies

WHO Weekly Epidem Rec No. 21, 2014; 89: 221-36

Strategic Advisory Group of Experts WHO Working Group

Current Paradigm

o Vaccines have targeted effects against the vaccine disease only

o Males and females respond similarly to vaccines

o Vaccines can be given in any order and combination regardless of sex, season, nutritional status

New Paradigm

o Vaccines have non-vaccine specific / non-targeted effects

o Males and females differ in their response to vaccines

o The order in which vaccines are given influences morbidity and mortality

Enhanced Abs and CMI to HBV, enhanced Abs to OPV at 2m and 4.5m– BCG Pasteur

Failed to show enhanced Abs to HBV, enhanced Abs to pneumococcal serotypes (PCV-7) at 7 months - BCG Denmark, Japan, Russia

Mechanism shown to be a reprogramming of innate inflammatory responses via a modification of the NOD2 receptor on mononuclear phagocytes

Epigenetic change at the level of histonemethylation

Process has been called “trained immunity”

BCG Denmark

BCG Denmark

Transcriptome data are combined with in vitro analyses e.g. cytokine multiplex, tetramer, flow cytometry; plus proteomics, metabolomics, microbiomics providing a very powerful tool to study vaccine effects

From Pulendran PNAS 2014

Described as the next ‘golden age’

in vaccinology

+1 week

+2 weeks

+6 weeks

+4 weeks

9 MONTH

OLD

GROUP 1

GROUP 2

MEASLES VACCINE

NO VACCINE

0.5mLs whole blood into PAXgeneTM tubes

RNA extraction in The Gambia

RNA hybridised to Illumina human arrays (HumanHT12_V3_0_R1_11283641_A) containing 48,771 features

n = 24

n = 11

Blood Samples

Baseline +1 week +2 weeks +4 weeks +6 weeks

32 sex-independent group-wise comparisons yielded 3,803 differentially

expressed genes (10.8% of probesets)

Largest Network

All comparisons

(Pearson

correlation >0.75)

Analysed by T Forster and A Ivens , University of Edinburgh

Vaccinated

Controls

KEGG Pathways

RIG-I-like receptor signaling

pathway, TLR signaling pathway

Antigen processing and presentation

Chemokine signaling pathways

GO terms

Response to virus / biotic stimulus /

other organisms, immune system

process, multi-organism process,

interspecies interaction between

organisms, and defence response.Major hubs around STAT1, IRF-7& 8,

& multiple IFN induced genes

2m 3m 4m 9m 18m

GROUP 1

GROUP 2

DTP3 MV

MV + DTP3MV

Challenge

Age

RNA / Immunology Immunology

10m

GROUP 3 DTP3

19m11m

MV

YF/OPV

YF/OPV

MV/DTP Study

303 Infants Randomized into the Study

Males and Females Randomized Separately

Vaccine antibodies

RNA for whole human transcriptome analysis

Cell culture for innate and T cell responses

5mL venous blood

Vaccine Antibodies

o No sex differences in titres to MV or DTwP

o No effect of combining vaccines

Noho-Konteh et al, submitted

Plasma Cytokines

(4 weeks Post-Vaccination)

All DonorsDTP Gp: IL-7 & TNF:IL-10 & PDGF

FemalesDTP Gp:TNF:IL-10, IP-10 MV+DTP gp: IFN-γ:IL-10

MalesDTP Gp:TNF:IL-10 and IFN-γ:IL-10 and PDGF

Noho-Konteh et al, submitted

TLR Agonist Cytokine Responses

Medium HKLM(TLR2) LPS(TLR4) Flagellin(TLR5) CLO-75(TLR8)

Whole Blood in Wells

IL-1b, IL-6, IL-10, IL-12(p70), TNF-a

Mean plus SEM shown. *p<0.05, **p<0.01 significantly different in linear mixed model analysis

