Even there are very limited immunological studies to date, Professor Flanagan explains the new paradigms in vaccinology exploring sex differences: - 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 Professor Flanagan concludes the slideset with the need to understand mechanisms in order to exploit beneficial and avoid harmful effects.

1. A/Prof Katie Flanagan
Infectious Diseases Physician and Clinical Associate Professor
Director of Clifford CraigVaccineTrial Centre, University ofTasmania, 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

2. 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

3. Studies conducted in Senegal,Guinea
Bissau, Haiti showed:
HTMV protects against measles infection
BUT
cf standard MV
females and cf males (both gps)
1992 HTMV withdrawn byWHO
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 byWHO at 6 mo in high measles endemic settings
Senegal Data
1987-92
Guinea-Bissau
1986-90

4. MV EfficacyAgainst
Death
10 studies (size of square
proportional to data
quality)
Aaby et al., BMJ 1995; 311: 481
RandomisedTrials
Extra 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

5. 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 mortality
Aaby et al. BMJ Open 2012; 2: e000707

6. 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
DTPVaccination
Deleterious 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
MeaslesVaccination
Beneficial in females cf males in
many observational studies
Aaby et al. Personal Communication

7. 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

8. 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):2349-54
Flanagan et al. Clin Infect Dis 2013; 57(2): 283-9 Benn et al.Trends Immunol 2013; 34: 431

9. 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 change
More studies needed powered by sex including detailed immunological
studies
WHO Weekly Epidem Rec No. 21, 2014; 89: 221-36
StrategicAdvisory Group of Experts
WHO Working Group

10. 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

12. 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

13.  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 histone
methylation
 Process has been called “trained immunity”
BCG Denmark

15. BCG Denmark

16. 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

17. +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 inThe Gambia
RNA hybridised to Illumina human arrays (HumanHT12_V3_0_R1_11283641_A)
containing 48,771 features
n = 24
n = 11
Blood
Samples

18. 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 byT Forster and A Ivens , University of Edinburgh
Vaccinated
Controls

19. 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

20. 2m 3m 4m 9m 18m
GROUP 1
GROUP 2
DTP3 MV
MV + DTP3
MV
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

21. Vaccine Antibodies
o No sex differences in titres
to MV or DTwP
o No effect of combining
vaccines
Noho-Konteh et al, submitted

22. Plasma
Cytokines
(4 weeks Post-
Vaccination)
All Donors
DTP Gp: IL-7 &
TNF:IL-10 &  PDGF
Females
DTP Gp: TNF:IL-10,
IP-10
MV+DTP gp:
 IFN-γ:IL-10
Males
DTP Gp: TNF:IL-10
and IFN-γ:IL-10 and 
PDGF
Noho-Konteh et al, submitted

23. 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 andTNF:IL-10 to LPS Noho-Konteh et al, submitted

24. InVitro Cytokine Responses toT 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

25. Females
DTP Females: type 1 pro-inflammatory responses toT cell stimulation
Mean plus SEM shown. *p<0.05 significantly different in linear mixed model analysis
Noho-Konteh et al, submitted

26. 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

27. 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 Profiling
Systems Biology Approach
Noho-Konteh et al, submitted

28. 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

29. 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-regulatedTCR
signalling, protein kinase
signalling
Down-regulated genes
of developmental
pathways, signalling
processes, amino acid
metabolism
!
!
A !
B
MV+DTP Males

30.  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 immunological
studies to date
Conclusions

31. Prof NigelCurtis, 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, Melbourne
Several ongoing studies of immunity to vaccines and
vaccine mechanisms using a systems vaccinology approach to
study seasonal inflluenza and DTP vaccination in the elderly.

32. ‘Optimmunize
Research Network’
International research
network established 2010 to
study heterologous and sex
differential effects
of vaccines
www.cviva.dk

33. Gambia Government
Yamu Ndow Jallow
MRC Gambia Staff Sarah Rowland-Jones,
HiltonWhittle, Kim Mulholland, Jayne Sutherland
Manchester Students
MyThanh Le, Fran Barker
DPM, University of Edinburgh
Peter Ghazal, Paul Dickinson,Thorsten Forster
SSI, Denmark/BHP, Guinea Bissau
Christine Benn, Peter Aaby
Monash University
Magdalena Plebanski, John Reynolds Funded by MRC(UK) &The Gambia
Acknowledgements

34. My Lab Group & FieldTeam
inThe Gambia

35. ShouldWe BeTreatingTheseTwo Differently
in Order toTreatThem Equally?

36. Transcriptional & translational
effectors
Immune receptors &
associated proteins
Immune response proteins
X-linked
miRNAs
that
regulate
immune
function
Pinheiro Bioess 2011; 33: 791
Fish Nat Rev Immunol
2008; 8: 737
X Chromosome Immune Response Genes

37. Testosterone Oestradiol Progesterone
Macrophage
activity
NK-κB activity
Proinflammatory
cytokines
Th1 cytokines
Th2 cytokines
Treg activity
Antibody
production
Broadly oestrogens improve the outcome of infections, while androgens
increase susceptibility

38. 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