Chapter+7: Discussion+
7.4 Limitations+of+the+study+
The!size!of!our!cohort!of!motherDinfant!pairs!was!moderate!and!was!set!up!to!principally!
assess!immunological!rather!than!clinical!outcomes.!We!were!able!to!identify!a!trend!
towards!increased!morbidity!amongst!HIVDexposed,!uninfected!infants!in!keeping!with!the!
published!literature!from!larger!studies!addressing!clinical!endpoints.!The!study!size!
however,!precluded!us!from!fully!assessing!clinical!outcomes,!which!was!not!the!focus!of!the!
study.!!
!
The!study!was!conducted!in!an!informal!periDurban!settlement.!It!was!an!ideal!environment!
to!address!our!study!questions!since!there!is!a!high!prevalence!of!HIV!infection!amongst!
women!attending!maternity!services!coupled!with!a!wellDestablished!PMTCT!programme.!
The!study!setting!does!not!necessarily!reflect!provision!of!care!in!many!other!high!HIV!
burden!countries!of!the!world.!One!unique!aspect!of!the!study!setting!was!the!provision!of!
free!replacement!feeding!for!HIVDexposed!infants!whose!mothers!chose!exclusive!
replacement!feeding.!In!many!other!contexts!breastDfeeding!is!the!most!commonly!practised!
and!most!appropriate!feeding!method!for!HIVDexposed!infants.!Since!all!HIVDexposed!infants!
received!replacement!feeds,!we!were!unable!to!assess!the!effect!of!breast!milk!on!immune!
responses.!!
!
In!this!study!peripheral!blood!was!collected!from!newborn!infants!and!a!repeat!sample!was!
collected!10!weeks!after!the!BCG!vaccine!was!administered.!This!limited!the!volume!of!
blood!available!and!therefore!restricted!the!number!of!assays!that!could!be!conducted.!
Peripheral!blood!sampling!was!preferred!over!cord!blood!sampling!in!this!study!since!it!
would!not!have!been!feasible!to!collect!cord!blood!samples!in!this!setting!for!a!number!of!
reasons.!Firstly,!we!demonstrated!that!an!antenatal!recruitment!strategy!was!not!efficient!in!
this!mobile!population,!we!would!therefore!needed!to!consent!mothers!in!labour,!which!is!
not!a!suitable!setting!for!truly!informed!consent.!Secondly!it!was!not!possible!to!be!present!
at!the!delivery!of!all!the!women!from!a!logistics!or!safety!point!of!view!in!a!township!setting,!
since!many!would!have!delivered!out!of!routine!working!hours.!All!assays!using!whole!blood!
required!to!be!set!up!in!the!laboratory!within!a!few!hours!of!obtaining!the!samples.!!
!
The!limited!blood!volumes!did!not!allow!for!assessment!of!the!memory!phenotypes!of!
proliferating!cells!in!this!study.!However,!it!is!likely!that!the!T!cells!proliferating!in!response!
to!antigen!were!central!memory!cells!given!that!central!memory!cells!show!vigorous!
proliferation!in!response!to!antigen,!whereas!effector!memory!cells!have!reduced!expansion!
potential.220!!!
!
Resource!constraints!coupled!with!limited!blood!volumes!precluded!the!ability!to!use!a!
shortDterm!stimulation!assay!to!complement!the!longerDterm!lymphoproliferation!assay.!We!
chose!the!longerDterm!assay!over!the!shorterDterm!assay!to!allow!a!better!assessment!of!the!
central!memory!response,!since!this!is!thought!to!be!critical!for!longDterm!protection!
induced!by!vaccines.252!During!the!6Dday!incubation!period,!cells!divide!in!response!to!
antigen.!!This!longer!incubation!time!may!therefore!increase!the!sensitivity!of!the!assay!as!
cells!that!are!“resting”!or!require!more!than!a!short!period!of!stimulation!to!divide,!can!be!
also!detected.!A!shorterDterm!assay!is!complementary!since!it!allows!the!direct!ex#vivo!
quantification!of!antigenDinduced!T!cells!that!produce!cytokines!and!may!reflect!an!effector!
memory!response.!However,!it!is!more!sensitive!to!processing!delays.252!Given!that!a!single!
investigator!was!performing!the!clinical!and!laboratory!aspects!of!the!study,!it!would!not!
have!been!feasible!to!incubate!the!blood!within!2!hours!of!sample!collection.!!In!addition,!
Mansoor!et#al!have!recently!used!a!shortDterm!assay!to!compare!the!BCGDinduced!T!cell!
response!in!infected!and!uninfected!HIVDexposed!infants!to!control!infants!without!
demonstrating!any!effect!of!HIVDexposure!in!uninfected!infants!on!responses!to!BCG!
vaccination.127!Studies!have!shown!that!results!from!the!shortDterm!and!longerDterm!assays!
do!not!correlate!and!confirm!that!they!assess!different!aspects!of!the!immune!
response.102,253!!
!
Given!unlimited!resources!it!would!have!been!ideal!to!assess!the!innate!immune!response!to!
mycobacterial!antigens!in!motherDinfant!pairs.!The!measurement!of!cytokines!and!
chemokines!in!day!1!supernatants!may!give!some!insight!into!early!innate!immune!
response,!however!to!further!understand!the!dynamic!at!a!cellular!level,!it!would!be!optimal!
to!identify!which!cells!are!producing!the!response.!!
