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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1992 HTMV withdrawn by WHO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
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