DTP Gp: TNF to LPS

MV Males: TNF and TNF:IL-10 to LPS Noho-Konteh et al, submitted

In Vitro Cytokine Responses to T Cell Antigens

Medium Measles Peptides TT PPD Anti-CD3/CD28

ICS CD4, CD8, IL-2, IFN-g, IL-10, IL-13

IL-4, IL1b, IL-10, IL-12(p70), Eotaxin, GMCSF, IFN-g, PDGFBB, TNF-a, VEGF

Whole Blood in Wells

Noho-Konteh et al, submitted

Females

DTP Females: type 1 pro-inflammatory responses to T cell stimulation

Mean plus SEM shown. *p<0.05 significantly different in linear mixed model analysis

Noho-Konteh et al, submitted

Males

Females

MV+DTP Females

PPD responses

MV+DTP Males

PPD responses

Mean plus SEM shown. *p<0.05, **p<0.01 significantly different in linear mixed model analysis

Noho-Konteh et al, submitted

MV

MV+DTP

DTP

All

M

F

All

M

F

All

M

F

70 differentially expressed genes (x-axis) with a 1.5 fold difference in expression in group & sex comparisons (right hand y-axis) (day of vacc vs 4 weeks post vacc).

Whole Human Genome ProfilingSystems Biology Approach

Noho-Konteh et al, submitted

MV/DTP Study: Transcriptome Profile

MV

MV+DTP

DTP

All

M

F

All

M

F

All

M

F

Opposite patterns in males and females which concur with the protein data

Noho-Konteh et al, submitted

DTP Females DTP Males

MV/DTP Study: Transcriptome Networks

NB No networks could be generated unless groups separated by sex

Down-regulated innate pathways,

recognition of bacteria & viruses

Up-regulated TCR signalling, protein kinase

signalling

Down-regulated genes of developmental

pathways, signalling processes, amino acid

metabolism

!

!A

!B

MV+DTP Males

Vaccines have non-targeted heterologous effects on innate and adaptive immunity

These can alter susceptibility to non-vaccine targeted infectious diseases and can alter all cause mortality

Females are more susceptible

We need to understand mechanisms in order to exploit beneficial and avoid harmful effects.

There are very limited immunologicalstudies to date

Conclusions

Prof Nigel Curtis, Prof of Paediatric ID, RCH, Melbourne. “BCG immunisation to prevent the development of allergy in infants: a randomised trial.” Prospective randomised trial of ~1,500 children to receive either BCG or no BCG at birth. Immunology studies to include transcriptome, proteome and microbiome analysis.

Future Directions

Professor Magdalena Plebanski, Vaccine & Infectious Diseases Laboratory, Dept of Immunology, Monash University, MelbourneSeveral ongoing studies of immunity to vaccines and vaccine mechanisms using a systems vaccinology approach to study seasonal inflluenza and DTP vaccination in the elderly.

‘Optimmunize Research Network’

International research network established 2010 to study heterologous and sex

differential effects of vaccines

www.cviva.dk

Gambia GovernmentYamu Ndow Jallow

MRC Gambia Staff Sarah Rowland-Jones, Hilton Whittle, Kim Mulholland, Jayne Sutherland

Manchester StudentsMy Thanh Le, Fran Barker

DPM, University of EdinburghPeter Ghazal, Paul Dickinson, Thorsten Forster

SSI, Denmark/BHP, Guinea BissauChristine Benn, Peter Aaby

Monash University Magdalena Plebanski, John Reynolds Funded by MRC(UK) & The Gambia

Acknowledgements

My Lab Group & Field Team in The Gambia

Should We Be Treating These Two Differently in Order to Treat Them Equally?

Transcriptional & translational effectors

Immune receptors & associated proteins

Immune response proteins

X-linked miRNAsthat regulate immune function

Pinheiro Bioess 2011; 33: 791

Fish Nat Rev Immunol 2008; 8: 737

X Chromosome Immune Response Genes

Testosterone Oestradiol Progesterone

Macrophage activity

NK-κB activity

Proinflammatorycytokines

Th1 cytokines

Th2 cytokines

Treg activity

Antibody production

Broadly oestrogens improve the outcome of infections, while androgens

increase susceptibility

Testosterone and oestrogen levels differ in males and females in the first

year of life

From Flanagan and Jensen, Sex & Gender Differences in Infection & Treatments for Infectious Diseases, Chapter 10, Eds Roberts & Klein, Springer-Verlag, in press