!
On!day!6!of!incubation,!PMA!and!ionomycin!are!added!to!assess!the!potential!of!the!
expanded!cells!to!produce!cytokine.!This!gave!a!measure!of!cell!function,!however!it!meant!
that!it!was!not!possible!to!use!a!CD4!marker!to!directly!identify!CD4+!T!cells,!rather!this!
population!of!cells!were!identified!by!the!lack!of!expression!of!CD8.!It!is!probable!therefore!
that!cells!which!were!CD8D!and!CD4D!were!included!in!the!population!of!putative!CD4+!cells,!
this!population!would!include!γδ!cells.!These!cells!are!likely!to!represent!a!small!proportion!
of!CD8D!cells!after!6!days!of!culture.!!!
!
A!viability!marker!was!used!to!discriminate!between!live!and!dead!cells.!This!was!a!major!
advantage!in!this!study,!since!a!number!of!newborn!infant!samples!displayed!high!
proportions!of!dead!cells!following!prolonged!culture.!There!is!a!high!turnDover!of!neonatal!T!
cells!which!show!an!increased!susceptibility!to!apoptosis,120!!explaining!the!need!to!exclude!
a!much!higher!number!of!newborn!infant!samples!compared!to!older!infant!or!adult!
samples.!The!exclusion!of!samples!with!high!proportions!of!dead!cells!from!the!analysis!may!
have!meant!that!we!underestimated!the!effect!of!maternal!HIV!or!Mtb!sensitisation!on!
newborn!infant!responses!to!BCG.!However,!this!approach!should!have!avoided!the!invalid!
identification!of!differences!between!groups!of!infants!since!samples!with!high!proportions!
of!dead!cells!tend!to!produce!erroneous!populations!of!cytokineDproducing!T!cells.!!
!
Many!statistical!tests!were!performed!in!the!analysis!of!the!immune!responses!to!BCG!
vaccination!in!mothers!and!infants,!so!it!is!possible!that!some!apparently!significant!findings!
could!have!occurred!by!chance.!A!cautious!approach!should!be!taken!to!results!where!only!a!
small!effect!was!shown.!Further!studies!will!be!required!to!confirm!or!refute!the!findings!of!
this!study.!In!order!to!account!for!the!multiple!comparisons,!in!a!number!of!cases!
adjustment!of!multiple!testing!was!applied!and!in!these!cases!adjusted!p!values!are!
reported.!!We!did!not!formally!adjust!for!multiple!comparisons!in!the!analysis!of!cytokine!
data.!!Individual!results!should!therefore!be!interpreted!with!caution,!the!analysis!does!
however!give!an!overview!of!patterns!and!we!have!demonstrated!consistency!between!
cytokine!responses!assessed!using!Luminex!and!multiDparameter!flow!cytometry.!
Polyfunctional!T!cells!form!a!small!proportion!of!the!overall!response!to!antigen!and!the!
total!number!of!cells!are!low.!These!results!should!also!be!interpreted!conservatively!and!
they!require!further!studies!to!confirm!the!findings.!!
!
This!study!assessed!humoral!responses!to!EPI!vaccines!in!both!mothers!and!infants.!We!did!
not!collect!data!on!maternal!vaccination!history,!due!to!limitations!in!recall!and!
documentation.!Vaccination!records!in!this!setting!are!typically!available!for!young!children!
only.!This!meant!that!we!were!unable!to!identify!the!contribution!of!maternal!vaccination!
versus!natural!exposure!to!infections!on!maternal!specificDantibody!responses.!Women!in!
the!study!had!comparable!ages;!therefore!similar!maternal!vaccination!history!between!
groups!could!be!inferred!based!on!the!date!of!the!introduction!of!the!EPI!schedule!in!South!
Africa!EPI!(1973).!Data!regarding!infant!vaccination!was!carefully!prospectively!recorded.!!!
!
Although!antibody!levels!can!be!used!to!indicate!potential!susceptibility!to!infection,!there!is!
some!uncertainty!regarding!the!functional!relevance!of!a!single!soDcalled!protective!level.!In!
addition,!protective!levels!for!collective!response!to!multiple!pneumococcal!serotypes!are!
unclear!and!there!is!paucity!of!evidence!for!defining!the!protective!levels!for!other!
antibodies!such!as!pertussis.254!Functional!assays!may!give!a!superior!assessment!of!the!
ability!of!the!immune!system!to!effectively!clear!a!pathogen.!!
!
We!were!unable!to!correlate!antibody!levels!with!longDterm!vaccine!responses!or!clinical!
outcomes!in!mothers!or!infants,!since!our!followDup!period!was!limited.!However,!this!data!
contributes!to!a!potential!explanation!for!the!higher!morbidity!and!mortality!observed!
amongst!African!HIVDexposed!infants.!For!example,!the!lower!pneumococcalDspecific!
antibody!amongst!HIVDexposed!infants!prior!to!vaccination!might!be!associated!with!
increased!severity!of!pneumonia!seen!in!this!group!of!infants.28!This!study!highlights!the!
need!for!larger!prospective!studies!to!determine!whether!the!lower!antibody!levels!in!HIVD exposed!infants!at!birth!translate!into!increased!morbidity!from!vaccineDpreventable!
infections.!!This!study!however,!can!begin!to!answer!novel!and!relevant!mechanistic!
questions!and!may!inform!future!clinical!and!vaccine!studies.!!
!